Search

Newborn anatomy, despite being distinctly different than adult anatomy, does not constitute a major component of a typical medical school course in gross anatomy. Accordingly, there is a perception that other than the well-known late 20th-century atlas and small textbook by Edmund Crelin on newborn anatomy, there is almost no information available for anatomists and clinicians to refer to on normal infant anatomy. This perception, as verbalized by Crelin in his books, is not correct. There is an amazing wealth of accurate descriptive and pictorial information on infant anatomy available from late 19th- and early 20th-century sources. One of these sources is a comprehensive 200-page chapter on pediatric anatomy by Richard Scammon that was published in 1923 and that is freely available. Because of some inconsistencies and inaccuracies we have identified in the Crelin works, we suggest that any anatomist or clinician who wishes to learn and teach about infant anatomy refer to Scammon's chapter before using any text or image from the Crelin books.

The inherent variable anatomy of the neonate and the uniquely-shaped maternal birth canal that is associated with the evolution of human bipedalism constitute risk factors for neonatal brachial plexus palsy (NBPP). For example, those neonates with a prefixed brachial plexus (BP) are at greater risk of trauma due to lateral neck traction during delivery than those with a normal or postfixed BP. Compared to adults, neonates also have extremely large and heavy heads (high head: body ratio) set upon necks with muscles and ligaments that are weak and poorly developed. Accordingly, insufficient cranial stability can place large torques on the cervical spinal nerves. In addition, the pelvic changes necessary for habitual bipedal posture resulted in a uniquely-shaped, obstruction-filled, sinusoidal birth canal, requiring the human fetus to complete a complicated series of rotations to successfully traverse it. Furthermore, although there are many risk factors that are known to contribute to NBPP, the specific anatomy and physiology of the neonate, except for macrosomia, is not considered to be one of them. In fact, currently, the amount of lateral traction applied to the neck during delivery is the overwhelming legal factor that is used to evaluate whether a birth attendant is liable in cases of permanent NBPP. Here, we suggest that the specific anatomy and physiology of the neonate and mother, which are clearly not within the control of the birth attendant, should also be considered when assessing liability in cases of NBPP.

Progression of the first meiotic division in male germ cells is regulated by a variety of factors, including androgens and possibly estrogens. When this regulation fails, meiosis is arrested and primary spermatocytes degenerate by apoptosis. Earlier studies showed that overexpression of rat androgen-binding protein (ABP) in the testis of transgenic mice results in a partial meiotic arrest and apoptosis of pachytene spermatocytes. In view of the recent localization of estrogen receptor β (ERβ) in primary spermatocytes and data suggesting the ability of ERβ to repress cellular proliferation, we tested the hypothesis that variations in the testicular steroid microenvironment caused by excess ABP produce changes in ERβ expression in this cellular type that could be associated to the meiotic arrest and, eventually, to the induction of germ cell apoptosis observed in the ABP transgenic mice. Increased levels of ERβ mRNA and protein were demonstrated in the testis of rat ABP transgenic mice compared wit...

Degeneration of primary spermatocytes by apoptosis occurs during normal spermatogenesis, as well as in several pathological conditions, including exposure to specific testicular toxicants. The mechanisms that regulate the death and survival of primary spermatocytes, however, are still not well understood. The recent localization of estrogen receptor beta (ERbeta) and P450 aromatase in pachytene spermatocytes suggests a role for estrogens in this step of spermatogenesis. Using a well-known model of pachytene spermatocyte apoptosis in adult rats consisting of the administration of methoxyacetic acid (MAA), we investigated the participation of ERbeta during the initial phase of apoptosis, prior to germ cell loss. Adult rats were treated with a single intraperitoneal dose of MAA, and DNA laddering analysis confirmed apoptotic cell death in the testis. In enriched germ cell fractions and testis from MAA-treated animals, ERbeta mRNA increased significantly at 3 and 6 hours, respectively. Next, stage-specific induction of ERbeta mRNA was demonstrated by use of laser capture microdissection of seminiferous tubules in combination with semiquantitative reverse transcription-polymerase chain reaction. The ERbeta protein also increased significantly after 6 hours and was mainly immunolocalized in the cytoplasm of pachytene spermatocytes of afflicted tubules. The cytoplasmic localization was confirmed by Western blot analysis of isolated cytoplasmic and nuclear fractions of testicular extracts. Finally, the MAA activation of ERbeta was tested in vitro in HepG2 cells cotransfected with ERbeta and a reporter construct that contained a consensus estrogen responsive element. Addition of MAA at similar doses used in vivo elicited a similar estrogenic activation as did estradiol at 1 nmol/L concentration. The present results raise the possibility that cytoplasmic ERbeta participates in the apoptotic process of pachytene spermatocytes induced by MAA. Whether MAA interacts with ERbeta in the cytoplasm of primary spermatocytes, preventing the progression of the first meiotic division, however, remains to be determined.

