Search

Intraflagellar transport protein 74 (IFT74) is a component of the core intraflagellar transport complex, a bidirectional movement of large particles along the axoneme microtubules for cilia formation. In this study, we investigated its role in sperm flagella formation and discovered that mice deficiency in Ift74 gene in male germ cells were infertile with low sperm count and immotile sperm. The few developed spermatozoa displayed misshaped heads and short tails. Transmission electron microscopy revealed abnormal flagellar axonemes in the seminiferous tubules where sperm are made. Clusters of unassembled microtubules were present in the spermatids. Testicular expression levels of IFT27, IFT57, IFT81, IFT88, and IFT140 proteins were significantly reduced in the conditional Ift74 mutant mice, with the exception of IFT20 and IFT25. The levels of outer dense fiber 2 and sperm-associated antigen 16L proteins were also not changed. However, the processed A-Kinase anchor protein, a major component of the fibrous sheath, a unique structure of sperm tail, was significantly reduced. Our study demonstrates that IFT74 is essential for mouse sperm formation, probably through assembly of the core axoneme and fibrous sheath, and suggests that IFT74 may be a potential genetic factor affecting male reproduction in man.

Estrogens have traditionally been considered female hormones. Nevertheless, the presence of estrogen in males has been known for over 90 years. Initial studies suggested that estrogen was deleterious to male reproduction because exogenous treatments induced developmental abnormalities. However, demonstrations of estrogen synthesis in the testis and high concentrations of 17β-estradiol in rete testis fluid suggested that the female hormone might have a function in normal male reproduction. Identification of estrogen receptors and development of biological radioisotope methods to assess estradiol binding revealed that the male reproductive tract expresses estrogen receptor extensively from the neonatal period to adulthood. This indicated a role for estrogens in normal development, especially in efferent ductules, whose epithelium is the first in the male reproductive tract to express estrogen receptor during development and a site of exceedingly high expression. In the 1990s, a paradigm shift occurred in our understanding of estrogen function in the male, ushered in by knockout mouse models where estrogen production or expression of its receptors was not present. These knockout animals revealed that estrogen's main receptor (estrogen receptor 1 [ESR1]) is essential for male fertility and development of efferent ductules, epididymis, and prostate, and that loss of only the membrane fraction of ESR1 was sufficient to induce extensive male reproductive abnormalities and infertility. This review provides perspectives on the major discoveries and developments that led to our current knowledge of estrogen's importance in the male reproductive tract and shaped our evolving concept of estrogen's physiological role in the male.

Ring finger protein 216 (RNF216) belongs to the RING family of E3 ubiquitin ligases that are involved in cellular protein degradation. Mutations in human Rnf216 gene have been identified in Gordon Holmes syndrome, which is defined by ataxia, dementia, and hypogonadotropism. However, the gene function of Rnf216 in mammalian species remains unknown. Here, we show that targeted deletion of Rnf216 in mice results in disruption in spermatogenesis and male infertility. RNF216 is not required for female fertility. These findings reveal an essential function of RNF216 in spermatogenesis and male fertility and suggest a critical role for RNF216 in human gonadal development.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2−/−), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2−/− round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2−/− spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2−/− spermatids. The presence of proacrosomal vesicles but lack of a develope...

Estrogen's presence in the male reproductive system has been known for over 60 years, but its potential function in the epididymis remains an important area of investigation. Estrogen is synthesized by germ cells, producing a relatively high concentration in rete testis fluid. There are two estrogen receptors (ESR), the presence of which in the head of the epididymis is well documented and consistent between species; however, in other regions of the epididymis, their expression appears to be isotype, species, and cell specific. ESR1 is expressed constitutively in the epididymis; however, its presence is downregulated by high doses of estrogen, making the design of experiments complicated, as the phenotype of the Cyp19a1−/− mouse does not resemble that of the Esr1−/− mouse. Ligand-independent and DNA-binding Esr1 mutant models further demonstrate the complexity and importance of both signaling pathways in maintenance of efferent ductules and epididymis. Data now reveal the presence of not only classical nuclear receptors, but also cytoplasmic ESR and rapid responding membrane receptors; however, their importance in the epididymis remains undetermined. ESR1 regulates ion transport and water reabsorption in the efferent ducts and epididymis, and its regulation of other associated genes is continually being uncovered. In the male, some genes, such as Aqp9 and Slc9a3, contain both androgen and estrogen response elements and are dually regulated by these hormones. While estrogen pathways are a necessity for fertility in the male, future studies are needed to understand the interplay between androgens and estrogens in epididymal tissues, particularly in cell types that contain both receptors and their cofactors.

