114 results on '"Sertoli Cells ultrastructure"'
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2. Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells.
- Author
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Lyon K, Adams A, Piva M, Asghari P, Moore ED, and Vogl AW
- Subjects
- Animals, Gene Expression Regulation, Male, Rats, Rats, Sprague-Dawley, Sertoli Cells ultrastructure, Calcium metabolism, Calcium Signaling physiology, Endoplasmic Reticulum physiology, Intercellular Junctions physiology, Sertoli Cells physiology
- Abstract
The endoplasmic reticulum (ER) in Sertoli cells is a component of unique adhesion junctions (ectoplasmic specializations-ESs) and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca2+ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. Using electron microscopy, we define two new ER-plasma membrane (PM) contact sites in apical Sertoli cell processes. One of these sites occurs at TBCs where flattened lamellar cisternae of ER envelope the swollen bulb regions of the complexes, and where the gap between adjacent membranes is 12 nm. The other is at the periphery of apical processes where the gap between membranes is 13-14 nm. Using immunolocalization at the light and electron microscopic levels, we demonstrate that Ca2+ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. Sarco/endoplasmic reticulum Ca2+-ATPase 2 (ATP2A2, SERCA2) is present at ESs; transient receptor potential channel subfamily M member 6 (TRPM6), Homer1 (HOMER1), and inositol 1,4,5-trisphosphate receptor (ITPR, IP3R) are present at ER-PM contacts associated with TBC bulbs; and stromal interacting molecule 1 (STIM1), Orai1 (ORAI1), and ATP2A2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. In Sertoli cells, the molecular machinery associated with ER generated Ca2+ fluxes is present in regions and structures directly related to junction remodeling-a process necessary for sperm release., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2017
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3. The death of sertoli cells and the capacity to phagocytize elongated spermatids during testicular regression due to short photoperiod in Syrian hamster (Mesocricetus auratus).
- Author
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Seco-Rovira V, Beltrán-Frutos E, Ferrer C, Sáez FJ, Madrid JF, and Pastor LM
- Subjects
- Animals, Cricetinae, Immunohistochemistry veterinary, In Situ Nick-End Labeling veterinary, Male, Microscopy, Electron, Transmission veterinary, Phagocytosis physiology, Photoperiod, Sertoli Cells cytology, Sertoli Cells ultrastructure, Spermatids cytology, Spermatids ultrastructure, Testis cytology, Apoptosis physiology, Mesocricetus physiology, Sertoli Cells physiology, Spermatids physiology, Testis physiology
- Abstract
In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells., (© 2014 by the Society for the Study of Reproduction, Inc.)
- Published
- 2014
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4. Palladin is a regulator of actin filament bundles at the ectoplasmic specialization in adult rat testes.
- Author
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Qian X, Mruk DD, Wong EW, Lie PP, and Cheng CY
- Subjects
- Actin Cytoskeleton genetics, Age Factors, Animals, Blood-Testis Barrier drug effects, Blood-Testis Barrier metabolism, Blood-Testis Barrier physiology, Cell Differentiation drug effects, Cell Differentiation genetics, Cells, Cultured, Cytoskeletal Proteins genetics, Cytoskeletal Proteins metabolism, Hydrazines pharmacology, Indazoles pharmacology, Male, Phosphoproteins genetics, Phosphoproteins metabolism, Rats, Rats, Sprague-Dawley, Rats, Transgenic, Sertoli Cells drug effects, Sertoli Cells metabolism, Sertoli Cells physiology, Sertoli Cells ultrastructure, Testis drug effects, Testis ultrastructure, Tight Junctions drug effects, Tight Junctions genetics, Tight Junctions physiology, Actin Cytoskeleton metabolism, Cytoskeletal Proteins physiology, Phosphoproteins physiology, Testis metabolism, Testis physiology, Tight Junctions metabolism
- Abstract
In rat testes, the ectoplasmic specialization (ES) at the Sertoli-Sertoli and Sertoli-spermatid interface known as the basal ES at the blood-testis barrier and the apical ES in the adluminal compartment, respectively, is a testis-specific adherens junction. The remarkable ultrastructural feature of the ES is the actin filament bundles that sandwiched in between the cisternae of endoplasmic reticulum and apposing plasma membranes. Although these actin filament bundles undergo extensive reorganization to switch between their bundled and debundled state to facilitate blood-testis barrier restructuring and spermatid adhesion/transport, the regulatory molecules underlying these events remain unknown. Herein we report findings of an actin filament cross-linking/bundling protein palladin, which displayed restrictive spatiotemporal expression at the apical and the basal ES during the epithelial cycle. Palladin structurally interacted and colocalized with Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein) and Arp3 (actin related protein 3, which together with Arp2 form the Arp2/3 complex to induce branched actin nucleation, converting bundled actin filaments to an unbundled/branched network), illustrating its role in regulating actin filament bundle dynamics at the ES. A knockdown of palladin in Sertoli cells in vitro with an established tight junction (TJ)-permeability barrier was found to disrupt the TJ function, which was associated with a disorganization of actin filaments that affected protein distribution at the TJ. Its knockdown in vivo also perturbed F-actin organization that led to a loss of spermatid polarity and adhesion, causing defects in spermatid transport and spermiation. In summary, palladin is an actin filament regulator at the ES.
- Published
- 2013
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5. A Wt1-Dmrt1 transgene restores DMRT1 to sertoli cells of Dmrt1(-/-) testes: a novel model of DMRT1-deficient germ cells.
- Author
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Agbor VA, Tao S, Lei N, and Heckert LL
- Subjects
- Animals, Cell Adhesion Molecules metabolism, Infertility, Male genetics, Infertility, Male metabolism, Infertility, Male pathology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Microfilament Proteins metabolism, Microscopy, Electron, Transmission, Nectins, Sertoli Cells pathology, Sertoli Cells ultrastructure, Sperm Motility physiology, Testis pathology, Testis ultrastructure, Transcription Factors metabolism, Models, Animal, Sertoli Cells metabolism, Testis metabolism, Transcription Factors deficiency, Transcription Factors genetics, Transgenes genetics
- Abstract
DMRT1 is an evolutionarily conserved transcriptional factor expressed only in the postnatal testis, where it is produced in Sertoli cells and germ cells. While deletion of Dmrt1 in mice demonstrated it is required for postnatal testis development and fertility, much is still unknown about its temporal- and cell-specific functions. This study characterized a novel mouse model of DMRT1-deficient germ cells that was generated by breeding Dmrt1-null (Dmrt1(-/-)) mice with Wt1-Dmrt1 transgenic (Dmrt1(+/-;tg)) mice, which express a rat Dmrt1 cDNA in gonadal supporting cells by directing it from the Wilms tumor 1 locus in a yeast artificial chromosome transgene. Like Dmrt1(-/-) mice, male Dmrt1(-/-) transgenic mice (Dmrt1(-/-;tg)) were infertile, while female mice were fertile. Immunohistochemistry and Western blot analysis showed transgenic DMRT1 expressed in supporting cells of the newborn gonads of both sex and in Sertoli cells of the testis afterbirth. Sertoli cells were evaluated by electron microscopy, revealing that maturation of Dmrt1(-/-;tg) Sertoli cells was incomplete. Morphological analysis of testes from 42-day-old mice showed that, compared to Dmrt1(-/-) mice, Dmrt1(-/-;tg) mice have improved seminiferous tubule structure, with lumens present in many. Immunohistochemistry of the polarity markers ESPIN and NECTIN-2 showed that DMRT1 in Sertoli cells is required for NECTIN-2 expression and influences organization of ectoplasmic specializations. Further functional analyses of the transgene on a Dmrt1(-/-) background showed that it did not rescue the decrease in Dmrt1(-/-) testis size, but when expressed on a wild-type background, exogenous DMRT1 prevented the normal age-related decline in testis size and enhanced sperm progressive motility. The studies suggest that DMRT1 in Sertoli cells regulates tubule morphology, spermatogenesis, and sperm function via its effects on Sertoli cell maturation and polarity. Furthermore, expression and function of transgenic DMRT1 in Sertoli cells establishes a novel mouse model of DMRT1-deficient germ cells generated by breeding Dmrt1-null mice with Wt1-Dmrt1 transgenic mice (rescue; Dmrt1(-/-;tg)).
- Published
- 2013
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6. Loss of occludin expression and impairment of blood-testis barrier permeability in rats with autoimmune orchitis: effect of interleukin 6 on Sertoli cell tight junctions.
- Author
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Pérez CV, Sobarzo CM, Jacobo PV, Pellizzari EH, Cigorraga SB, Denduchis B, and Lustig L
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- Animals, Autoimmune Diseases metabolism, Cell Membrane Permeability, Cell Proliferation, Cells, Cultured, Male, Orchitis metabolism, Rats, Rats, Sprague-Dawley, Testis chemistry, Testis drug effects, Testis immunology, Tight Junctions drug effects, Blood-Testis Barrier metabolism, Interleukin-6 pharmacology, Interleukin-6 physiology, Occludin metabolism, Orchitis immunology, Sertoli Cells ultrastructure, Tight Junctions physiology
- Abstract
Inflammation of the male reproductive tract is accepted as being an important etiological factor of infertility. Experimental autoimmune orchitis (EAO) is characterized by interstitial lymphomononuclear cell infiltration and severe damage of seminiferous tubules with germ cells that undergo apoptosis and sloughing. Because the blood-testis barrier (BTB) is relevant for the protection of haploid germ cells against immune attack, the aim of this study was to analyze BTB permeability and the expression of tight junction proteins (occludin, claudin 11, and tight junction protein 1 [TJP1]) in rats during development of autoimmune orchitis. The role of IL6 as modulator of tight junction dynamics was also evaluated because intratesticular content of this cytokine is increased in EAO rats. Orchitis was induced in Sprague-Dawley adult rats by active immunization with testicular homogenate and adjuvants. Control rats (C) were injected with saline solution and adjuvants. Untreated (N) rats were also studied. Concomitant with early signs of germ cell sloughing, a reduced expression of occludin and delocalization of claudin 11 and TJP1 were detected in the testes of rats with EAO compared to C and N groups. The use of tracers showed increased BTB permeability in EAO rats. Intratesticular injection of IL6 induced focal testicular inflammation, which is associated with damaged seminiferous tubules. Rat Sertoli cells cultured in the presence of IL6 exhibited a redistribution of tight junction proteins and reduced transepithelial electrical resistance. These data indicate the possibility that IL6 might be involved in the downregulation of occludin expression and in the modulation of BTB permeability that occur in rats undergoing autoimmune orchitis.
- Published
- 2012
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7. Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.