Chemical agents can disrupt the balance between survival and apoptosis during spermatogenesis and thus give rise to reduced counts of spermatozoa (oligospermia). One such agent that produces significant germ cell apoptosis at specific stages of the cycle of the seminiferous epithelium is methoxy acetic acid (MAA), the active metabolite of a commonly used solvent, methoxyethanol. Although MAA gives rise to apoptosis of pachytene spermatocytes, it is not known whether MAA exerts a direct effect on germ cells or whether it also affects other testicular cell types such as the Sertoli cells. In the present investigation, we tested the hypothesis that MAA has direct effects on Sertoli cells in vivo. In MAA-treated rats, stage-specific expression of androgen receptor (AR) protein in Sertoli cells was significantly altered, as determined by AR immunohistochemistry. In MAA-treated animals, high AR expression was found in Sertoli cells coincident with the MAA-induced apoptosis of late-stage pachytene spermatocytes. The altered expression of AR in MAA-treated animals was also seen in seminiferous tubules harvested by laser capture microdissection. In addition to effects on AR expression, androgen-binding protein (ABP) mRNA levels were also altered in a stage-specific manner. Using a different system for mouse Sertoli cell lines TM4 and MSC-1, positive for either AR or ABP, respectively, we found a direct effect of MAA on ABP protein and mRNA expression in the MSC-1 cell but did not detect an effect on AR protein or mRNA expression in TM4 cells. Mouse fibroblasts that express endogenous AR were stably transfected with two AR promoter/reporter systems (MMTV-CAT and probasin-luciferase, respectively). We used these fibroblasts to examine the ability of MAA to potentiate dihydrotestosterone (DHT) activation of AR. Although MAA did not activate AR directly, it did potentiate DHT activation of the AR by 2- to 4-fold. MAA altered the expression level of AR and ABP in vivo and increased AR transcriptional activity in tissue culture cells. The abnormal spermatogenesis generated by MAA is at least partly due to direct effects on Sertoli cells. It is still unclear whether MAA elicits a proapoptotic signal from Sertoli cells or diminishes a prosurvival signal required by germ cells downstream to altering AR and ABP expression in a stage-specific fashion.

Erythropoietin, known for its role in erythroid differentiation, has been shown to be neuroprotective during brain ischaemia in adult animal models. Although high levels of erythropoietin receptor are produced in embryonic brain, the role of erythropoietin during brain development is uncertain. We now provide evidence that erythropoietin acts to stimulate neural progenitor cells and to prevent apoptosis in the embryonic brain. Mice lacking the erythropoietin receptor exhibit severe anaemia and defective cardiac development, and die at embryonic day 13.5 (E13.5). By E12.5, in addition to apoptosis in foetal liver, endocardium and myocardium, the erythropoietin receptor null mouse shows extensive apoptosis in foetal brain. Lack of erythropoietin receptor affects brain development as early as E10.5, resulting in a reduction in the number of neural progenitor cells and increased apoptosis. Corresponding in vitro cultures of cortical cells from Epor–/– mice also exhibited decreases in neuron generation compared with normal controls and increased sensitivity to low oxygen tension with no surviving neurons in Epor–/– cortical cultures after 24 hour exposure to hypoxia. The viability of primary Epor+/+ rodent embryonic cortical neurons was further increased by erythropoietin stimulation. Exposure of these cultures to hypoxia induced erythropoietin expression and a tenfold increase in erythropoietin receptor expression, increased cell survival and decreased apoptosis. Cultures of neuronal progenitor cells also exhibited a proliferative response to erythropoietin stimulation. These data demonstrate that the neuroprotective activity of erythropoietin is observed as early as E10.5 in the developing brain, and that induction of erythropoietin and its receptor by hypoxia may contribute to selective cell survival in the brain.

Androgen receptor (AR) immunohistochemistry was performed in an archival collection of adult human cryptorchid testes to determine whether AR cellular distribution and intensity of immunostaining were functions of the severity of cellular dysgenesis. The seminiferous tubule histology of cryptorchid testes collected from adults is marked by three specific patterns. 1) Seminiferous tubules are characterized as maintaining focal areas of germinal cell differentiation (albeit incomplete) that are interspersed with 2) tubules composed of Sertoli cells only, these latter cells being principally of the adult type, although dysgenetic and immature Sertoli cells may also be detected. 3) In contrast, there is a class of tubule that is characterized as being composed exclusively of Sertoli cells that are extremely dysgenetic in appearance. The majority of adult-type Sertoli cells found in the first types of tubules exhibited either robust or moderate AR staining intensity. Peritubular cells of these tubules also expressed a similar AR staining intensity. In contrast, in the more dysgenetic and immature type Sertoli cells found in the second type of tubules, the intensity of AR staining was significantly less, if not missing altogether. Finally, in the most dysgenetic tubules, Sertoli cell AR staining was never detected. To our knowledge, this is the first report in the literature that addresses the intensity of AR immunostaining in Sertoli cells of cryptorchid testes. The results presented herein are consistent with the interpretation that the intensity of AR staining in Sertoli cells diminishes as a function of the severity to which the cells are afflicted within a cryptorchid testis and that focal absence of AR expression in Sertoli cells correlates with a lack of local spermatogenesis in the tubules.

We have developed a new form of laser capture microdissection (LCM) optimized for isolating and concentrating single cells from a tissue slide for subsequent molecular analysis. In LCM an infrared laser diode is used together with a microscope to locally melt a thin film of ethylene vinyl acetate (EVA) placed in contact or close proximity to microscopically targeted cells. Since the desired cells adhere to the film where it has been melted, they are extracted from their adjacent tissue in the specimen when the film is separated from the slide. In our system the EVA is bonded to a cylindrical substrate resulting in a very small area of contact between the EVA and the slide which minimizes contamination to levels consistent with analyzing a few cells of a selected cell type. By collecting cells at different portions about the cylinder periphery, the novel geometry also allows the concentration of cells from different slides onto a single substrate, enabling very sensitive molecular analysis to be performed ...