Male mice deficient in ESR1 (ERalpha) (Esr1KO mice) are infertile, and sperm recovered from the cauda epididymis exhibit reduced motility and fail to fertilize eggs in vitro. These effects on sperm appear to result from defective epididymal function and not a direct effect on spermatogenesis, as Esr1KO germ cells transplanted into wild-type testes yield normal offspring. We hypothesized that the previously described defect in efferent duct fluid reabsorption would lead to alterations in the epididymal fluid milieu, which would negatively impact sperm function. Analysis of the epididymal fluid revealed that the Esr1KO maintains a higher luminal pH throughout the epididymis, confirming an inability of the efferent ducts and/or epididymis to properly acidify the luminal contents. Subsequent studies showed that these abnormalities were not the result of global defects in epididymal function since protein secretion by the Esr1KO epididymis appeared normal as judged by SDS-PAGE of total secreted proteins and by immunoblotting of candidate secreted proteins. To gain insight into the basis of the aberrant fluid homeostasis in the Esr1KO epididymis, the expression of several enzymes and transporters known to be involved in acid/base regulation were analyzed. The levels of SLC9A3 (NHE3) as well as carbonic anhydrase XIV and SLC4A4 (NBC1) were all reduced in the proximal portion of the Esr1KO epididymis, while other components appeared unaffected, including other ion transporters and ATP6V0A1 (V-ATPase). The altered luminal milieu of the Esr1KO epididymis was shown to lead to a corresponding increase in the intracellular pH of Esr1KO sperm, relative to sperm from control animals. Since pH and bicarbonate ions are critical regulators of sperm cAMP levels and motility, we attempted to bypass the abnormal luminal and intracellular environment by supplementing sperm with exogenous cAMP. This treatment rescued all defective motility parameters, as assayed by CASA, further showing that motility defects are not intrinsic to the sperm but, rather, result from the abnormal epididymal milieu.

The blood-testis barrier (BTB) is formed by tight junctions between Sertoli cells. Results of previous studies suggested that the barrier is deficient in ets variant 5 (ETV5) gene-deleted mice; therefore, microarray data were examined for changes in tight junction-associated genes. The tight junctional protein claudin 5 (CLDN5) was decreased in testes of 8-day-old Etv5(-/-) pups. The study reported herein examined the expression of CLDN5 in wild-type (WT) and Etv5(-/-) mice and evaluated its contribution to BTB function. CLDN5 protein expression was evaluated in 8-day-old WT and Etv5(-/-) and adult WT, Etv5(-/-), and W/W(v) testes by immunohistochemistry and in 8-day-old WT Sertoli cell-enriched and germ cell-enriched fractions by immunocytochemistry. Cldn5 mRNA expression was evaluated in 0- to 20-day-old and adult WT mice and in 8-day-old and adult Etv5(-/-) mice via quantitative PCR. Tracer studies were performed in adult WT, Etv5(-/-), and W/W(v) mice. The results indicate the following: 1) CLDN5 was expressed in Sertoli cells, spermatogonia, and preleptotene spermatocytes. 2) Seminiferous epithelial CLDN5 expression depended upon both the presence of germ cells and ETV5. 3) CLDN5 expression in testicular vascular endothelium and rete testis epithelium was ETV5 independent. 4) Cldn5 mRNA expression increased in the testes of juvenile mice at the time of BTB formation. 5) Testes of Etv5(-/-) and W/W(v) mice, which are both deficient in seminiferous epithelial CLDN5 expression, had biotin tracer leakage from the interstitial space into the seminiferous tubule lumen. In conclusion, CLDN5 is expressed in the seminiferous epithelium, appears to be regulated by multiple influences, and contributes to BTB function.