- Author
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Dias FF, Chiarini-Garcia H, Parreira GG, and Melo RC
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- Animals, Cell Count, Cell Differentiation, Cell Movement, Cell Proliferation, Cytokines biosynthesis, Macrophages cytology, Macrophages immunology, Macrophages ultrastructure, Male, Mice, Monocytes cytology, Monocytes immunology, Monocytes ultrastructure, Organelles immunology, Organelles ultrastructure, Phagocytosis immunology, Seminiferous Epithelium immunology, Seminiferous Tubules immunology, Sertoli Cells immunology, Spermatogenesis, Spermatogonia cytology, Spermatogonia immunology, Spermatogonia transplantation, Stem Cells cytology, Time Factors, Microscopy, Electron, Transmission methods, Seminiferous Epithelium ultrastructure, Seminiferous Tubules ultrastructure, Sertoli Cells ultrastructure, Spermatogonia ultrastructure, Stem Cell Transplantation methods, Stem Cells immunology
- Abstract
Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.
- Published
- 2011
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8. Sertoli cell-specific deletion of the androgen receptor compromises testicular immune privilege in mice.
- Author
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Meng J, Greenlee AR, Taub CJ, and Braun RE
- Subjects
- Animals, Antigens immunology, Autoantibodies, Gene Expression Regulation physiology, Germ Cells immunology, Immunoglobulin G metabolism, Male, Mice, Receptors, Androgen genetics, Sertoli Cells ultrastructure, Tight Junctions ultrastructure, Blood-Testis Barrier physiology, Gene Deletion, Receptors, Androgen metabolism, Sertoli Cells metabolism, Testis immunology
- Abstract
In the mammalian testis, meiotic and postmeiotic germ cell antigens are granted immune privilege. Both local immune suppression and specialized intercellular junctions between somatic Sertoli cells have been proposed to contribute to a highly restricted and effective blood-testis barrier (BTB) that helps maintain tolerance to germ cell antigens. Several studies have suggested that androgens play a role in immune suppression, although direct evidence for this is lacking. We previously reported that Sertoli cell-specific ablation of the androgen receptor (Ar) decreases expression of Cldn3, an androgen-regulated gene and component of Sertoli cell tight junctions, and increases the permeability of the BTB to biotin, a small-molecular-weight tracer. The physiological consequences of Sertoli cell-specific Ar (S-Ar) ablation on immune privilege are unknown. Here we show that in the testes of S-Ar mutant mice, the ultrastructure of Sertoli cell tight junctions is defective and testicular IgG levels are elevated. The interstitium of S-Ar mutant testes becomes populated with macrophages, neutrophils, plasma cells, and eosinophils, and serum samples of mutant mice contain antibodies against germ cell antigens. Together, these results suggest that Sertoli cell-specific deletion of the androgen receptor results in loss of testicular immune privilege. Suppressed levels of androgen signaling may be a contributing factor in idiopathic male infertility.
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- 2011
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9. A novel approach for the derivation of putative primordial germ cells and sertoli cells from human embryonic stem cells.
- Author
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Bucay N, Yebra M, Cirulli V, Afrikanova I, Kaido T, Hayek A, and Montgomery AM
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- Animals, Biomarkers metabolism, Cell Differentiation, Cell Line, Cell Movement, Cell Shape, Cell Survival, Coculture Techniques, Colony-Forming Units Assay, Embryonic Stem Cells metabolism, Embryonic Stem Cells ultrastructure, Gene Expression Regulation, Developmental, Germ Cells metabolism, Germ Cells ultrastructure, Humans, Male, Mice, Phenotype, Receptors, CXCR4 metabolism, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Cell Culture Techniques methods, Embryonic Stem Cells cytology, Germ Cells cytology, Sertoli Cells cytology
- Abstract
Using human embryonic stem cells (hESCs), we describe a novel method for the rapid derivation and enrichment of cells that are comparable to primordial germ cells (PGCs) and Sertoli cells. The methodology described is based on modest changes to the growth conditions commonly used to expand hESCs and does not require genetic manipulation or complex three-dimensional culture. Remarkably, we have determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli-like support cells. Such cells normally provide trophic support and immunoprotection to developing germ cells and may have significant clinical utility in the prevention of graft rejection. The putative Sertoli-germ cell cocultures generated in this study may ultimately be developed to study and manipulate interactions and processes involved in human gametogenesis.
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- 2009
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10. Effect of heat stress on expression of junction-associated molecules and upstream factors androgen receptor and Wilms' tumor 1 in monkey sertoli cells.
- Author
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Chen M, Cai H, Yang JL, Lu CL, Liu T, Yang W, Guo J, Hu XQ, Fan CH, Hu ZY, Gao F, and Liu YX
- Subjects
- Androgen Antagonists pharmacology, Androgens pharmacology, Animals, Cells, Cultured, Flutamide pharmacology, Heat Stress Disorders pathology, Hot Temperature, Humans, Immunohistochemistry, Intercellular Junctions drug effects, Intercellular Junctions ultrastructure, Macaca mulatta, Male, Microscopy, Electron, Rats, Rats, Sprague-Dawley, Receptors, Androgen genetics, Sertoli Cells ultrastructure, Spermatocytes metabolism, Spermatocytes ultrastructure, Testosterone pharmacology, Transfection, Heat Stress Disorders metabolism, Intercellular Junctions metabolism, Receptors, Androgen metabolism, Sertoli Cells metabolism, WT1 Proteins metabolism
- Abstract
Sertoli cells are important in determining the fate of spermatogenic cells by providing nutrition and structural support via cell junctions. In this study, we sought to examine the effect of 43 C warming on cell junctions in seminiferous epithelium and the expression of junction-associated molecules in Sertoli cells. Electron microscopy showed the appearance of large vacuoles between Sertoli and germ cells and adjacent Sertoli cells, leading to disruption of corresponding cell junctions 24 h after terminating the heat treatment. Using primary Sertoli cells isolated from pubertal monkey testes, we demonstrated that expression of adherens junction-associated molecules, such as N-cadherin and beta-catenin, and tight junction-associated molecule zonula occludens protein 1 was significantly reduced in 24-48 h after heat treatment. In contrast, intermediate filament vimentin expression was up-regulated in 6-48 h. Androgen receptor (AR) and Wilms' tumor gene 1 expression dramatically decreased after heat treatment. Both proteins completely disappeared immediately after terminating heat treatment and began to recover after 6 h. Treatment of the monkey Sertoli cells with an AR antagonist, flutamide, could mimic the heat-induced changes in the expression of junction-associated molecules in Sertoli cells. Furthermore, overexpression of AR in the Sertoli cells up-regulated the expression of N-cadherin, beta-catenin, and zonula occludens protein 1 and down-regulated vimentin expression. Their expression after heat treatment could be rescued by the AR overexpression. These results indicate that the decreased AR expression after heat treatment is involved in heat-induced cell junction disruption.
- Published
- 2008
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11. A kinesin is present at unique sertoli/spermatid adherens junctions in rat and mouse testes.
- Author
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Vaid KS, Guttman JA, Singaraja RR, and Vogl AW
- Subjects
- Adherens Junctions ultrastructure, Animals, Antibodies metabolism, Cytoplasm metabolism, Kinesins immunology, Male, Mice, Peptides metabolism, Rats, Sertoli Cells ultrastructure, Species Specificity, Spermatids ultrastructure, Adherens Junctions metabolism, Kinesins metabolism, Sertoli Cells metabolism, Spermatids metabolism
- Abstract
During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20.
- Published
- 2007
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12. An overview of cell renewal in the testis throughout the reproductive cycle of a seasonal breeding teleost, the gilthead seabream (Sparus aurata L).
- Author
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Chaves-Pozo E, Mulero V, Meseguer J, and García Ayala A
- Subjects
- Animals, Apoptosis physiology, Cell Proliferation, Germ Cells physiology, Hermaphroditic Organisms, In Situ Nick-End Labeling, Male, Reproduction physiology, Sea Bream physiology, Seasons, Sertoli Cells physiology, Sex Determination Processes, Spermatogonia ultrastructure, Testis physiology, Cell Differentiation physiology, Germ Cells ultrastructure, Sea Bream anatomy & histology, Sertoli Cells ultrastructure, Testis ultrastructure
- Abstract
The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the testicular regression process that occurs in both seasonal testicular involution and sex change. Insofar as fish reproduction is concerned, little is known about cell renewal and elimination during the reproductive cycle of seasonal breeding teleosts with asynchronous spermatogenesis. We have previously described how acidophilic granulocytes infiltrate the testis during postspawning where, surprisingly, they produce interleukin-1beta, a known growth factor for mammalian spermatogonia, rather than being directly involved in the elimination of degenerative germ cells. In this study, we are able to discriminate between spermatogonia stem cells and primary spermatogonia according to their nuclear and cytoplasmic diameters and location in the germinal epithelium, finding that these two cell types, together with Sertoli cells, proliferate throughout the reproductive cycle with a rate that depends on the reproductive stage. Thus, during spermatogenesis the spermatogonia stem cells, the Sertoli cells, and the developing germ cells (primary spermatogonia, A and B spermatogonia, and spermatocytes) in the germinal compartment, and cells with fibroblast-shaped nuclei in the interstitial tissue proliferate. However, during spawning, the testis shows few proliferating cells. During postspawning, the resumption of proliferation, the occurrence of apoptotic spermatogonia, and the phagocytosis of nonshed spermatozoa by Sertoli cells lead to a reorganization of both the germinal compartment and the interstitial tissue. Finally, the proliferation of spermatogonia increases during resting when, unexpectedly, both oogonia and oocytes also proliferate. This proliferative pattern was correlated with the gonadosomatic index, testicular morphology, and testicular and gonad areas, suggesting that complex mechanisms operate in the regulation of gonocyte proliferation in hermaphrodite fish.
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- 2005
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13. Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions.
- Author
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Guttman JA, Takai Y, and Vogl AW
- Subjects
- Adherens Junctions metabolism, Animals, Biomarkers, Cell Adhesion physiology, Cell Adhesion Molecules metabolism, Cell Communication physiology, Cell Membrane metabolism, Endosomes metabolism, Lysosomes metabolism, Male, Microscopy, Electron, Transmission, Nectins, Protein Kinase C metabolism, Protein Kinase C-alpha, Rats, Rats, Sprague-Dawley, Sertoli Cells metabolism, Spermatids metabolism, Spermatozoa metabolism, Transport Vesicles physiology, Adherens Junctions physiology, Seminiferous Epithelium cytology, Seminiferous Epithelium physiology, Sertoli Cells ultrastructure, Spermatids ultrastructure, Spermatogenesis physiology
- Abstract
Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.
- Published
- 2004
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14. 2,5-hexanedione and carbendazim coexposure synergistically disrupts rat spermatogenesis despite opposing molecular effects on microtubules.