The distribution of the androgen receptor (AR) in archival human testes was determined immunocytochemically using an affinity-purified peptide-specific rabbit antibody, PG21, and employing a modified biotin-streptavidin-immunoperoxidase method that incorporated a biotin amplification step. In combination with microwave epitope retrieval, the biotin amplification step increased the sensitivity of the immunostaining assay approximately 20-fold. Thus, the useful range at which PG21 rendered a robust, specific immunostaining signal without also increasing nonspecific background was extended dramatically. Broadening the useful range of the PG21 antibody made it possible to resolve the relative amounts of immunopositive AR in different cell types of the human testis. At a high PG21 concentration, for example, all AR-positive cells exhibited a robust immunostaining intensity, but it was not possible to distinguish between nuclei exhibiting either high or moderate immunostaining intensities. In contrast, as the concentration of PG21 was decreased, distinct populations of testicular cells exhibited differential AR immunostaining intensities in their nuclei. AR immunostaining of Sertoli cell nuclei was present at low PG21 concentrations at which no immunostaining of peritubular myoid cells or Leydig cells could be detected. In turn, AR immunostaining of peritubular myoid cells was detected at PG21 concentrations that did not immunostain Leydig cells. Moreover, within the seminiferous epithelium, Sertoli cell nuclear AR staining intensity was less at stages V and VI of the cycle of the seminiferous epithelium than that at stage III, and stage III staining intensity was greater than that at stages I and II. This AR immunostaining pattern in human Sertoli cell nuclei as a function of the cycle of the seminiferous epithelium is reminiscent of the pattern observed in rodent species. Finally, no AR immunostaining of germ cells was observed at any of the PG21 concentrations examined.

La question de savoir si les cellules de la lignee germinale possedent ou non des recepteurs aux androgenes reste debattue. Dans ce travail nous avons determine la distribution des recepteurs aux androgenes dans le testicule de rat et de souris a l'aide d'un anticorps polyclonal de lapin purifie par affinite, dirige contre un peptide de 21 acides amines de la region terminale du recepteur des androgenes (RA), marque par le systeme biotine-streptavidine immunoperoxidase. La specificite de l'anticorps est attestee par les points suivants: 1) Presence d'une bande specifique approximativement a 110 kD en Western immunoblot; 2) Elimination specifique du marquage par pre-adsorption de l'anticorps avec le peptide de 21 acides amines; 3) Mise en evidence d'une relation entre l'intensite du marquage dans toutes les cellules RA positives en fonction de la dilution des antiserums; 4) Mise en evidence dans tous les tissus connus pour exprimer de facon importante des RA (par exemple l'epididyme, la prostate, les vesicules seminales) d'un marquage extremement intense au niveau du noyau des cellules epitheliales; 5) Obtention d'un reaction RA positive avec les anticorps dans les lignees de cellules prostatiques connues pour exprimer les recepteurs aux androgenes; 6) Positivation de cellules COS-7 apres leur transfection par un vecteur exprimant le recepteur aux androgenes de rat. De plus la distribution intracellulaire des RA dans les cellules COS-7 est fonction de la presence d'androgenes dans le milieu. L'immunolocalisation des recepteurs aux androgenes montre les points suivants: a l'interieur du compartiment interstitiel chez le rat adulte des RA sont detectes dans quelques cellules de Leydig et dans toutes les cellules musculaires lisses formant la paroi des vaisseaux sanguins, alors que les cellules endotheliales restent negatives. Dans les tubes seminiferes les RA sont observes dans tous les noyaux des cellules myoides peritubulaires, mais pas dans les couches plus peripheriques correspondant aux cellules endotheliales des vaisseaux lymphatiques. Dans les cellules de Sertoli le marquage nucleaire des RA est specifique du stade de l'epithelium seminifere: la positivite du marquage commence a devenir evidente vers le stade IV tardif du cycle, atteint un pic tres fort aux stades VII et VIII puis disparait completement. Un immunomarquage specifique a egalement ete mis en evidence dans les noyaux des spermatides allongees stade XI, stade auquel l'elongation nucleaire est deja apparente mais sans que la condensation de la chromatine soit deja commencee. Des le debut de la condensation de la chromatine des spermatides allongees, l'immunomarquage nucleaire disparait en meme temps que le cytoplasme devient positif. Globalement les memes observations ont ete faites sur le testicule de Souris adulte, excepte le fait que, dans cette espece, toutes les cellules de Leydig sont tres fortement RA positives. Afin de confirmer l'immunomarquage des RA nous avons realise des etudes d'hybridation in situ dans le testicule de Rat adulte a l'aide d'une sonde antisens ARNc de 236 paires de bases (I'ADNc de RA nous a ete donne par le Docteur Chang, Universite du Wisconsin, Madison, WI). Un important signal d'hybridation a ete observe a la base de l'epithelium seminifere, dans la region occupee par les cellules de Sertoli et les spermatogonies. Ceci nous a conduit a reexaminer les resultats de l'immunomarquage afin de determiner si les spermatogonies etaient egalement positives pour les anticorps anti RA. Nos resultats preliminaires sont en faveur de la presence de RA dans certaines populations spermatogoniales. Au total, ces resultats concordent avec des observations anterieures suggerant que les RA sont presents dans les cellules somatiques du testicule; de ce fait, ce sont ces types cellulaires qui vraisemblablement repondent aux androgenes circulants pour controler la spermatogenese. Cependant nos resultats montrent egalement un immunomarquage de certains elements de la lignee germinale ce qui reactualise la controverse concernant le fait de savoir si la lignee germinale peut repondre ou non directement aux androgenes.