Mice that are ets variant gene 5 (ETV5) null (Etv5 � /� ) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5 � /� mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5 � /� germ cells failed to establish spermatogenesis in W/W v mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal

Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI–VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.

The transcription factor ets variant gene 5 (ETV5; also known as ERM) is essential for self-renewal of spermatogonial stem cells (SSCs). Mice with targeted disruption of Etv5 (Etv5−/−) undergo the first wave of spermatogenesis, but all SSCs are lost during this time, causing a Sertoli cell-only phenotype. This study examined body and testis growth and the time course of SSC loss in Etv5−/− mice to understand how loss of ETV5 impacts testicular and somatic development. Body weights were reduced in postnatal Etv5−/− males, indicating a role of ETV5 in growth. Testis weights and histology in Etv5−/− and wild-type (WT) males were similar at Postnatal Day 4, but testis weights were reduced at d8 and subsequently, indicating that ETV5 impacts postnatal testis growth. SSC density (SSCs per μm2 of seminiferous tubule), estimated using an antibody against GFRA1, was similar in 4d WT and Etv5−/− mice. By 8 and 12d, GFRA1-positive cell density in Etv5−/− mice was decreased 17% and 32%, respectively, vs. WT. By 28d, GFRA1-positive cell density in Etv5−/− was reduced 95%, and GFRA1-positive cells were absent in 36d Etv5−/− males. In contrast to WT, 35- to 56-day-old Etv5−/− mice were infertile as assessed by natural breeding, artificial insemination, and in vitro fertilization, although motile sperm were present in epididymides of Etv5−/− mice during this time. In summary, initial testis development is normal in Etv5−/− mice despite decreased body weight, but SSC loss begins between 4 and 8d of age, indicating that ETV5 has effects beginning in the early neonatal period. Etv5−/− mice are infertile even when sperm is produced, indicating that ETV5 loss has other effects besides lack of SSC self-renewal that impair fertility.

Thyroid hormone inhibits neonatal Sertoli cell proliferation and recent results have shown that thyroid hormone upregulates cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 and p21Cip1 (also known as CDKN1B and CDKN1A, respectively) in neonatal Sertoli cells. This suggests that these CDKIs, which negatively regulate the cell cycle, could be critical in Sertoli cell proliferation. Consistent with this hypothesis, mice lacking p27Kip1 develop testicular organomegaly, but Sertoli cell numbers have not been determined. Likewise, effects of loss of p21Cip1 or both p27 and p21 on Sertoli cell number and testicular development were unknown. To determine if p27 and/or p21 regulate Sertoli cell proliferation, we measured Sertoli cell proliferation at Postnatal Day 16 and testis weight, Sertoli cell number, and daily sperm production (DSP) in 4-mo-old wild-type (WT), p21 knockout (p21KO), p27 knockout (p27KO), and p27/p21 double-knockout (DBKO) mice. Testis weights were increased 27%, 42%, and 86% in adult p21KO, p27KO, and DBKO mice, respectively, compared with WT. Sertoli cell number also was increased 48%, 126%, and 126% in p21KO, p27KO, and DBKO mice, respectively, versus WT. DSP in p21KO, p27KO, and DBKO testes also showed significant increases compared with WT mice. Although DSP was increased, there were increased spermatogenic defects observed in both p27KO and DBKO mice compared with WT. These data indicate that both p27 and p21 play an inhibitory role in regulating adult Sertoli cell number such that loss of either CDKI produces primary increases in Sertoli cell number and secondary increases in DSP and testis weight. Furthermore, loss of both CDKIs causes additive effects on DSP and testis weight, suggesting a central role for these CDKIs in testis development.