- Author
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Markelewicz RJ Jr, Hall SJ, and Boekelheide K
- Subjects
- Animals, Antispermatogenic Agents administration & dosage, Benzimidazoles administration & dosage, Body Weight drug effects, Carbamates administration & dosage, Drug Synergism, Hexanones administration & dosage, Male, Microtubules drug effects, Organ Size drug effects, Rats, Rats, Inbred F344, Seminiferous Tubules drug effects, Seminiferous Tubules pathology, Sertoli Cells drug effects, Sertoli Cells ultrastructure, Testis pathology, Time Factors, Antispermatogenic Agents toxicity, Benzimidazoles toxicity, Carbamates toxicity, Hexanones toxicity, Spermatogenesis drug effects, Testis drug effects
- Abstract
2,5-Hexanedione (2,5-HD), a taxol-like promoter of microtubule assembly, and carbendazim (CBZ), a colchicine-like inhibitor of microtubule assembly, are two environmental testicular toxicants that target and disrupt microtubule function in Sertoli cells. At the molecular level, these two toxicants have opposite effects on microtubule assembly, yet they share the common physiologic effect of inhibiting microtubule-dependent functions of Sertoli cells. By studying a combined exposure to 2,5-HD and CBZ, we sought to determine whether CBZ would antagonize or exacerbate the effects of an initial 2,5-HD exposure. In vitro, 2,5-HD-treated tubulin had a decreased lag time and an increased maximal velocity of microtubule assembly. These 2,5-HD-induced in vitro alterations in microtubule assembly were normalized by CBZ exposure. In vivo, adult male rats were exposed to a 1% solution of 2,5-HD in the drinking water for 2.5 weeks. CBZ was administered by gavage (200 mg/kg body weight) at the same time as unilateral surgical ligation of the efferent ducts, 24 h before evaluation of the testis. Measures of testicular effect (testis weight, histopathologic changes [sloughing and vacuolization], and seminiferous tubule diameters) were all significantly altered with combined exposure. The testicular effects in the combined exposure group were either different (seminiferous tubule diameters), additive (% vacuolization), or greater than additive (% sloughing) compared to the effects of the individual toxicant exposure groups referenced to the controls. Therefore, CBZ coexposure does not antagonize the effects of an initial 2,5-HD exposure, as might be expected if their molecular effects on microtubule assembly were solely responsible for their combined toxicity; instead, 2,5-HD and CBZ act together to exacerbate the testicular injury.
- Published
- 2004
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15. Non-muscle cofilin is a component of tubulobulbar complexes in the testis.
- Author
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Guttman JA, Obinata T, Shima J, Griswold M, and Vogl AW
- Subjects
- Actin Cytoskeleton metabolism, Actin Depolymerizing Factors, Animals, Antibodies, Monoclonal, Cell Communication physiology, Fluorescent Antibody Technique, Male, Microfilament Proteins genetics, Microfilament Proteins immunology, Oligonucleotide Array Sequence Analysis, Phosphorylation, RNA, Messenger analysis, Rats, Rats, Sprague-Dawley, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Spermatids metabolism, Spermatids ultrastructure, Spermatogenesis physiology, Microfilament Proteins metabolism, Testis cytology, Testis metabolism
- Abstract
Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.
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- 2004
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16. Nuclear factor-kappaB activates transcription of the androgen receptor gene in Sertoli cells isolated from testes of adult rats.
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Zhang L, Charron M, Wright WW, Chatterjee B, Song CS, Roy AK, and Brown TR
- Subjects
- Animals, Binding Sites, CREB-Binding Protein, Cell Nucleus chemistry, Cells, Cultured, DNA metabolism, DNA Footprinting, Deoxyribonuclease I, Gene Expression, Luciferases genetics, Male, Mutagenesis, Site-Directed, NF-kappa B metabolism, Nuclear Proteins genetics, Nuclear Proteins pharmacology, Promoter Regions, Genetic genetics, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins, Sertoli Cells ultrastructure, Trans-Activators genetics, Trans-Activators pharmacology, Transfection, NF-kappa B pharmacology, Receptors, Androgen genetics, Sertoli Cells metabolism, Transcription, Genetic
- Abstract
The androgen receptor (AR) in Sertoli cells mediates the actions of testosterone on spermatogenesis. However, the transcription factors responsible for AR gene regulation in Sertoli cells remain unknown. In this study, we determined that nuclear factor-kappaB (NF-kappaB) regulates transcription of AR in primary cultures of Sertoli cells isolated from testes of adult rats. Electrophoretic mobility shift and antibody supershift assays with nuclear extracts prepared from Sertoli cells identified two binding sites, termed kappaB1 at -491/-482 bp and kappaB2 at -574/-565 bp, upstream of the transcription start site of the AR gene that bind the NF-kappaB subunits, p50 and p65. DNAse I footprint analyses showed that binding of the p50 NF-kappaB subunit protected the same regions on the rat AR promoter. Analyses of AR promoter-luciferase reporter gene activity after transfection of primary cultures of Sertoli cells demonstrated that mutation of the kappaB2 site or combined mutation of the kappaB1 and kappaB2 sites reduced activity by 40%. Preferential binding of the transcriptionally active p65/p50 heterodimer to the kappaB2 site rather than to the kappaB1 site supported these observations. Overexpression of the NF-kappaB p65 and p50 subunits in Sertoli cells increased activity from the wild-type AR promoter and the promoter with mutation of the kappaB1 site, but not the kappaB2 site. Activity was further stimulated by CBP (CREB binding protein), a coactivator of p65 transcriptional activity. Taken together, our data show that NF-kappaB is an activator of AR gene transcription in Sertoli cells and may be an important determinant of androgen activity during spermatogenesis.
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- 2004
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17. gamma-Tubulin overexpression in Sertoli cells in vivo. II: Retention of spermatids, residual bodies, and germ cell apoptosis.
- Author
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Fleming SL, Shank PR, and Boekelheide K
- Subjects
- Adenoviridae genetics, Animals, Apoptosis, Genetic Vectors, Green Fluorescent Proteins, Luminescent Proteins genetics, Male, Microtubules metabolism, Rats, Rats, Inbred F344, Recombinant Fusion Proteins genetics, Seminiferous Tubules cytology, Seminiferous Tubules metabolism, Sertoli Cells ultrastructure, Spermatogenesis, Transfection, Sertoli Cells metabolism, Spermatids cytology, Tubulin genetics
- Abstract
The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, gamma-tubulin, were delivered to Sertoli cells in vivo. gamma-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.
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- 2003
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18. gamma-Tubulin overexpression in Sertoli cells in vivo: I. Localization to sites of spermatid head attachment and alterations in Sertoli cell microtubule distribution.
- Author
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Fleming SL, Shank PR, and Boekelheide K
- Subjects
- Animals, Binding Sites, Cell Adhesion, Cells, Cultured, Gene Expression, Green Fluorescent Proteins, Luminescent Proteins genetics, Luminescent Proteins metabolism, Male, Microtubules metabolism, Microtubules ultrastructure, Rats, Rats, Inbred F344, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Seminiferous Epithelium cytology, Seminiferous Epithelium metabolism, Spermatids cytology, Spermatogenesis genetics, Spermatogenesis physiology, Transfection, Tubulin metabolism, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Spermatids metabolism, Tubulin genetics
- Abstract
Sertoli cells play a number of roles in supporting spermatogenesis, including structural organization, physical and paracrine support of germ cells, and secretion of factors necessary for germ cell development. Studies with microtubule disrupting compounds indicate that intact microtubule networks are crucial for normal spermatogenesis. However, treatment with toxicants and pharmacologic agents that target microtubules lack cell-type selectivity and may therefore elicit direct effects on germ cells, which also require microtubule-mediated activities for division and morphological transformation. To evaluate the importance of Sertoli cell microtubule-based activities for spermatogenesis, an adenoviral vector that overexpresses the microtubule nucleating protein, gamma-tubulin, was used to selectively disrupt microtubule networks in Sertoli cells in vivo. gamma-Tubulin overexpression was observed to cause redistribution of Sertoli cell microtubule networks, and overexpression of a gamma-tubulin-enhanced green fluorescent protein fusion protein was observed to localize to the site of elongate spermatid head attachment to the seminiferous epithelium.
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- 2003
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19. Sertoli cell tight junction dynamics: their regulation during spermatogenesis.
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Lui WY, Mruk D, Lee WM, and Cheng CY
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- Animals, Cell Adhesion Molecules metabolism, Humans, Junctional Adhesion Molecules, Male, Membrane Proteins metabolism, Models, Biological, Occludin, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Testis physiology, Tight Junctions metabolism, Tight Junctions ultrastructure, Sertoli Cells physiology, Spermatogenesis physiology, Tight Junctions physiology
- Abstract
During spermatogenesis, developing preleptotene and leptotene spermatocytes must translocate from the basal to the adluminal compartment of the seminiferous epithelium so that fully developed spermatids (spermatozoa) can be released to the tubular lumen at spermiation. It is conceivable that the opening and closing of the inter-Sertoli tight junctions (TJs) that constitute the blood-testis barrier are regulated by an array of intriguingly coordinated signaling pathways and molecules. Several molecules have been shown to regulate Sertoli cell TJ dynamics; they include, for example, transforming growth factor beta3 (TGFbeta3), occludin, protein kinase A, protein kinase C, and signaling pathways such as the TGFbeta3/p38 mitogen-activated protein kinase pathway. Yet the mechanisms that regulate these events are essentially not known. This minireview summarizes some of the recent advances in the study of TJ dynamics in the testis and reviews several models that can be used to study TJ dynamics. It also highlights specific areas for future research toward understanding the precise physiological relationship between junction dynamics and spermatogenesis.
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- 2003
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20. Isolation of sertoli cells from adult rat testes: an approach to ex vivo studies of Sertoli cell function.
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Anway MD, Folmer J, Wright WW, and Zirkin BR
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- Animals, Blotting, Northern, Cathepsin L, Cathepsins genetics, Cathepsins metabolism, Clusterin, Cysteine Endopeptidases, Glycoproteins genetics, Glycoproteins metabolism, Male, Microscopy, Fluorescence, Molecular Chaperones genetics, Molecular Chaperones metabolism, RNA, Messenger biosynthesis, RNA, Messenger genetics, Rats, Rats, Sprague-Dawley, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Testis metabolism, Testis physiology, Transferrin genetics, Transferrin metabolism, Sertoli Cells cytology, Sertoli Cells physiology, Testis cytology
- Abstract
Much of what is known about the molecular regulation and function of adult Sertoli cells has been inferred from in vitro studies of immature Sertoli cells. However, adult and immature cells differ in significant ways and, moreover, many Sertoli cell functions are regulated by conditions that are difficult to replicate in vitro. Our objective was to develop a procedure to isolate Sertoli cells rapidly and in sufficient number and purity to make it possible to assess Sertoli cell function immediately after the isolation of the cells. The isolation procedure described herein takes less than 4 h and does not require culturing the cells. From a single 4-mo-old adult rat, we routinely obtain 7.0 +/- 0.4 x 10(6) Sertoli cells per testis, and from a 21-mo-old rat, 7.2 +/- 0.4 x 10(6) Sertoli cells per testis. The purity, determined by morphologic analyses of plastic-embedded cells or after staining for tyrosine-tubulin or vimentin, averaged 80%. The contaminants typically included germ cells (10%) and myoid cells (10%). The germ cell-expressed genes protamine-2 and hemiferrin were not detected in the Sertoli cell preparations by Northern blot analyses, but the Sertoli cell-expressed genes clusterin, cathepsin L, and transferrin were highly expressed. Transferrin mRNA levels were greater in Sertoli cells isolated from aged than from young adult rats, consistent with previous analyses of whole testes; and cathepsin L mRNA levels were far more highly expressed in Sertoli cells isolated from stages VI-VII than from other stages of the cycle of the seminiferous epithelium, also consistent with previous analyses of whole testes and isolated tubules. These studies indicate that the freshly isolated cells retain differentiated function, and thus it should be possible to assess the in vivo function of adult Sertoli cells by isolating the Sertoli cells and immediately assessing their function.