The epididymis participates in the post-testicular maturation and storage of spermatozoa by secreting proteins into the tubule lumen in a region-specific fashion. The underlying molecular mechanisms leading to biogenesis of these region-specific differences, however, are not known, although components of the Golgi complex membrane container must undoubtedly be intimately involved. Two monoclonal antibodies raised against Golgi integral membrane proteins, recognizing either the cis (GIMPc) or trans Golgi (GIMPt) cisternae, were used as molecular probes of these regions to begin the characterization of the Golgi complex of in vivo and in vitro epididymal cells. Immunolocalization of GIMPs was performed on frozen sections and in cultured cells using biotin-streptavidin-peroxidase immunocytochmistry. In tissue sections, immunostaining of GIMPt was extremely robust in the supranuclear cytoplasm throughout the epididymis. In contrast, no GIMPc immunostaining was detected in the initial segment or in clear cells of the distal caput, corpus, and cauda. Immunodetection of GIMPc and GIMPt in epididymal cells in vitro revealed a reticular, perinuclear pattern, and NH4Cl treatment preferentially disrupted the GIMPt immunolocalization. These results characterizing the molecular components of the Golgi complex will form the basis of additional studies to gain further insight into mechanisms leading to generation of regional differences in epididymal function. © 1994 Wiley-Liss, Inc.

The distribution of the androgen receptor (AR) in the adult rat testis was determined by biotin-streptavidin immunoperoxidase, employing tissue embedded in polyester wax which preserves antigenicity without compromising tissue preservation. The antibody probe used, which has been characterized previously, was an affinity purified, rabbit polyclonal antibody raised to the amino terminus peptide of the rat AR. Within the interstitial compartment, AR immunostaining was detected in some Leydig cells and all smooth muscle cells forming the walls of blood vessels, but endothelial cells of blood vessels were negative. Furthermore, in those Leydig cells that were clearly identified as exhibiting AR immunostaining, the intensity of the reaction varied. In the seminiferous tubules AR immunostaining was observed in all peritubular myoid cell nuclei, but not in the distal layer of lymphatic endothelial cells. In Sertoli cells, nuclear AR immunostaining was stage specific. Moderate AR immunostaining first became evident at late stage IV or early stage V of the cycle, reached a robust peak at stages VII-VIII, and then disappeared completely. Specific AR immunostaining was also discerned in the nuclei of stage XI elongated spermatids, the spermatids in which nuclear elongation is apparent but chromatin condensation has not yet begun. Next, with onset of chromatin condensation, nuclear AR immunostaining in elongated spermatids was not discerned concomitant with its detection in the cytoplasm of the germ cells. These results are interpreted in the following manner: 1) The presence of AR in Leydig cells is consistent with the hypothesis that androgens modify Leydig cell activity in an autocrine fashion. Further, that not all Leydig cells exhibited AR immunostaining at steady state suggests a differential, functional activity of these cells within the population. 2) The intense AR immunostaining of smooth muscle cells present in the interstitium indicates that these cells are targets for androgens. 3) AR immunoreactivity in both Sertoli and peritubular myoid cells suggests their involvement in the androgenic control of spermatogenesis. The stage specific AR immunoreactivity in Sertoli cells, however, may be more indicative of a specific androgen response during these stages, whereas peritubular cells may participate in the tonal maintenance of spermatogenesis. 4) The specific presence of AR in step 11 elongated spermatids may suggest that androgens can act directly on germ cells to regulate spermatogenesis.

Immunocytochemistry is a compromise between maintaining antigenicity and preserving tissue morphology. In the testis, successful immunostaining results at the level of resolution provided by the light microscope have been obtained through use of either frozen or paraffin sections, although both techniques are fraught with limitations. With freezing, tissue preservation is not optimum, whereas with paraffin embedding, antigenicity is often destroyed. These limitations are not trivial and have led to numerous ambiguous results in the literature. In the present study we wish to report the results of immunocytochemical localization of various proteins in testis fixed by perfusion with Bouin's fluid and embedded in polyester wax, a ribboning embedding medium for histology. The advantages of this medium are that it does not require clearing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38°C. Because of these two properties, the polyester was appears to adequately maintain antigenicity as compared to that observed in frozen sections, yet because it is a ribboning wax, it preserves detailed structure as well as paraffin does. Proteins that were immunolocalized included cytoskeletal proteins (tubulin, actin, vinculin, vimentin) and cell-specific markers: 1) androgen-binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiazepine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ cells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and both rabbit antisera and mouse monoclonal antibodies were used with equal success. In addition, fluorochrome-labeled second antibodies combined with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguously immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubulin or vimentin immunostaining patterns in Sertoli cells are altered during the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report that anti-PBR is a specific probe for Leydig cells in vivo and that an anti-nuclear lamin antibody appears to serve as a specific probe for spermatogonia and pachytene spermatocytes, but that the commercially available anti-smooth muscle a-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule. We would like to suggest the use of this embedding protocol as a means for immunocytochemical examination of protein antigens in the testis.