The objective of the present study was to characterize and investigate potential mechanisms for the male reproductive toxicity of trichloroethylene (TCE). Male rats exposed to TCE in drinking water exhibited a dose-dependent decrease in the ability to fertilize oocytes from untreated females. This reduction in fertilizing ability occurred in the absence of treatment-related changes in combined testes/epididymides weight, sperm concentration, or sperm motility. In addition, flow cytometric analysis showed that there were no treatment-related differences in sperm mitochondrial membrane potential or acrosomal stability. TCE caused slight histological changes in efferent ductule epithelium, coinciding with the previously reported ductule localization of cytochrome P450 2E1. However, no alterations were noted in the testis or in any segment of the epididymis. Because there were no treatment-related changes to sperm indices and no clear pathological lesions to explain the reduced fertilization, the present study investigated TCE-mediated sperm oxidative damage. Oxidized proteins were detected by immunochemical techniques following the derivatization of sperm protein carbonyls with dinitrophenyl hydrazine. Immunochemical staining of whole, intact sperm showed the presence of halos of oxidized proteins around the head and midpiece of sperm from TCE-treated animals. The presence of oxidized sperm proteins was confirmed by Western blotting using in vitro-oxidized sperm as a positive control. Thiobarbituric acid reactive substances analyses showed a dose-dependent increase in the level of lipid peroxidation in sperm from treated animals, as well. Oxidative damage to sperm may explain the diminished fertilizing capacity of exposed animals and provide another mechanism by which TCE can adversely affect reproductive capabilities in the male.

The estrogen receptor-alpha (ERalpha) knockout mouse (alphaERKO) lacks ERalpha throughout development; therefore, an adult model for the study of estrogen effects in male mice was recently developed using the antiestrogen ICI 182,780. However, differences between species have been noted during immunostaining for ERalpha in the male tract as well as in response to treatments with antiestrogens. Therefore, we developed the antiestrogen model in the adult male rat to test, in another species, the hypothesis that estrogen regulates fluid reabsorption in efferent ductules. Estrogen receptor in the rat was blocked using ICI 182,780 for 100-150 days. Male Sprague-Dawley rats were treated weekly with s.c. injections of ICI 182,780 (10 mg) or castor oil (as control). The effects of ICI included testicular atrophy and infertility, similar to terminal effects in the alphaERKO male. Additionally, ICI induced dilations of the rete testis and efferent ductules and a reduction in the height of the ductule epithelium, which are changes similar to those in both alphaERKO and ICI-treated mice. One difference between species was a large variation in effects on the rat efferent ductule epithelium, including a transient increase in the number of periodic acid-Schiff-positive, lysosomal-like granules. These data confirm that estrogen is required for normal function of the efferent ductules and is essential for long-term fertility in the male rodent.

We recently found that cytochrome P450 aromatase (P450arom) is present in germ cells of the mammalian testis and is capable of converting androgens to estrogens in the male reproductive tract. The objective of the present study was to determine whether testicular germ cells and epididymal sperm of an avian species are also capable of synthesizing estrogen. P450arom was localized in the rooster testis and epididymal region by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. Immunostaining was present in pachytene spermatocytes, round spermatids, elongated spermatids, flagella of late spermatids, and sperm in the epididymal region. A positive reaction was also found in nonciliated cells of the epididymal region. However, the absence of mRNA for P450arom in the epididymal region indicated that the immunoreactive protein present in the epididymal region is not synthesized in this region. The immunoreactive P450arom found in epididymal sperm was shown to be active through use of a 3H2O assay. On the basis of these data, we conclude that rooster testicular germ cells and epididymal sperm are sites for the synthesis of estrogen, a potential regulator or modulator of germinal epithelium in the testis and the epithelium of the epididymal region of the avian species.

Propylthiouracil- (PTU) induced transient neonatal hypothyroidism increases adult rat testis weight 80-100%; this effect involves prolongation of Sertoli cell proliferation. To gain insight into developmental effects of PTU on the testis, we used Northern analysis to examine chronological expression of Sertoli cell mRNA in postnatal rat testes from rats that were untreated (controls) or were given PTU from birth to Day 25. Treated rats showed prolonged early expression of genes associated with dividing Sertoli cells such as MIS (Mullerian inhibiting substance) and c-erbA alpha (thyroid hormone receptor). Expression of several other Sertoli cell mRNAs (androgen-binding protein [ABP], clusterin, and inhibin-beta B) was delayed, as was that of hemiferrin, a spermatid-specific mRNA. Temporal expression patterns for other mRNAs (sulfated glycoprotein [SGP]-1, transferrin, and inhibin-alpha) were similar in control and treated animals. Additionally, thyroid hormone replacement in PTU-treated animals decreased MIS and c-erbA alpha mRNA expression to control levels. The altered developmental pattern of expression of a number of major Sertoli cell genes reflects a prolonged mitogenesis and delayed maturation of Sertoli cells in neonatally hypothyroid animals. Furthermore, our results suggest that thyroid hormone may directly potentiate molecular events associated with cessation of Sertoli cell proliferation and maturation during early testis development.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