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- 2003
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21. The interplay of collagen IV, tumor necrosis factor-alpha, gelatinase B (matrix metalloprotease-9), and tissue inhibitor of metalloproteases-1 in the basal lamina regulates Sertoli cell-tight junction dynamics in the rat testis.
- Author
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Siu MK, Lee WM, and Cheng CY
- Subjects
- Animals, Collagen Type IV genetics, Extracellular Matrix physiology, Gene Expression, Humans, Immunoblotting, Immunohistochemistry, Male, Matrix Metalloproteinase 2 genetics, Matrix Metalloproteinase 9 analysis, Matrix Metalloproteinase 9 genetics, RNA, Messenger analysis, Rats, Rats, Sprague-Dawley, Recombinant Proteins pharmacology, Reverse Transcriptase Polymerase Chain Reaction, Sertoli Cells chemistry, Spermatozoa chemistry, Testis chemistry, Testis ultrastructure, Tissue Inhibitor of Metalloproteinase-1 genetics, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha pharmacology, Collagen Type IV physiology, Matrix Metalloproteinase 9 physiology, Sertoli Cells ultrastructure, Tight Junctions physiology, Tissue Inhibitor of Metalloproteinase-1 physiology, Tumor Necrosis Factor-alpha physiology
- Abstract
During spermatogenesis, preleptotene and leptotene spermatocytes must translocate across the blood-testis barrier formed by inter-Sertoli cell-tight junctions (TJs) from the basal compartment of the seminiferous epithelium adjacent to the basement membrane to the adluminal compartment at stages VIII-IX for further development. Because of the close proximity between extracellular matrix (ECM) that constitutes the basement membrane and the blood-testis barrier, we sought to investigate the role of ECM in Sertoli cell TJ dynamics. When Sertoli cells were cultured in vitro to initiate the assembly of the Sertoli cell TJ-permeability barrier, the presence of an anticollagen IV antibody indeed perturbed the barrier. Because ECM is known to maintain a pool of cytokines and TNFalpha has been shown to regulate TJ dynamics in other epithelia, we investigated whether TNFalpha can regulate Sertoli cell TJ function via its effects on collagen alpha3(IV) and other proteins that maintain the homeostasis of ECM. As expected, recombinant TNFalpha perturbed the Sertoli cell TJ-barrier assembly in vitro dose dependently. TNFalpha also inhibited the timely induction of occludin, which is known to associate with the Sertoli cell TJ-barrier assembly. Furthermore, TNFalpha induced the expression of Sertoli cell collagen alpha3(IV), gelatinase B (matrix metalloprotease-9, MMP-9) and tissue inhibitor of metalloproteases-1 but not gelatinase A (matrix metalloprotease-2), and promoted the activation of pro-MMP-9. These results thus suggest that the activated MMP-9 induced by TNFalpha is used to cleave the existing collagen network in the ECM, thereby perturbing the TJ-barrier. This in turn creates a negative feedback that causes TNFalpha to induce collagen alpha3(IV) and tissue inhibitor of metalloproteases-1 expression so as to replenish the collagen network in the disrupted TJ-barrier and limit the activity of MMP-9. Taken collectively, these observations strengthen the notion that ECM is involved in the regulation of junction dynamics in addition to its structural role in the testis.
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- 2003
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22. Relationship of sertoli-sertoli tight junctions to ectoplasmic specialization in conventional and en face views.
- Author
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Parreira GG, Melo RC, and Russell LD
- Subjects
- Animals, Arvicolinae, Dogs, Endoplasmic Reticulum, Rough ultrastructure, Ferrocyanides, Fixatives, Freeze Fracturing, Frozen Sections, Gerbillinae, Male, Mice, Microscopy, Electron, Opossums, Osmium Compounds, Rats, Species Specificity, Actin Cytoskeleton ultrastructure, Endoplasmic Reticulum ultrastructure, Sertoli Cells ultrastructure, Tight Junctions ultrastructure
- Abstract
Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.
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- 2002
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23. Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis.
- Author
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Noguchi J, Toyama Y, Yuasa S, Kikuchi K, and Kaneko H
- Subjects
- Animals, Cell Differentiation, Cytochrome c Group metabolism, Cytoplasm ultrastructure, Inclusion Bodies ultrastructure, Male, Mice, Mice, Inbred BALB C, Mice, Nude, Microscopy, Electron, Permeability, Rats, Rats, Mutant Strains, Seminiferous Epithelium ultrastructure, Seminiferous Tubules cytology, Sertoli Cells ultrastructure, Spermatogonia ultrastructure, Testis ultrastructure, Tight Junctions metabolism, Tight Junctions ultrastructure, Transplantation, Heterologous, Blood-Testis Barrier genetics, Mutation, Spermatogenesis genetics, Spermatogonia transplantation, Spermatozoa ultrastructure
- Abstract
The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.
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- 2002
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24. Gap junctions with varied permeability properties establish cell-type specific communication pathways in the rat seminiferous epithelium.
- Author
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Risley MS, Tan IP, and Farrell J
- Subjects
- Animals, Cadaverine, Cell Membrane Permeability, Coculture Techniques, Fluorescein-5-isothiocyanate, Fluorescent Dyes, Isoquinolines, Male, Microscopy, Fluorescence, Rats, Rats, Sprague-Dawley, Sertoli Cells ultrastructure, Spermatozoa ultrastructure, Biotin analogs & derivatives, Cell Communication, Gap Junctions physiology, Seminiferous Epithelium ultrastructure
- Abstract
Dye coupling experiments were performed to determine whether the gap junctions connecting Sertoli cells with other Sertoli cells and different germ cell stages in rats showed functional variations. Chop loading of adult rat seminiferous tubules was conducted using fluorescent dextran controls and a variety of low-molecular-weight tracers (lucifer yellow, biotin-X-cadaverine, biotin cadaverine, and neurobiotin) to evaluate dye coupling in situ, and scrape loading was used to study dye coupling in Sertoli-germ cell cocultures established using prepuberal rats. Sertoli-Sertoli coupling is relatively short range and nonselective in situ, whereas coupling between Sertoli cells and chains of spermatogonia is strongly selective for the positively charged biotin tracers relative to negatively charged lucifer yellow. Coupling between Sertoli cells and spermatogonia was also asymmetric; lucifer yellow in germ cells never diffused into Sertoli cells, and biotinylated tracers only weakly diffused from spermatogonia to Sertoli cells. Asymmetric coupling would facilitate the concentration in germ cells of molecules diffusing through junctions from Sertoli cells. Dye coupling between Sertoli cells and adluminal germ cells was too weak to detect by fluorescence microscopy, suggesting that the junctional communication between these cells may be functionally different from that between Sertoli and basal germ cells. The results show that there are multiple routes of gap junction communication in rat seminiferous tubules that differ in permeability properties and show alternative gating states. Functional diversity of gap junctions may permit regulated communication among the many interacting Sertoli cells and germ cell stages in the seminiferous epithelium.
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- 2002
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25. Follicle-stimulating hormone amplifies insulin-like growth factor I-mediated activation of AKT/protein kinase B signaling in immature rat Sertoli cells.
- Author
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Khan SA, Ndjountche L, Pratchard L, Spicer LJ, and Davis JS
- Subjects
- Animals, Aromatase metabolism, Blotting, Western, Cell Survival drug effects, Cells, Cultured, Cyclic AMP Response Element-Binding Protein metabolism, DNA biosynthesis, DNA genetics, Humans, Male, Phosphorylation, Proto-Oncogene Proteins c-akt, Rats, Recombinant Proteins pharmacology, Sertoli Cells ultrastructure, Follicle Stimulating Hormone pharmacology, Insulin-Like Growth Factor I physiology, Protein Serine-Threonine Kinases physiology, Proto-Oncogene Proteins, Sertoli Cells drug effects, Signal Transduction drug effects
- Abstract
FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 microM), (Bu)(2)cAMP (40 microM), or forskolin (10 microM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10-20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1-4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 microM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells.
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- 2002
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26. Sertoli cell vacuolization and abnormal germ cell adhesion in mice deficient in an inositol polyphosphate 5-phosphatase.
- Author
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Hellsten E, Bernard DJ, Owens JW, Eckhaus M, Suchy SF, and Nussbaum RL
- Subjects
- Animals, Apoptosis physiology, Cadherins biosynthesis, Cell Adhesion genetics, Cell Membrane physiology, Cytoskeletal Proteins metabolism, Endocytosis physiology, Epithelial Cells physiology, Female, Fertility genetics, Germ Cells ultrastructure, Immunohistochemistry, Inositol Polyphosphate 5-Phosphatases, Intercellular Junctions physiology, Intercellular Junctions ultrastructure, Male, Mice, Mice, Inbred Strains, Microscopy, Electron, Phenotype, Phosphoric Monoester Hydrolases genetics, Testis ultrastructure, Trans-Activators metabolism, beta Catenin, Germ Cells physiology, Phosphoric Monoester Hydrolases deficiency, Sertoli Cells ultrastructure, Vacuoles ultrastructure
- Abstract
The dynamic nature of cellular interactions during differentiation of germ cells and their translocation from the basement membrane to the lumen of the seminiferous tubules requires the existence of complex and well-regulated cellular adhesion mechanisms in the testis. Successful migration of the developing germ cells is characterized by dynamic breakage and reformation of cadherin-containing adherens junctions between the germ cells and Sertoli cells, the polarized somatic cells of the testis that support and nourish the developing gametes. Here, we demonstrate the accumulation of abnormally swollen, actin-coated, endosome-like structures that contain intact adherens junctions and stain positive for N-cadherin and beta-catenin in the Sertoli cell cytosol of mice deficient in Inpp5b, an inositol polyphosphate 5-phosphatase. Simultaneous to the formation of these abnormal structures, developing germ cells are prematurely released from the seminiferous epithelium and sloughed into the epididymis. Our results demonstrate a role for Inpp5b in the regulation of cell adhesion in the testis and in the formation of junctional complexes with neighboring cells, and they emphasize the important and essential role of phosphoinositides in spermatogenesis.
- Published
- 2002
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27. Heat shock-initiated apoptosis is accelerated and removal of damaged cells is delayed in the testis of clusterin/ApoJ knock-out mice.