The epididymis, a post-testicular site required for maturation and storage of spermatozoa, is actively involved in exocytic and endocytic events, two phenomena likely to depend on the integrity of the lysosomal system. To study the lysosomal system of the epididymis, five monoclonal antibodies, previously characterized as recognizing five distinct lysosomal integral membrane proteins (LIMPs 1-5), were used as molecular probes of lysosome distribution in cells lining the epithelium. Immunocytochemical localization of LIMPs, using biotin-streptavidin immunoperoxidase methodology, was performed on frozen sections of adult rat epididymides and in cell cultures prepared from either the caput or cauda epididymis. In frozen sections, a heterogeneous distribution of the different LIMPs along the length of the epididymis was observed. For example, the distribution of LIMP 1 (35-50 K) was detected in all cells of the caput and quite dramatically in clear cells of the distal caput, corpus, and cauda epididymis, but specifically not in the principal cells of the distal caput, corpus, and cauda. In contrast, LIMP 2 (64-71 K) was present in all cells of the epididymis, except clear cells. LIMPs 4 and 5 (93 K and 93 K) were detected in all epididymal cells, including the clear cells. Finally, whereas the regional and cell type distribution of LIMP 3 (74 K) in the epididymis was identical to that of LIMPs 4 and 5, the nature of the vesicles immunostained was distinct. In cultured cells, the general immunostaining patterns observed in vivo were maintained during the duration of the primary cultures for all five LIMPs. Our results begin to address the molecular heterogeneity of the lysosomal system along the length of the epididymis, and may suggest in part a basis for underlying structural and functional characteristics of the epididymis leading to the sequential maturation of sperm.

A morphological and immunocytochemical study of the Golgi apparatus in pachytene spermatocytes was performed in an effort to correlate the structure and function of this organelle during meiotic prophase. In stages I-III of the cycle, the Golgi complex of pachytene spermatocytes is a flattened discoid, 0.5-1 microns in diameter, composed of vesicles interspersed with classically described Golgi cisternae. During subsequent maturation of pachytene spermatocytes (stages IV-XIII), the size of the Golgi complex increases significantly, attaining a size of 2-3 microns. However, unlike pachytene spermatocytes of stages I-III, the majority of the Golgi complex of more mature spermatocytes is characterized by an abundance of distinct stacks of cisternae interspersed with numerous vesicles and tubules. The composition of the Golgi complex was also studied by using two monoclonal antibodies that recognize either the cis or the trans Golgi cisternae, respectively, and employing biotin-streptavidin-peroxidase immunocytochemistry in 5 micron frozen sections of testes. Immunodetection of the distinct cisternae revealed that the increase in size of the Golgi complex during maturation of pachytene spermatocytes was due predominantly to an accumulation of trans Golgi; the amount of cis Golgi remained unchanged. The morphological data presented in this study are consistent with an heightened secretory activity of pachytene spermatocytes during their maturation. In addition, the increase in size of the Golgi apparatus during the extensive prophase of pachytene spermatocytes may suggest that the mechanism employed by germ cells to partition the Golgi complex during the first division of meiosis varies significantly from that of somatic cells undergoing mitosis.

The distribution of the epidermal growth factor receptor (EGFR) in mouse testis was ascertained by immunocytochemical methodology using a polyclonal antibody (RK2) shown previously to recognize the cytoplasmic domain of the human (A431 cells), murine (Swiss 3T3 cells), and chicken (CK 109 cells) EGFR. Initial studies performed to determine the usefulness of this antibody as a probe of the murine EGFR in testis employed two murine cell lines, TM4 and MA10, of Sertoli cell and Leydig cell origin, respectively, in which a physiological response of EGF and specific binding of iodinated EGF has been demonstrated. Western blotting in membrane preparations of TM4 and MA10 revealed only one prominent band at 170 kDa. Immunocytochemical localization in TM4 and MA10 cells illustrated a plasma membrane distribution of the receptor. Western blotting of membrane fractions prepared from testis also revealed a specific band at 170 kDa. In the intact testis, the EGFR was immunolocalized specifically in Leydig cells and Sertoli cells only. These results suggest that the involvement of EGF action in spermatogenesis may occur at the level of the somatic components of the testes, principally in the Leydig and Sertoli cells.

Sertoli cells from immature rats (18 days old) were cultured on Millipore filters impregnated with reconstituted basement membrane in bicameral chambers. Three types of cultures were obtained: 1) confluent monolayer cultures that formed a permeability barrier (impermeable), 2) confluent monolayer cultures that did not form a permeability barrier (permeable), and 3) subconfluent cultures (permeable). The relationships among fluid equilibrium, electrical resistance, and [3H]inulin transport between the apical and basal reservoirs of the chambers were examined. An impermeable confluent monolayer is defined when the cells of the Sertoli cell epithelial sheet are able to prevent hydrodynamic equilibration of fluid levels between the apical and basal reservoirs of a bicameral chamber. That is, a permeability barrier is present between the two sides of the chamber when fluid levels (volumes) do not change. In the impermeable confluent Sertoli cell monolayers, 7.5 +/- 0.6% of added [3H]inulin diffused across the monolayer during a 6-h collection period versus 13.7 +/- 0.5% in permeable cultures. Conversely, the electrical resistance was higher in the impermeable monolayers (41-71 ohm.cm2) than in the permeable layers (less than 33 ohm.cm2). A reciprocal linear relationship (Y = -4.68(X) + 91.50, r = 0.808) exists between inulin flux and electrical resistance, and this relationship is a function of cell density. Transferrin (Tf) was one of a few proteins detected in the basal medium of bicameral chambers, whereas most de novo synthesized proteins were secreted into the apical reservoir of the chamber. No significant differences in the total amount of Tf secreted by impermeable or permeable monolayers of Sertoli cells were observed. However, the Sertoli cell secretion ratios (apical/basal) of Tf during a 15-20-h collection period were 2.03 and 1.57 for impermeable monolayers plated at 2.4 x 10(6) and 3.6 x 10(6) cells/well, respectively, but less than 1.0 in permeable layers of cells. When fewer than 2 x 10(6) Sertoli cells were plated, the apical/basal polarity of Tf secretion declined to below 1 in a 24-h culture period, even though those chambers contained impermeable monolayers (recognized by the lack of hydrodynamic equilibrium). These results indicate that polarized secretion by Sertoli cells is dependent on (1) plating density and (2) formation of an impermeable epithelial sheet.