Somatic and testis-specific cytochromes c were localized ultrastructurally in the seminiferous epithelium by immunocytochemistry using monospecific antibodies. Cytochrome cS was lost from the mitochondria as spermatogenesis advanced, while there was a relative increase in cytochrome cT during the zygotene-to-pachytene transition; this was in agreement with other studies that have suggested activation of the cytochrome cT gene during prophase of the first meiotic division. Cytochrome cT was highly concentrated in mitochondria that were being degraded within cytoplasmic lobes of spermatids and in residual bodies that were phagocytized by Sertoli cells. The two isoforms were found to coexist within the same mitochondrion during the transitional period from cytochrome cS to cytochrome cT predominance. In addition, both cytochromes c were present in the chromatoid bodies of spermatocytes and round spermatids; this suggests that the chromatoid body may be involved in the storage of these isozymes and possibly in their differential expression within germ cell mitochondria. Apocytochrome c was concentrated in mitochondria and chromatoid bodies of the germ cells and also scattered in the cytoplasm. The presence of the holoprotein and apoprotein immunoprobes within the chromatoid bodies of spermatocytes and spermatids was an interesting observation that raises questions regarding the precise location of the synthesis of cytochromes c in spermatogenic cells.

We have previously shown that treating rats with the reversible goitrogen 6-propyl-2-thiouracil (PTU) from birth to Day 25 increases testis size and sperm production in adulthood by up to 80% and 140%, respectively. The purpose of this study was to determine the critical period(s) during development when PTU treatment can increase adult testis size and sperm production. Rats were treated with PTU beginning on Days 0, 8, 16, or 24 for periods of 9, 17, or 25 days. To further define the critical period, additional rats were treated with PTU prenatally or on Days 4-24. PTU treatments of 9, 17, or 25 days beginning at birth increased testis weight 18%, 38%, and 69%, respectively, by 135 days of age, while daily sperm production (DSP) increased 35%, 65%, and 94%, respectively. Efficiency of sperm production (DSP/g testis) also increased by approximately 25% in these rats. There was an inverse relationship between testis and body weights: increasing lengths of PTU treatment increased testis weight but decreased body weight. PTU treatment starting on Day 8 or later did not increase testis weight or DSP regardless of duration. Treatment on Days 4-24 increased adult testis weight and DSP similarly to treatment from birth to Day 24, but prenatal PTU treatment was ineffective. Testosterone concentrations were not altered in treated rats, even those with increased testis weight. These results indicate that the later part of the first postnatal week (Days 4-8) is the crucial period during which PTU treatment must begin in order to increase adult testis size and sperm production.(ABSTRACT TRUNCATED AT 250 WORDS)

An estimated 25%–40% of infertile men have idiopathic infertility associated with deficient sperm numbers and quality. Here, we identify the membrane-anchored serine protease PRSS21, also known as testisin, to be a novel proteolytic factor that directs epididymal sperm cell maturation and sperm-fertilizing ability. PRSS21-deficient spermatozoa show decreased motility, angulated and curled tails, fragile necks, and dramatically increased susceptibility to decapitation. These defects reflect aberrant maturation during passage through the epididymis, because histological and electron microscopic structural analyses showed an increased tendency for curled and detached tails as spermatozoa transit from the corpus to the cauda epididymis. Cauda epididymal spermatozoa deficient in PRSS21 fail to mount a swelling response when exposed to hypotonic conditions, suggesting an impaired ability to respond to osmotic challenges facing maturing spermatozoa in the female reproductive tract. These data suggest that aberrant regulation of PRSS21 may underlie certain secondary male infertility syndromes, such as “easily decapitated” spermatozoa in humans.