- Author
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Bailey RW, Aronow B, Harmony JA, and Griswold MD
- Subjects
- Acetates pharmacology, Animals, Clusterin, Cytoplasm chemistry, DNA Fragmentation, Female, Glycoproteins analysis, Glycoproteins physiology, Immunohistochemistry, In Situ Nick-End Labeling, Male, Mice, Mice, Knockout, Molecular Chaperones analysis, Molecular Chaperones physiology, Organ Size, Sertoli Cells ultrastructure, Sperm Count, Spermatogenesis, Spermatozoa drug effects, Spermatozoa ultrastructure, Testis drug effects, Apoptosis, Glycoproteins deficiency, Hot Temperature, Testis cytology
- Abstract
The secretion and localization of clusterin in the testis has led to the hypothesis that clusterin plays a role in spermatogenesis. Furthermore, the association of clusterin with apoptosis, cellular injury, disease, and regression of nongonadal tissues has led to the hypothesis that clusterin acts to protect cells from apoptosis or may be involved in tissue remodeling. To investigate the role of clusterin in the testis, we analyzed clusterin knock-out (cluKO) mice to determine the impact of the absence of clusterin on spermatogenesis. Furthermore, we investigated the cellular response to injury caused by methoxyacetic acid (MAA) toxicity and mild heat exposure in the cluKO mice to determine the extent to which clusterin protects against apoptosis or participates in tissue remodeling. We found that cluKO mice were fertile and had essentially normal spermatogenesis with the exception of some incomplete spermiation after stage VIII. No differences in testicular morphology or the incidence of apoptosis in the testis were seen between the cluKO and clusterin wild-type (cluWT) mice after MAA treatment. In contrast, apoptosis was delayed in the cluWT mice compared with the cluKO mice after heat exposure, suggesting that clusterin does have a slight protective effect against apoptosis under some conditions. Also, a dramatic loss of germ cells after heat stress occurred earlier in the cluWT testes than in the cluKO testes. Clusterin is clearly acting in a dual role in that cells can be protected from damage and dead cells can be more easily removed after some types of cellular damage but not after others.
- Published
- 2002
- Full Text
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28. Dynamic testicular adhesion junctions are immunologically unique. I. Localization of p120 catenin in rat testis.
- Author
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Johnson KJ and Boekelheide K
- Subjects
- Actins analysis, Animals, Antibodies, Monoclonal, Blotting, Western, Catenins, Cell Differentiation, Desmosomes chemistry, Fluorescent Antibody Technique, Immunosorbent Techniques, Intercellular Junctions chemistry, Intermediate Filament Proteins analysis, Male, Plectin, Rats, Rats, Inbred F344, Sertoli Cells ultrastructure, Spermatids ultrastructure, Spermatozoa ultrastructure, Tissue Distribution, Delta Catenin, Cell Adhesion Molecules analysis, Phosphoproteins analysis, Testis chemistry
- Abstract
In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact, but the molecular composition of such junctions is not well known. At prototypical adherens junctions, proteins termed catenins bind to the intracellular domain of classic cadherins and regulate the strength of adhesion. Using a panel of monoclonal antibodies (5A7, 8D11, and 15D2), p120 catenin (p120) was localized in postnatal and adult rat testis cryosections and touch preparations by immunofluorescence. Immunoprecipitation of testis homogenates showed that at least four p120 isoforms were expressed from Postnatal Day 7 through adulthood. Both inter-Sertoli and Sertoli-germ cell junctions were p120-positive, however, individual p120 monoclonals were localized to specific junctions. The 5A7 and 8D11 antibodies colocalized with beta-catenin and plectin at inter-Sertoli and Sertoli-spermatocyte junctions. At inter-Sertoli junctions, p120 was juxtaposed to but did not colocalize with f-actin. Thus, p120 is likely a component of inter-Sertoli desmosome-like junctions. In contrast, the 15D2 monoclonal antibody specifically immunostained Sertoli-round spermatid and inter-Sertoli cell junctions in a dynamic pattern. From the time that round spermatids form to their differentiation into elongate spermatids, Sertoli-round spermatid 15D2 immunostaining cycled from a single mass to a curvilinear pattern, and finally to punctate structures scattered throughout the epithelium. This localization and stage-specific immunostaining pattern indicated that 15D2 recognized Sertoli-round spermatid desmosome-like junctions. Between Sertoli cells, 15D2 immunostained newly formed junctions (at Postnatal Days 21 through 43), but not mature junctions in the adult. From these data, we conclude that p120 is a component of most, if not all, desmosome-like junctions, and that desmosome-like junctions between different cell types contain a unique molecular composition.
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- 2002
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29. Dynamic testicular adhesion junctions are immunologically unique. II. Localization of classic cadherins in rat testis.
- Author
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Johnson KJ and Boekelheide K
- Subjects
- Animals, Antibodies, Monoclonal, Blotting, Western, Male, Peptide Fragments analysis, Rats, Rats, Inbred F344, Sertoli Cells ultrastructure, Spermatids ultrastructure, Testis chemistry, Testis growth & development, Cadherins analysis, Cell Adhesion, Intercellular Junctions chemistry, Testis ultrastructure
- Abstract
In the seminiferous epithelium, morphologically diverse junctions mediate inter-Sertoli and Sertoli-germ cell adhesive contact and likely transmit signals between contacting cells. Defining the molecular composition of testicular cell-cell junctions is an important step in determining their function. Proteins belonging to the cadherin superfamily are important mediators of cell-cell adhesion, as well as cell signaling. Here, we determined the spatial and temporal protein expression of four classic cadherins in rat testis: N-cadherin, cadherin-6, cadherin-11, and a cadherin defined by an antiserum generated against a conserved classic cadherin peptide (L4). Through Western blot analysis, all antibodies recognized unique proteins. Similarly, each cadherin displayed unique, cell-type specific immunostaining patterns. Whereas N-cadherin, cadherin-11, and L4-positive cadherin were expressed from Postnatal Day 7 through adulthood, cadherin-6 protein was not present at Postnatal Day 7 and first appeared at Day 21. Immunostaining of testis cryosections on Postnatal Days 7, 21, 31, 43, and those of adults indicated that cadherin-11 localized to peritubular cell junctions. N-cadherin immunostaining localized to basal inter-Sertoli junctions, Sertoli-spermatocyte junctions, and at about stages I-VII in Sertoli-elongate spermatid junctions. Cadherin-6 immunostaining was restricted to Sertoli-round spermatid and in Sertoli-elongate spermatid junctions at approximately stages XII-I. Finally, L4-positive immunostaining also detected Sertoli-round spermatid junctions in addition to Sertoli-elongate spermatid junctions at approximately stages XII-I. These data show that the various testicular cell-cell junctions are molecularly unique and dynamic complexes.
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- 2002
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30. GLI1 localization in the germinal epithelial cells alternates between cytoplasm and nucleus: upregulation in transgenic mice blocks spermatogenesis in pachytene.
- Author
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Kroft TL, Patterson J, Won Yoon J, Doglio L, Walterhouse DO, Iannaccone PM, and Goldberg E
- Subjects
- Animals, Apoptosis, Epithelial Cells chemistry, Epithelial Cells ultrastructure, Gene Expression, Hedgehog Proteins, Isoenzymes analysis, L-Lactate Dehydrogenase analysis, Male, Mice, Mice, Knockout, Mice, Transgenic, Microtubules chemistry, Mitosis, Oncogene Proteins genetics, Sertoli Cells ultrastructure, Spermatozoa enzymology, Trans-Activators genetics, Transcription Factors genetics, Zinc Finger Protein GLI1, Zinc Fingers, Cell Nucleus chemistry, Cytoplasm chemistry, Oncogene Proteins analysis, Spermatogenesis, Testis ultrastructure, Transcription Factors analysis
- Abstract
The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.
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- 2001
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31. Disruption of spermatogenesis and Sertoli cell structure and function by the indenopyridine CDB-4022 in rats.
- Author
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Hild SA, Reel JR, Larner JM, and Blye RP
- Subjects
- Androgen-Binding Protein analysis, Animals, Apoptosis drug effects, Epididymis chemistry, Epididymis drug effects, Female, Fertility drug effects, Inhibins blood, Leydig Cells drug effects, Leydig Cells physiology, Male, Organ Size drug effects, Rats, Rats, Sprague-Dawley, Seminiferous Tubules cytology, Seminiferous Tubules drug effects, Sertoli Cells physiology, Sertoli Cells ultrastructure, Sperm Count, Spermatids drug effects, Spermatogonia cytology, Spermatogonia drug effects, Spermatozoa cytology, Spermatozoa drug effects, Testis anatomy & histology, Vacuoles drug effects, Antispermatogenic Agents pharmacology, Indenes pharmacology, Piperidines pharmacology, Sertoli Cells drug effects, Spermatogenesis drug effects
- Abstract
The present studies were undertaken to determine the testicular cell type(s) affected by the antispermatogenic indenopyridine CDB-4022. At the oral threshold dose (2.5 mg/kg), CDB-4022 induced infertility in all males. CDB-4022 did not alter (P > 0.05) Leydig cell function as assessed by circulating testosterone, seminal vesicle, and ventral prostate weights or body weight gain compared to controls. Conversely, CDB-4022 reduced (P < 0.05) testicular weight, spermatid head counts, and percentage of seminiferous tubules undergoing spermatogenesis. In a second study, adult male rats received a maximally effective oral dose of CDB-4022 (12.5 mg/kg), dipentylphthalate (DPP; 2200 mg/kg; a Sertoli cell toxicant), or vehicle and were necropsied 3, 6, or 12 h after dosing to determine acute effects. Serum inhibin B levels were suppressed (P < 0.05) by 6 h after CDB-4022 or DPP treatment, but epididymal androgen-binding protein (ABP) levels were not altered (P > 0.05), compared to controls. CDB-4022 and DPP increased (P < 0.05) the percentage of tubules with apoptotic germ cells, particularly differentiating spermatogonia and spermatocytes, by 12 h after dosing. Microscopic examination of the testis indicated a greater degree of vacuolation in Sertoli cells and initial signs of apical germ cell sloughing/shedding by 3 or 12 h after CDB-4022 or DPP treatment, respectively. In a third study, prepubertal male rats were treated with vehicle, 12.5 mg/kg of CDB-4022, or 2200 mg/kg of DPP, and the efferent ducts of the right testis were ligated 23 h before necropsy. Seminiferous tubule fluid secretion (difference in weight of testes), serum inhibin B levels, and ABP levels in the unligated epididymis were reduced (P < 0.05) at 24 and 48 h after dosing in CDB-4022- and DPP-treated rats compared to controls. Collectively, these data suggest that CDB-4022 disrupts spermatogenesis by inducing apoptosis in early stage germ cells via a direct action on the Sertoli cell.
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- 2001
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32. Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice.