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. Thein vivoprocollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies inin vitroculture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.

Molecular chaperones mediate multiple aspects of steroid receptor function, but the physiological importance of most receptor-associated cochaperones has not been determined. To help fill this gap, we targeted for disruption the mouse gene for the 52-kDa FK506 binding protein, FKBP52, a 90-kDa heat shock protein (Hsp90)-binding immunophilin found in steroid receptor complexes. A mouse line lacking FKBP52 (52KO) was generated and characterized. Male 52KO mice have several defects in reproductive tissues consistent with androgen insensitivity; among these defects are ambiguous external genitalia and dysgenic prostate. FKBP52 and androgen receptor (AR) are coexpressed in prostate epithelial cells of wild-type mice. However, FKBP52 and AR are similarly coexpressed in testis even though testis morphology and spermatogenesis in 52KO males are usually normal. Molecular studies confirm that FKBP52 is a component of AR complexes, and cellular studies in yeast and human cell models demonstrate that FKBP52 can enhance AR-meditated transactivation. AR enhancement requires FKBP52 peptidylprolyl isomerase activity as well as Hsp90-binding ability, and enhancement probably relates to an affect of FKBP52 on AR-folding pathways. In the presence of FKBP52, but not other cochaperones, the function of a minimally active AR point mutant can be dramatically restored. We conclude that FKBP52 is an AR folding factor that has critically important physiological roles in some male reproductive tissues.

Progression of the first meiotic division in male germ cells is regulated by a variety of factors, including androgens and possibly estrogens. When this regulation fails, meiosis is arrested and primary spermatocytes degenerate by apoptosis. Earlier studies showed that overexpression of rat androgen-binding protein (ABP) in the testis of transgenic mice results in a partial meiotic arrest and apoptosis of pachytene spermatocytes. In view of the recent localization of estrogen receptor beta (ERbeta) in primary spermatocytes and data suggesting the ability of ERbeta to repress cellular proliferation, we tested the hypothesis that variations in the testicular steroid microenvironment caused by excess ABP produce changes in ERbeta expression in this cellular type that could be associated to the meiotic arrest and, eventually, to the induction of germ cell apoptosis observed in the ABP transgenic mice. Increased levels of ERbeta mRNA and protein were demonstrated in the testis of rat ABP transgenic mice compared with nontransgenic littermates by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments, Northern blotting, and Western Blotting. The major differences were found when isolated germ cells of transgenic and nontransgenic littermates were analyzed by RT-PCR. In keeping with this finding, ERbeta was strongly immunolabeled in pachytene spermatocytes of rat ABP transgenic mice and localized in tubular stages in which TUNEL labeling was maximal. Confocal microscopy analysis of a fluorescent TUNEL assay and ERbeta immunohistochemistry revealed that degenerating pachytene spermatocytes overexpressed ERbeta. The present results are consistent with the interpretation that ERbeta is associated with the events that regulate negatively the progression of meiosis or that lead to spermatocyte apoptosis.

Chemical agents can disrupt the balance between survival and apoptosis during spermatogenesis and thus give rise to reduced counts of spermatozoa (oligospermia). One such agent that produces significant germ cell apoptosis at specific stages of the cycle of the seminiferous epithelium is methoxy acetic acid (MAA), the active metabolite of a commonly used solvent, methoxyethanol. Although MAA gives rise to apoptosis of pachytene spermatocytes, it is not known whether MAA exerts a direct effect on germ cells or whether it also affects other testicular cell types such as the Sertoli cells. In the present investigation, we tested the hypothesis that MAA has direct effects on Sertoli cells in vivo. In MAA-treated rats, stage-specific expression of androgen receptor (AR) protein in Sertoli cells was significantly altered, as determined by AR immunohistochemistry. In MAA-treated animals, high AR expression was found in Sertoli cells coincident with the MAA-induced apoptosis of late-stage pachytene spermatocytes. The altered expression of AR in MAA-treated animals was also seen in seminiferous tubules harvested by laser capture microdissection. In addition to effects on AR expression, androgen-binding protein (ABP) mRNA levels were also altered in a stage-specific manner. Using a different system for mouse Sertoli cell lines TM4 and MSC-1, positive for either AR or ABP, respectively, we found a direct effect of MAA on ABP protein and mRNA expression in the MSC-1 cell but did not detect an effect on AR protein or mRNA expression in TM4 cells. Mouse fibroblasts that express endogenous AR were stably transfected with two AR promoter/reporter systems (MMTV-CAT and probasin-luciferase, respectively). We used these fibroblasts to examine the ability of MAA to potentiate dihydrotestosterone (DHT) activation of AR. Although MAA did not activate AR directly, it did potentiate DHT activation of the AR by 2- to 4-fold. MAA altered the expression level of AR and ABP in vivo and increased AR transcriptional activity in tissue culture cells. The abnormal spermatogenesis generated by MAA is at least partly due to direct effects on Sertoli cells. It is still unclear whether MAA elicits a proapoptotic signal from Sertoli cells or diminishes a prosurvival signal required by germ cells downstream to altering AR and ABP expression in a stage-specific fashion.