Mice that are ets variant gene 5 (ETV5) null (Etv5(-/-)) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5(-/-) mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5(-/-) germ cells failed to establish spermatogenesis in W/W(v) mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal Etv5(-/-) mice by Postnatal Days 4-8, indicating that disrupted GDNF/RET/GFRA1 signaling may occur before initial spermatogonial stem/progenitor cell decrease. Etv5(-/-) spermatogonia had reduced proliferation in vivo and in vitro. Decreased cell proliferation may cause the observed decreases in the number of type A spermatogonia (Postnatal Day 17) and daily sperm production (Postnatal Day 30) in Etv5(-/-) mice, indicating quantitative impairments in the first wave of spermatogenesis. In conclusion, ETV5 is expressed beginning in fetal Sertoli cells and can potentially have effects on neonatal Sertoli cells and germ cells. In addition, ETV5 has critical effects on neonatal spermatogonial proliferation, which may involve impaired signaling through the RET receptor.

The dfcct on the dcvdoping fetal testis of in utero c:xposurc to 1-MHz, continuous-wave ultrasound in the spatial peak, temporal average intensity range 0.5-10 W /em• for durations of 400-30 sec on Day 9, 12, or 15 of gestation was determined Results show that two subde, yet potentially ddctcrious, dfccts occurred: a reduction in the ScrtoU cell population and an apparent delay in the cessation of gonocyte mitosJs. An increase was also seen in the number of fetal resorptions and stillborn pups per number of implantation sites in the exposed specimens as compared to the sham and cage controls. Because the reduction in testis weight was proportional to dccrcascd body weight and because there was no dJlfcrcncc in ScrtoU cell numbers due to day of treatment, the testicular dfccts may reflect a gcncralizcd delay in growth. Whether this dfcct of ultrasound on fetal testis will be translated into an equal reduction in germ cell numbers in the adult testis remains to be determined

Vitamin A is required for male fertility and normal spermatogenesis. Retinoic acid (RA), an active metabolite of vitamin A, is necessary for spermatogonial maturation and proper entry of germ cells into meiotic prophase in the postnatal testes. The expression of Stra8, which is essential for successful meiosis in both male and female gonads and normal spermatogenesis, is directly related to the availability of RA. This study examined the developmental expression pattern of Stra8 transcript in both male and female gonads, provided specific cellular localization of STRA8 protein in the postnatal and adult testis, and investigated RA actions in adult germ cells in a vitamin A-sufficient condition. The peak of Stra8 mRNA expression coincided with the onset of meiosis in postnatal testes. STRA8 protein was detected in gonocytes as early as 5 days postpartum. The expression of STRA8 protein in the neonatal testes was not uniform among spermatogonia, perhaps heralding the asynchronous beginning of spermatogenesis. In adult testes, the highest level of Stra8 mRNA and protein was found in seminiferous epithelial stages VI–VIII. STRA8 protein was localized to some type A and B spermatogonia, preleptotene spermatocytes, and early leptotene spermatocytes. In the vitamin A-sufficient adult testes, RA but not retinol acetate stimulated Stra8 mRNA expression. STRA8 protein expression in adult spermatogonia was induced by RA stimulation, suggesting its role in spermatogonial differentiation. Retinoic acid also increased the number of preleptotene spermatocytes exhibiting 5-bromo-2-deoxyuridine incorporation, indicating a more synchronized premeiotic DNA replication.

The stages of the cycle inthe rat seminiferous epitheium are illustrated for testes fixed by vascular perfusion and embedded in plastic resins.Improved cellular resolution in plastic sections permitted a clearer demarcation of the than in paraffin. Quantitative data are presented to the recognition of stages, particularly those in transition. Stages IV, V, VII, XI,and XII had the highest frequencies of transitional characteristics. Stage IV was redefined to be more consistent with the occurrence of a high percentage of mitotic figures and to clarify transitions in this stage. Although the resolution of cellular detail was greatly improved withthe use of plastics, the thinner sections contained fewer identifying features together within a single tubule cross section and sometimes major characteristics were absent. Therefore, additional characteristics were used for stage classification, SUCh as nuclear diameter and the presence or absence of mitotic figures. A binarydecision key isprovided to improve consistency among laboratories in the identification of the stages in plastic-embedded testes.