- Author
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Jeffs B, Meeks JJ, Ito M, Martinson FA, Matzuk MM, Jameson JL, and Russell LD
- Subjects
- Animals, DAX-1 Orphan Nuclear Receptor, DNA-Binding Proteins deficiency, Infertility, Male etiology, Infertility, Male metabolism, Infertility, Male pathology, Leydig Cells ultrastructure, Male, Mice, Mice, Knockout, Receptors, Retinoic Acid deficiency, Rete Testis ultrastructure, Sertoli Cells ultrastructure, Transcription Factors deficiency, DNA-Binding Proteins genetics, Infertility, Male genetics, Leydig Cells physiology, Receptors, Retinoic Acid genetics, Repressor Proteins, Rete Testis physiology, Sertoli Cells physiology, Transcription Factors genetics
- Abstract
DAX-1, an X-linked member of the orphan nuclear receptor superfamily of transcription factors, plays a key role in sex determination and gonadal differentiation. Dax1-deficient male mice are infertile and have small testes despite normal serum levels of T and gonadotropins. Examination of Dax1-deficient testes reveals dilated seminiferous tubules and abnormal parameters of sperm fertilizing capability consistent with a possible obstruction in the testis. To test this hypothesis, we performed a comprehensive evaluation of the male reproductive tract in Dax1-deficient mice. Light and electron microscopic examination revealed the rete testis is blocked by aberrantly located Sertoli cells, creating a tailback of necrosing sperm in the testis. Sertoli cells also obstruct the proximal and middle efferent ductules, and this is accompanied by an overgrowth of the efferent duct epithelium. Seminiferous tubules close to the rete testis contain ectopic Leydig cells, distinct from the hyperplastic Leydig cells present in the interstitial space. The peritubular tissue surrounding these tubules is frequently abnormal, containing relatively undifferentiated myoid cells and no basement membrane between the myoid cells and Sertoli cells. A third of aged (>1-yr-old) Dax1-deficient male mice develop sex cord-stromal tumors, derived from cells of the Sertoli/granulosa cell or Leydig cell lineages. Combined, these observations reveal abnormal differentiation and proliferation of Leydig cells and Sertoli cells in Dax1-deficient male mice, leading to obstruction of the rete testis and infertility.
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- 2001
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33. Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11.
- Author
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Lui WY, Lee WM, and Cheng CY
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cells, Cultured, Claudins, Male, Membrane Proteins chemistry, Membrane Proteins genetics, Molecular Sequence Data, Occludin, Permeability, Rats, Rats, Sprague-Dawley, Recombinant Proteins pharmacology, Sertoli Cells ultrastructure, Tight Junctions metabolism, Zonula Occludens-1 Protein, Membrane Proteins physiology, Nerve Tissue Proteins, Phosphoproteins physiology, Sertoli Cells metabolism, Tight Junctions drug effects, Transforming Growth Factor beta pharmacology
- Abstract
Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that create the blood-testis barrier in the rat must be disassembled and reassembled to permit the timely passage of preleptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism(s) and the participating molecules that regulate this event are largely unknown. Although there is no in vitro model to study the event and regulation of inter-Sertoli TJ disassembly, primary cultures of Sertoli cells in vitro can be used to study junction assembly. In this study, we sought to investigate whether cytokines are involved in the inter-Sertoli TJ assembly in vitro. Sertoli cells isolated from 20-day-old rats were cultured at a density of 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units for 8-9 days. The steady-state messenger RNA levels of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta2, and TGF-beta3 at different time points were assessed by semiquantitative RT-PCR. In selected experiments, the assembly of inter-Sertoli TJs was monitored by transepithelial electrical resistance measurement. It was found that there was no change in the expression of basic fibroblast growth factor throughout the entire culture period. However, there was a 2-fold reduction in the expression of TGF-beta2 and TGF-beta3 at the time inter-Sertoli TJs were being assembled. On days 5-8, after the inter-Sertoli TJs had been assembled, the Sertoli cell steady-state messenger RNA levels of TGF-beta2 and TGF-beta3 increased by as much as 3- and 6-fold, respectively, when compared with Sertoli cells on days 1-3 when TJs were being assembled. Also, it was found that recombinant TGF-beta3 added to Sertoli cells cultured in vitro at 1.2 x 10(6) cells/cm(2) on Matrigel-coated bicameral units perturbed the inter-Sertoli TJ permeability barrier dose-dependently. Moreover, the presence of TGF-beta3 also inhibited the transient and/or basal expression of several TJ-associated proteins, which include occludin, zonula occludens-1, and claudin-11 when inter-Sertoli TJs were being assembled in vitro. These results suggest that TGF-beta plays a crucial role in regulating the complicated biochemical events of junction assembly in the testis.
- Published
- 2001
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34. Is cadmium chloride-induced inter-sertoli tight junction permeability barrier disruption a suitable in vitro model to study the events of junction disassembly during spermatogenesis in the rat testis?
- Author
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Chung NP and Cheng CY
- Subjects
- Animals, Cadherins genetics, Cells, Cultured, Follicle Stimulating Hormone pharmacology, Male, Membrane Proteins genetics, Occludin, Permeability, Rats, Rats, Sprague-Dawley, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Testosterone pharmacology, Tight Junctions metabolism, Urokinase-Type Plasminogen Activator genetics, Cadmium Chloride toxicity, Sertoli Cells drug effects, Spermatogenesis drug effects, Testis drug effects, Tight Junctions drug effects
- Abstract
The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl(2)-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 x 10(6) cells/cm(2)) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl(2) at 5-10 microM for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl(2) (0.1-5 microM) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl(2) also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl(2), the presence of testosterone (T) at 1 x 10(-9) M allowed resealing of the inter-Sertoli TJ barrier after CdCl(2) was removed, whereas the presence of 2 x 10(-7) M testosterone even protected Sertoli cells from CdCl(2)-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl(2)-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl(2) treatment. Based on these results, it is apparent that CdCl(2)-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.
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- 2001
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35. Specific staining of Sertoli cell nuclei and evaluation of Sertoli cell number and proliferative activity in Meishan and White Composite boars during the neonatal period.
- Author
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McCoard SA, Lunstra DD, Wise TH, and Ford JJ
- Subjects
- Animals, Animals, Newborn, Cell Count, Cell Division physiology, Coloring Agents, DNA-Binding Proteins metabolism, Female, GATA4 Transcription Factor, Germ Cells physiology, Immunohistochemistry, Male, Organ Size physiology, Pregnancy, Species Specificity, Swine, Testis growth & development, Transcription Factors metabolism, Cell Nucleus ultrastructure, Sertoli Cells ultrastructure, Testis cytology, Testis ultrastructure
- Abstract
The positive relationship between Sertoli cell number and testicular size emphasizes the importance of determining factors involved in the regulation of the Sertoli cell population. Based on data from other species and indirect evidence in the boar, it is generally accepted that porcine Sertoli cells proliferate rapidly throughout the early postnatal period. However, direct evaluation of Sertoli cell number and the proliferative activity of Sertoli cells during the early postnatal period in boars have not been reported. Stereological enumeration of Sertoli cells is a labor-intensive process and would be greatly facilitated by a marker for these cells especially in the sexually mature male. Thus, the first objective of this study was to determine if expression of the transcription factor GATA-4 is an effective marker for fetal, postnatal, and adult Sertoli cells to facilitate enumeration procedures. The second objective was to evaluate the proliferative activity and growth of the Sertoli cell population in neonatal White Composite and Meishan boars, known to differ in mature testis size and Sertoli cell number, to determine the importance of this developmental period for the adult Sertoli cell population. GATA-4 was abundantly expressed by Sertoli cells throughout fetal and prepubertal stages of development and specifically stained both type A and B Sertoli cell nuclei in the sexually mature boar. Immunoreactivity was never observed in the germ cells regardless of their stage of development, illustrating that GATA-4 is a useful marker for both developing and adult Sertoli cells in the boar. Testicular size did not differ between breeds on Day 1 postpartum, but by 14 days postpartum White Composite boars had significantly larger testes compared to Meishan boars (P: < 0.001). Similarly, Sertoli cell number did not differ between breeds at 1 day postpartum; however, at 14 days postpartum White Composite boars had a significantly larger Sertoli cell population compared to Meishan boars (P: < 0.05). Surprisingly, despite having more Sertoli cells than Meishan boars at 14 days postpartum, the proportion of actively proliferating Sertoli cells in the White Composite boars was almost 50% less than the Meishan boars. This result illustrates that rapid rates of Sertoli cell proliferation probably occurred prior to 14 days postpartum in the White Composite boars. Collectively, these results illustrate that the relationship between testicular size and Sertoli cell number is manifested very early in the postnatal period for these two breeds. The substantial difference in the size of the Sertoli cell population and their proliferative activity between Meishan and White Composite boars during the early postnatal period emphasizes the importance of this early period for the establishment of the Sertoli cell population and subsequent adult testicular size.
- Published
- 2001
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36. Rat seminiferous epithelium contains a unique junction (Ectoplasmic specialization) with signaling properties both of cell/cell and cell/matrix junctions.
- Author
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Mulholland DJ, Dedhar S, and Vogl AW
- Subjects
- Actins analysis, Animals, Cadherins analysis, Cytoskeletal Proteins analysis, Cytoskeletal Proteins metabolism, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Integrins analysis, Male, Microscopy, Immunoelectron, Paxillin, Phosphoproteins analysis, Phosphoproteins metabolism, Phosphotyrosine analysis, Protein Serine-Threonine Kinases analysis, Protein-Tyrosine Kinases analysis, Protein-Tyrosine Kinases metabolism, Rats, Rats, Sprague-Dawley, Sertoli Cells ultrastructure, Spermatogenesis, Spermatozoa physiology, Vinculin analysis, Vinculin metabolism, beta Catenin, Intercellular Junctions physiology, Seminiferous Epithelium ultrastructure, Signal Transduction, Trans-Activators
- Abstract
The seminiferous epithelium contains unique actin related cell-cell junctions, termed ectoplasmic specializations (ESs). Turnover of these junctions is fundamental to sperm release and to movement of spermatocytes from basal to adluminal compartments of the epithelium during spermatogenesis. In this study we report several novel observations related to the spatial and temporal distribution of integrin-related signaling molecules at ESs. We confirm the presence of beta(1)-integrin at these sites and further demonstrate co-localization of integrin linked kinase (ILK). beta(1)-Integrin and ILK were shown by immunoprecipitation to associate in whole cell lysates of seminiferous epithelium. This observation provides the first evidence for a direct beta(1)-integrin/ILK interaction in noncultured epithelium. Pan-cadherin and beta-catenin antibodies did not react at ESs. Rather, antibodies reacted with desmosome-like junctions that are present both at basal junctional complexes between Sertoli cells and at sites of attachment to spermatogenic cells. Focal adhesion kinase (FAK), a known integrin-associated molecule, did not codistribute with beta(1)-integrins and did not associate with these adhesion molecules in immunoprecipitation studies. Although FAK was expressed in the epithelium, it appeared to be limited to the cytoplasm of early spermatogenic cells. Significantly, polyclonal antibodies against phosphotyrosine-containing residues reacted strongly at ESs, with highest levels detected during sperm release and turnover of basal junction complexes. Our observations indicate that ESs share cell signaling features both of cell-cell junctions and of cell-extracellular matrix junctions.