The fundamental role of androgen-binding protein (ABP) in spermatogenesis remains obscure after nearly 25 yr since its first characterization. In the present investigation, we used a transgenic mouse model that overexpresses rat ABP to examine the potential involvement of this protein in the regulation of processes occurring during spermatogenesis. Specifically, homozygous or heterozygous transgenic mice were analyzed in terms of spermatogenic progression, DNA fragmentation pattern, and germinal cell ploidy status. All animals homozygous for transgenic ABP exhibited an increased accumulation of primary spermatocytes and cells at metaphase with abnormal morphology and localization within the seminiferous epithelium. Analysis of DNA fragmentation by in situ techniques and agarose gel electrophoresis provided evidence for an increased occurrence of apoptosis in the transgenic animals, principally involving pachytene spermatocytes and cells at metaphase. Flow cytometric analysis of the DNA content of isolated germ cells revealed a reduction in the number of haploid cells, an increase in the number of tetraploid cells, and the appearance of a hypotetraploid cell population, consistent with degenerating primary spermatocytes. In mice heterozygous for the transgene, the effects were less prominent, and the degree to which spermatogenesis was compromised correlated with the levels of ABP messenger RNA in individual animals. The present results are interpreted to suggest that ABP can act as a modulator of spermatogenesis by regulating completion of the first meiotic division of primary spermatocytes.

Various proteins contain EGF-like domains that are not ligands for the EGF receptor. In the present study a cognate polypeptide for residues 361-406 of the mouse EGF precursor was synthesized by the solid-phase method. The product was renatured under oxidative conditions since it probably has an EGF-like array of three cystine disulfide bonds in its native state. HPLC analysis of the renaturation reaction revealed formation of a peak material with no apparent free-SH groups. Accordingly, the HPLC retention time of this product was readily increased by treatment (reduction of disulfides) with dithiothreitol. The renatured 46-mer (PEGF-1) did not displace 125I-EGF bound to rat liver membranes and 125I-PEGF-1 did not exhibit specific binding to membrane preparations from the mouse liver, mammary gland, or kidney, with or without Ca2+ in the binding medium. Although PEGF-1 contains a putative Ca2+ binding motif, specific binding of this cation by the polypeptide could not be demonstrated by electromobility shiff or incubation with 45Ca2+. Immunoassay of PEGF-1 and EGF in fractions obtained following gel filtration of mouse urine revealed multiple peaks of PEGF-1 immunoreactivity with the major peaks eluting at an Mr30 kDa. In contrast, virtually all the EGF immunoreactivity eluted at a volume similar to that of 125I-EGF. These data suggest that selective cleavage of the PEGF-1 domain from the precursor does not occur with the proclivity known for that of EGF. Instead, the PEGF-1 probably functions coordinately with other EGF-like domains while tethered to the precursor backbone. Finally, localization of PEGF-1 immunoreactivity occurred only in cell populations of the mouse previously demonstrated as sites for EGF/EGF precursor, which suggests that PEGF-1 is exclusively a domain of the EGF precursor.

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 μm. A computer-controlled arm precisely positions a 40-μm-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Germ cells contain androgen receptor (AR). Germ cells do not contain androgen receptor. These diametrically opposed views are at the center of a long-running dispute regarding the cell site of androgen action within the testis (reviewed in 1, 2). In this chapter we examine AR distribution in the rat and mouse testis using an affinity-purified rabbit polyclonal antibody made to a 21 amino acid peptide of the amino terminus of the rat AR (3–5). Results reveal the presence of AR in testicular somatic cells, as well as in elongated spermatids.

Epidermal growth factor (EGF), a potent mitogen produced primarily in the submandibular gland of adult male mice, has been implicated in modulating processes known to be of vital importance in the regulation of spermatogenesis. In the present investigation we demonstrate that submandibular gland EGF from adult male mice is indeed capable of displacing radiolabeled EGF from testicular membranes. Scatchard analysis of this binding site reveals that it is of high affinity (K d = 0.77nM) and low capacity (B max = 8.15 fmol/mg protein). Cross-linking of 125 I-EGF to the identical membrane preparation resulted in the SDS-PAGE/ autoradiography identification of a single band at approximately 170 kDa. Next, we examined the cellular distribution of the EGF receptor in the testis using biotin-streptavidin immunoperoxidase and employing different antisera probes generated to a conserved sequence of the EGF receptor. The Scatchard and cross-linking data described above, along with the immunocytochemistry results, suggest strongly that there is only one functional binding site for EGF in the adult testis and that this receptor is present in Sertoli and Leydig cells.

Cell surface expression of the epidermal growth factor (EGF) receptor in several cell lines declines as a function of increased cell density and is associated with diminished responsiveness to EGF. However, the mechanism whereby this density-induced down regulation of receptors occurs has not been discerned. In the present study the distribution of the EGF receptor in A-431 cells as a function of cell density using (1) two polyclonal antibodies raised against peptide specific sequences of the EGF receptor that recognize either the cytoplasmic or extracellular domains of the receptor, respectively, and (2) biotinylated EGF, a specific probe for the cell surface receptor is now investigated. Immunolocalization of the receptor using the polyclonal antibodies or the biotin-EGF revealed that the receptor was homogeneously distributed on the cell surface of individual cells, or in cells plated at low density. In contrast, as cell density increased, prominent EGF immunoreactivity and biotin-EGF staining became limited to the periphery of the cells, at sites of cell-cell apposition, and was characterized by a honeycomb pattern, typical of a basolateral distribution. The effects of low Ca++ treatment, known to cause cells to round up and detach from one another, on EGF receptor distribution in cells at high cell density were then examined. Confocal microscopy of immunostained preparations revealed that incubation of high density cultures in Ca++-free media for as little as 10 min restored the homogeneous distribution of the EGF receptor and resulted in strong intracellular staining. Three-dimensional reconstruction of serial optical sections revealed that redistribution of the EGF receptor following low Ca++ treatment involved a heretofore undetected ‘ruffling’, an immunostaining pattern characterized by stripes of intense fluorescence signal interspersed with complete absence of fluorescence. Next, cell-cell adhesion was disrupted with antisera to the cell adhesion molecule E-cadherin. Although the antisera caused cells to detach from one another, eventually leading to cell rounding and redistribution of the EGF receptor, the receptor ‘ruffling’ immunostaining pattern rendered by the low Ca++ treatment was not detected. These results suggest that an association may exist between the plasma membrane EGF receptor distribution, density-induced EGF receptor down regulation, and the growth effects of low Ca++ observed in previous studies.

Previous studies demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) regulates steroid biosynthesis. In this study we investigate further PBR action by examining its subcellular localization in mouse adrenal gland using anti-peptide PBR antiserum and employing biotin-strep-tavidin peroxidase immunocytochemistry. Results demonstrated PBR immunostaining exclusively in the cortex. Within this region, however, PBR staining was homogeneously distributed in cells of the zona glomerulosa, whereas in cells of the zona fasciculata both cytoplasmic and prominent plasma membrane immunostaining was evident. Next, PBR distribution was examined using confocal microscopy. Confocal optical sections were obtained, 3-D reconstructions of these sections generated, and vertical, z-sections of the 3-D reconstruction recreated. The immunostaining pattern observed was consistent with a cell surface distribution of PBR. The demonstration of a subset of PBR at the plasma membrane may account for actions of PBR ligands not related to mitochondrial function.

We examined the disassembly and reformation of the Golgi apparatus as a function of the cycle of the seminiferous epithelium in adult rats during stages XIII and XIV, i. e., just prior to and during meiosis I and II. Serial section analysis of primary spermatocytes at metaphase I demonstrated the presence of two Golgi complexes. At the ultrastructural level, these Golgi complexes were shown to be composed of stacks of cisternae and vesicles, with each stack having a varying number of saccules. Although Golgi complex intermediates resulting from the process of organelle disassembly were not clearly identified in diplotene spermatocytes immediately prior to nuclear envelope vesiculation, we did observe clusters of vesicles resembling the “nuage,” with each cluster varying in size and number of vesicles. Meoisis I results in the formation of secondary spermatocytes that exhibit a well-formed spherical Golgi complex approximately half the size of the diplotene spermatocyte Golgi. Next, secondary spermatocytes enter meiosis II. In contrast to metaphase I, during metaphase II reformation of the Golgi apparatus into stacks was not observed and only small clusters of vesicles at two poles of dividing cells were detected. In addition, “nuage”-like structures were not identified during meiosis II. Our results begin to characterize the process by which Golgi apparatus partitioning is accomplished during meiosis, presumably resulting in the delivery of equal complements of this organelle to four round spermatids. We suggest that partitioning of the Golgi apparatus takes place prior to metaphase I and that the two steps of meiosis may exhibit subtle differences with respect to Golgi partitioning. © 1992 Wiley-Liss, Inc.

Sertoli cell nuclei are characterized by deep invaginations and, in addition, the orientation of the nuclei with respect to the wall of the seminiferous tubules varies during the cycle of the seminiferous epithelium. These events may be the result of cytoplasmic filaments acting at the level of the nuclear capsule and may represent significant changes in Sertoli cell activity. Thus, a study was performed to characterize the nature of the perinuclear filaments of Sertoli cells in vivo and in vitro. In Sertoli cells in vivo, microtubules and microfilaments were often detected in the perinuclear cytoplasm, and these cytoskeletal components were observed to course either parallel to, or abut at, the nuclear capsule. In Sertoli cells in vitro, the nuclear infoldings are retained and the perinuclear cytoskeleton was shown to contain microtubules, f-actin, and intermediate filaments. A fixation-permeabilization protocol employing tannic acid–saponin was used and it significantly enhanced the preservation of cytoskeletal components. The presence of f-actin was demonstrated by using the S1 fragment of muscle myosin to decorate the microfilaments. Treatment of the cultured cells with either microtubule or f-actin depolymerizing agents had no effect on nuclear shape. Thus, at present, the function of the prominent perinuclear cytoskeletal components remains unknown.