- Published
- 2001
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37. Cell proliferation and hormonal changes during postnatal development of the testis in the pig.
- Author
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França LR, Silva VA Jr, Chiarini-Garcia H, Garcia SK, and Debeljuk L
- Subjects
- Animals, Cell Count, Cell Division physiology, Germ Cells physiology, Germ Cells ultrastructure, Leydig Cells physiology, Leydig Cells ultrastructure, Male, Orchiectomy, Sertoli Cells physiology, Sertoli Cells ultrastructure, Swine, Animals, Newborn physiology, Follicle Stimulating Hormone blood, Testis cytology, Testis growth & development, Testosterone blood
- Abstract
Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.
- Published
- 2000
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38. Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations.
- Author
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Lyng FM, Jones GR, and Rommerts FF
- Subjects
- Androgen Antagonists pharmacology, Animals, Cell Line, Cyproterone Acetate pharmacology, Dihydrotestosterone pharmacology, Dose-Response Relationship, Drug, Flutamide pharmacology, Gap Junctions physiology, Humans, Kinetics, Male, Metribolone administration & dosage, Metribolone pharmacology, Prostate drug effects, Prostate ultrastructure, Prostatic Neoplasms, Rats, Rats, Wistar, Sertoli Cells drug effects, Sertoli Cells ultrastructure, Testosterone administration & dosage, Testosterone pharmacology, Tumor Cells, Cultured, Androgens administration & dosage, Androgens pharmacology, Calcium Signaling drug effects, Flutamide analogs & derivatives, Prostate metabolism, Sertoli Cells metabolism
- Abstract
Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2-3 min duration) increase in intracellular calcium levels was observed within 20-30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1-1000 pM induced transient calcium increases with ED(50) values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED(50) values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17beta were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.
- Published
- 2000
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39. Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study.
- Author
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Zhu LJ, Hardy MP, Inigo IV, Huhtaniemi I, Bardin CW, and Moo-Young AJ
- Subjects
- Animals, Cell Nucleus metabolism, Epididymis chemistry, Estrenes pharmacology, Follicle Stimulating Hormone blood, Gonadotropin-Releasing Hormone analogs & derivatives, Gonadotropin-Releasing Hormone antagonists & inhibitors, Gonadotropin-Releasing Hormone pharmacology, Hormone Antagonists pharmacology, Immunohistochemistry, Luteinizing Hormone blood, Male, Progesterone Congeners pharmacology, Rats, Rats, Sprague-Dawley, Receptors, Androgen analysis, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Testis chemistry, Testosterone blood, Androgens pharmacology, Epididymis metabolism, Gene Expression drug effects, Receptors, Androgen genetics, Testis metabolism
- Abstract
Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.
- Published
- 2000
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40. Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat.
- Author
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O'Donnell L, Stanton PG, Bartles JR, and Robertson DM
- Subjects
- Actins metabolism, Age Factors, Animals, Cell Membrane drug effects, Epithelial Cells drug effects, Epithelial Cells metabolism, Epithelial Cells ultrastructure, Immunohistochemistry, Male, Microfilament Proteins immunology, Microfilament Proteins metabolism, Microscopy, Confocal, Microscopy, Electron, Rats, Rats, Sprague-Dawley, Seminiferous Tubules metabolism, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Spermatids ultrastructure, Testis drug effects, Testis metabolism, Vinculin metabolism, Cell Membrane ultrastructure, Seminiferous Tubules cytology, Seminiferous Tubules drug effects, Sertoli Cells drug effects, Testosterone pharmacology
- Abstract
The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.
- Published
- 2000
- Full Text
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41. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells.
- Author
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Ogura A, Inoue K, Ogonuki N, Noguchi A, Takano K, Nagano R, Suzuki O, Lee J, Ishino F, and Matsuda J
- Subjects
- Animals, Cells, Cultured, Embryo Transfer, Embryonic and Fetal Development, Female, Male, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Nuclear Transfer Techniques, Oocytes ultrastructure, Pregnancy, Pseudopregnancy, Cloning, Organism, Cryopreservation, Sertoli Cells ultrastructure
- Abstract
Although it is generally accepted that relatively high efficiencies of somatic cell cloning in mammals can be achieved by using donor cells from the female reproductive system (e.g., cumulus/granulosa, oviduct, and mammary gland cells), there is little information on the possibility of using male-specific somatic cells as donor cells. In this study we injected the nucleus of immature mouse Sertoli cells isolated from the testes of newborn (Days 3-10) males into enucleated mature oocytes in order to examine the ability of their nuclei to support embryonic development. After activation of the oocytes that had received the freshly recovered immature Sertoli cells, some developed into the morula/blastocyst stage, depending on the age of the donor cells (22.0-37.4%). When transferred into pseudopregnant females, 7 (3.3%, 7 of 215) developed into normal pups at term. Nuclear transfer of immature Sertoli cells after 1 wk in culture also produced normal pups after embryo transfer (3.1%, 2 of 65). Even after cryopreservation in a conventional cryoprotectant solution, their ability as donor cells was maintained, as demonstrated by the birth of cloned young (6.7%, 7 of 105). Immature Sertoli cells transfected with green fluorescent protein gene also supported embryo development into morulae/blastocysts, which showed specific fluorescence. This study demonstrates that immature Sertoli cells, male-specific somatic cells, are potential donors for somatic cell cloning.
- Published
- 2000
- Full Text
- View/download PDF
42. Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes.
- Author
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Boulogne B, Levacher C, Durand P, and Habert R
- Subjects
- Animals, Animals, Newborn, Cell Count, Cell Nucleus chemistry, Cells, Cultured, Cytoplasm chemistry, Immunohistochemistry, Leydig Cells chemistry, Leydig Cells ultrastructure, Male, Rats, Rats, Wistar, Retinoid X Receptors, Retinoids pharmacology, Sertoli Cells chemistry, Sertoli Cells ultrastructure, Spermatozoa ultrastructure, Testis embryology, Testis ultrastructure, Receptors, Retinoic Acid analysis, Receptors, Retinoic Acid physiology, Sperm Count, Testis growth & development, Transcription Factors analysis, Transcription Factors physiology
- Abstract
Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.
- Published
- 1999
- Full Text
- View/download PDF
43. Cellular and subcellular localization of six retinoid receptors in rat testis during postnatal development: identification of potential heterodimeric receptors.
- Author
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Dufour JM and Kim KH
- Subjects
- Amino Acid Sequence, Animals, Animals, Newborn, Blotting, Western, Cell Nucleus metabolism, Cell Nucleus ultrastructure, Germ Cells metabolism, Germ Cells ultrastructure, Immunohistochemistry, Male, Mice, Mice, Knockout, Molecular Sequence Data, Rats, Rats, Sprague-Dawley, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Spermatocytes metabolism, Spermatocytes ultrastructure, Subcellular Fractions metabolism, Testis growth & development, Testis ultrastructure, Receptors, Retinoic Acid metabolism, Testis metabolism
- Abstract
Vitamin A is required in the testis for germ cell development. It acts through two families of retinoid receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR), each with three subtypes alpha, beta, and gamma. These receptors are postulated to dimerize and regulate the transcription of retinoid-responsive genes that are crucial for germ cell development. In this study, we determined the cellular and subcellular localization of six retinoid receptors in the developing rat testis to identify the specific cellular sites and times of receptor expression. Immunohistochemical results revealed the expression of RARalpha, RARbeta, RXRalpha, and RXRgamma proteins in somatic and germ cells throughout postnatal development. In contrast, the expression of RARgamma and RXRbeta did not increase until 30-35 days of age in somatic cells from the testis. Interestingly, RARalpha and RXRalpha had a similar subcellular localization pattern in Sertoli cells throughout postnatal testis development, while RARalpha and RXRgamma were both present in the nucleus of spermatocytes and elongating spermatids. These results suggest that RARalpha may potentially dimerize with RXRalpha in Sertoli cells and with RXRgamma in germ cells. In addition, we demonstrate that the only RAR in the nucleus of early meiotic germ cells is RARalpha.
- Published
- 1999
- Full Text
- View/download PDF
44. Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa.
- Author
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Pelletier R, Trifaro JM, Carbajal ME, Okawara Y, and Vitale ML
- Subjects
- Animals, Cattle, Ejaculation, Electrophoresis, Polyacrylamide Gel, Epididymis anatomy & histology, Epididymis cytology, Gelsolin, Germ Cells metabolism, Germ Cells ultrastructure, Immunoblotting, Immunohistochemistry, In Vitro Techniques, Male, Microscopy, Fluorescence, Seminiferous Tubules metabolism, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Spermatozoa ultrastructure, Testis anatomy & histology, Testis cytology, Calcium physiology, Epididymis metabolism, Microfilament Proteins metabolism, Spermatozoa metabolism, Testis metabolism
- Abstract
We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.
- Published
- 1999
- Full Text
- View/download PDF
45. Spermatid translocation in the rat seminiferous epithelium: coupling membrane trafficking machinery to a junction plaque.
- Author
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Beach SF and Vogl AW
- Subjects
- Animals, Fluorescent Dyes, Intercellular Junctions ultrastructure, Male, Microscopy, Electron, Microtubules metabolism, Rats, Rats, Sprague-Dawley, Sertoli Cells physiology, Spermatogenesis, Cell Membrane ultrastructure, Seminiferous Epithelium ultrastructure, Sertoli Cells ultrastructure, Sperm Transport physiology, Spermatids physiology
- Abstract
In this study, we demonstrate that specialized junction plaques that occur between Sertoli cells and spermatids in the rat testis support microtubule translocation in vitro. During spermatogenesis, Sertoli cells are attached to spermatids by specialized adhesion junctions termed ectoplasmic specializations (ESs). These structures consist of regions of the plasma membrane adherent to the spermatid head, a submembrane layer of tightly packed actin filaments, and an attached cistern of endoplasmic reticulum. It has been proposed that motor proteins on the endoplasmic reticulum interact with adjacent microtubules to translocate the junction plaques, and hence the attached spermatids, within the epithelium. If this hypothesis is true, then isolated junctions should support microtubule transport. To verify this prediction, we have mechanically isolated rat spermatids, together with their attached ESs, and tested them for their ability to transport microtubules in vitro. Most assays were done in the presence of 2 mg/ml testicular cytosol and at room temperature. ESs attached to spermatids supported microtubule translocation. In some cases in which motility events were detected, microtubules moved smoothly over the junction site. In others, the movement was slow but progressive, saltatory and "inch-worm-like." No motility was detected in the absence of exogenous ATP or in the presence of apyrase (an enzyme that catalyses the breakdown of ATP). Our results are consistent with the microtubule-based motility hypothesis of spermatid translocation.
- Published
- 1999
- Full Text
- View/download PDF
46. Germ cell genotype controls cell cycle during spermatogenesis in the rat.
- Author
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França LR, Ogawa T, Avarbock MR, Brinster RL, and Russell LD
- Subjects
- Animals, Cell Communication, Male, Mice, Mice, Inbred C57BL, Microscopy, Electron, Rats, Rats, Sprague-Dawley, Sertoli Cells physiology, Sertoli Cells ultrastructure, Spermatogonia transplantation, Spermatogonia ultrastructure, Testis cytology, Transplantation, Heterologous, Cell Cycle, Genotype, Spermatogenesis genetics, Spermatozoa physiology
- Abstract
Spermatogenesis is one of the most productive self-renewing systems in the body: on the order of 10(7) spermatozoa are produced daily per gram of testis tissue. In each mammalian species, the time required for completion of the process is unique and unalterable. Because the process is supported by somatic Sertoli cells, it has generally been thought that cell-cell interaction between germ and Sertoli cells controls the duration of cell cycles and cellular organization. We have used the newly developed technique of spermatogonial transplantation to examine which cell type(s) determines the rate at which germ cells proceed through spermatogenesis. Rat germ cells were transplanted into a mouse testis, and the mouse was killed 12.9-13 days after administration of a single dose of [3H]thymidine. The most advanced rat cell type labeled was the pachytene spermatocyte at stages VI-VIII of the spermatogenic cycle. In animals given only rat cells, some endogenous spermatogenesis of the mouse recovered. The most advanced labeled mouse cell types in recipients killed 12.9-13 days after administration of a single dose of [3H]thymidine were meiotic cells or young spermatids, which is consistent with a spermatogenic cycle length comparable to the 8.6 days reported for the mouse. The same results were obtained if a mixture of rat and mouse cells were transplanted. There existed two separate timing regimens for germ cell development in the recipient mouse testis; one of rat and one of mouse duration. Rat germ cells that were supported by mouse Sertoli cells always differentiated with cell cycle timing characteristic of the rat and generated the spermatogenic structural pattern of the rat, demonstrating that the cell differentiation process of spermatogenesis is regulated by germ cells alone.
- Published
- 1998
- Full Text
- View/download PDF
47. Characterization of a clonal Sertoli cell line using adult PyLT transgenic mice.
- Author
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Bourdon V, Lablack A, Abbe P, Segretain D, and Pointis G
- Subjects
- Animals, Antigens, Viral, Tumor biosynthesis, Blotting, Western, Cell Line, Clone Cells, Cyclic AMP metabolism, Indicators and Reagents, Male, Mice, Mice, Transgenic, Microscopy, Electron, Phagocytosis physiology, Polymerase Chain Reaction, Receptors, FSH biosynthesis, Receptors, FSH genetics, Sertoli Cells metabolism, Sertoli Cells ultrastructure, Tight Junctions physiology, Tight Junctions ultrastructure, Polyomavirus genetics, Sertoli Cells physiology
- Abstract
In the present study we report the isolation and characterization of a clonal Sertoli cell line (42GPA9) from sexually mature polyoma virus large T (PyLT) transgenic mice. The cells multiplied indefinitely and expressed large T antigen. The 42GPA9 cell line expressed biochemical features associated with normal Sertoli cells. Transferrin, sulfated glycoprotein-2, and the ligand of c-kit were detected by reverse transcription-polymerase chain reactions and Western blot analyses. Zymographic analysis indicated that the 42GPA9 cell line secreted tissue-type plasminogen activator. These cells also retained FSH receptors as suggested by their specific responsiveness to the gonadotropin (morphological and phagocytic changes, stimulation of cAMP production) and the detection of FSH receptor mRNAs. Another original aspect of the 42GPA9 cell line is its ability to form tight junctions at confluency as demonstrated by electron microscopic study and immunolocalization of the tight junction-associated protein zonula occludens 1. The 42GPA9 cell line, which has retained several important hallmarks of normal Sertoli cells, may prove useful for further studies on Sertoli cell behavior and on Sertoli-germ cell interactions in the mature testis.
- Published
- 1998
- Full Text
- View/download PDF
48. Detection of steroidogenic acute regulatory protein (stAR) in mitochondria of cultured rat Sertoli cells incubated with follicle-stimulating hormone.
- Author
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Gregory CW and DePhilip RM
- Subjects
- Animals, Blotting, Western, Cells, Cultured, Cytochrome P-450 Enzyme System metabolism, Fluorescent Antibody Technique, Immunohistochemistry, Leydig Cells metabolism, Male, Membrane Proteins chemistry, Mitochondria chemistry, Phosphoproteins chemistry, Rats, Rats, Sprague-Dawley, Sertoli Cells ultrastructure, Transcription, Genetic, Tumor Cells, Cultured, Follicle Stimulating Hormone, Membrane Proteins metabolism, Mitochondria metabolism, Phosphoproteins metabolism, Sertoli Cells metabolism
- Abstract
Previous work established biochemical homology between proteins named SCc1 and SCc2 in FSH-treated Sertoli cell cultures and a family of 30-kDa proteins, collectively referred to as Steroidogenic Acute Regulatory protein (StAR), in hormone-treated adrenal and gonadal cells. The purpose here was to establish the presence of StAR in FSH-treated Sertoli cell cultures using a StAR-specific antiserum. Immunofluorescence microscopy and immunoblot analysis were used to detect StAR in Sertoli cell cultures and in R2C rat Leydig tumor cell cultures that served as a positive control for StAR expression. FSH dramatically enhanced the appearance of StAR in mitochondria of Sertoli cells, whereas R2C cells expressed StAR constitutively. It has been proposed that StAR participates in steroidogenesis by facilitating the conversion of cholesterol to pregnenolone by the cytochrome P450 side-chain cleavage enzyme. FSH-treated Sertoli cells were shown here to lack detectable levels of side-chain cleavage enzyme protein and RNA, while R2C cells expressed both. We conclude that the appearance of StAR in Sertoli cell mitochondria is regulated by FSH, but that its function in Sertoli cells is not associated with the conversion of cholesterol to pregnenolone.
- Published
- 1998
- Full Text
- View/download PDF
49. Junctional contacts between Sertoli cells in normal and aspermatogenic rat seminiferous epithelium contain alpha6beta1 integrins, and their formation is controlled by follicle-stimulating hormone.
- Author
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Salanova M, Ricci G, Boitani C, Stefanini M, De Grossi S, and Palombi F
- Subjects
- Animals, CD59 Antigens genetics, Cell Communication physiology, Fluorescent Dyes, Follicle Stimulating Hormone biosynthesis, Integrin alpha6beta1, Integrins genetics, Laminin biosynthesis, Male, Microscopy, Electron, Microscopy, Fluorescence, Organ Culture Techniques, Rats, Rats, Wistar, Seminiferous Epithelium cytology, Sertoli Cells ultrastructure, Testis cytology, Testis growth & development, CD59 Antigens biosynthesis, Follicle Stimulating Hormone physiology, Integrins biosynthesis, Seminiferous Epithelium metabolism, Seminiferous Epithelium physiology, Sertoli Cells physiology, Spermatogenesis physiology
- Abstract
The distribution of alpha6beta1 integrins at the level of cell-to-cell contacts within the rat seminiferous epithelium was investigated. Double fluorescence experiments using phalloidin staining of actin filaments and anti-integrin subunit antibodies showed that the receptor belongs to the Sertoli cell lateral domains engaged in the characteristic junctional structures known as ectoplasmic specializations (ES), at the level both of inter-Sertoli junctions and of the contacts between Sertoli cells and elongating spermatids. In the seminiferous epithelium of aspermatogenic testes, obtained through X-irradiation in utero (Sertoli-cell-only testes), at the level of inter-Sertoli junctions both ES and alpha6beta1 integrins are present. In order to study the dependence of alpha6beta1 receptors and ES formation upon FSH stimulation during development, 9-day-old testes were grown in organ culture in basal as well as FSH-supplemented conditions. FSH stimulation, which is necessary for the progression of spermatogenesis to early meiotic stages, appears to be required for the development of inter-Sertoli junctional structures containing ES and alpha6beta1 integrins. These observations indicate that the receptor belongs to the inter-Sertoli junctional machinery and that its expression at that level is not dependent on active spermatogenesis but requires FSH stimulation.
- Published
- 1998
- Full Text
- View/download PDF
50. Differential distribution of the tight-junction-associated protein ZO-1 isoforms alpha+ and alpha- in guinea pig Sertoli cells: a possible association with F-actin and G-actin.
- Author
-
Pelletier RM, Okawara Y, Vitale ML, and Anderson JM
- Subjects
- Animals, Guinea Pigs, Humans, Immunoblotting, Immunoenzyme Techniques, Intercellular Junctions chemistry, Male, Rats, Sertoli Cells ultrastructure, Sexual Maturation, Tight Junctions chemistry, Zonula Occludens-1 Protein, Actins analysis, Membrane Proteins analysis, Phosphoproteins analysis, Sertoli Cells chemistry
- Abstract
To elucidate the significance of alpha- and alpha+ isoforms of the tight-junction-associated protein ZO-1 in Sertoli cell tight junction regulation, taking into consideration that different isoforms are expressed in cells with different junctional morphologies, we investigated whether alpha- and alpha+ are differentially associated with junctions forming the continuous occluding zonules responsible for the blood-testis barrier, and/or with junctions forming the focal discontinuous occluding zonules. In addition, since Sertoli cells contact Sertoli cells and germ cells, we investigated whether each isoform is differentially associated with distinct classes of germ cells. Our immunoblot analyses of isolated seminiferous tubules, using affinity-purified polyclonal antibodies recognizing rat and human alpha- and alpha+, showed that guinea pig testis contained the two ZO-1 isoforms initially described in rat and human kidneys, and that alpha+ and alpha- were predominantly expressed during puberty and adulthood, respectively, indicating that alpha+ was predominant during periods of increased junction assembly/disassembly. We used the same antibodies and immunoperoxidase labeling on fetal, neonatal, pubertal, and adult guinea pig testes sections. Both isoforms were expressed at the site of Sertoli cell-Sertoli cell and Sertoli cell-germ cell junctions in the seminiferous epithelium, before and after birth, and both were localized in continuous and in discontinuous tight junctions. However, the distribution of alpha- and alpha+ was not the same in different locations of the tight junctions. Only alpha- was incorporated into junctions joining the Sertoli cells to all classes of germ cells. The alpha+ involved junctions joining Sertoli cells to particular classes of germ cells, suggesting that Sertoli cell expression of ZO-1 isoforms could be regulated by unique germ cell-Sertoli cell contacts. Conversely, we found a correspondence between the distribution of F-actin and ZO-1alpha+, indicating that the spatial organization of the subsurface actin accompanying cell junctions may affect alpha+/alpha(-)-plasma membrane association.
- Published
- 1997
- Full Text
- View/download PDF
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