The transcription factor ets variant gene 5 (ETV5; also known as ERM) is essential for self-renewal of spermatogonial stem cells (SSCs). Mice with targeted disruption of Etv5 (Etv5(-/-)) undergo the first wave of spermatogenesis, but all SSCs are lost during this time, causing a Sertoli cell-only phenotype. This study examined body and testis growth and the time course of SSC loss in Etv5(-/-) mice to understand how loss of ETV5 impacts testicular and somatic development. Body weights were reduced in postnatal Etv5(-/-) males, indicating a role of ETV5 in growth. Testis weights and histology in Etv5(-/-) and wild-type (WT) males were similar at Postnatal Day 4, but testis weights were reduced at d8 and subsequently, indicating that ETV5 impacts postnatal testis growth. SSC density (SSCs per mum(2) of seminiferous tubule), estimated using an antibody against GFRA1, was similar in 4d WT and Etv5(-/-) mice. By 8 and 12d, GFRA1-positive cell density in Etv5(-/-) mice was decreased 17% and 32%, respectively, vs. WT. By 28d, GFRA1-positive cell density in Etv5(-/-) was reduced 95%, and GFRA1-positive cells were absent in 36d Etv5(-/-) males. In contrast to WT, 35- to 56-day-old Etv5(-/-) mice were infertile as assessed by natural breeding, artificial insemination, and in vitro fertilization, although motile sperm were present in epididymides of Etv5(-/-) mice during this time. In summary, initial testis development is normal in Etv5(-/-) mice despite decreased body weight, but SSC loss begins between 4 and 8d of age, indicating that ETV5 has effects beginning in the early neonatal period. Etv5(-/-) mice are infertile even when sperm is produced, indicating that ETV5 loss has other effects besides lack of SSC self-renewal that impair fertility.

Thyroid hormone inhibits neonatal Sertoli cell proliferation and recent results have shown that thyroid hormone upregulates cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 and p21Cip1 (also known as CDKN1B and CDKN1A, respectively) in neonatal Sertoli cells. This suggests that these CDKIs, which negatively regulate the cell cycle, could be critical in Sertoli cell proliferation. Consistent with this hypothesis, mice lacking p27Kip1 develop testicular organomegaly, but Sertoli cell numbers have not been determined. Likewise, effects of loss of p21Cip1 or both p27 and p21 on Sertoli cell number and testicular development were unknown. To determine if p27 and/or p21 regulate Sertoli cell proliferation, we measured Sertoli cell proliferation at Postnatal Day 16 and testis weight, Sertoli cell number, and daily sperm production (DSP) in 4-mo-old wild-type (WT), p21 knockout (p21KO), p27 knockout (p27KO), and p27/p21 double-knockout (DBKO) mice. Testis weights were increased 27%, 42%, and 86% in adult p21KO, p27KO, and DBKO mice, respectively, compared with WT. Sertoli cell number also was increased 48%, 126%, and 126% in p21KO, p27KO, and DBKO mice, respectively, versus WT. DSP in p21KO, p27KO, and DBKO testes also showed significant increases compared with WT mice. Although DSP was increased, there were increased spermatogenic defects observed in both p27KO and DBKO mice compared with WT. These data indicate that both p27 and p21 play an inhibitory role in regulating adult Sertoli cell number such that loss of either CDKI produces primary increases in Sertoli cell number and secondary increases in DSP and testis weight. Furthermore, loss of both CDKIs causes additive effects on DSP and testis weight, suggesting a central role for these CDKIs in testis development.

The objective of the present study was to characterize and investigate potential mechanisms for the male reproductive toxicity of trichloroethylene (TCE). Male rats exposed to TCE in drinking water exhibited a dose-dependent decrease in the ability to fertilize oocytes from untreated females. This reduction in fertilizing ability occurred in the absence of treatment-related changes in combined testes/epididymides weight, sperm concentration, or sperm motility. In addition, flow cytometric analysis showed that there were no treatment-related differences in sperm mitochondrial membrane potential or acrosomal stability. TCE caused slight histological changes in efferent ductule epithelium, coinciding with the previously reported ductule localization of cytochrome P450 2E1. However, no alterations were noted in the testis or in any segment of the epididymis. Because there were no treatment-related changes to sperm indices and no clear pathological lesions to explain the reduced fertilization, the present study investigated TCE-mediated sperm oxidative damage. Oxidized proteins were detected by immunochemical techniques following the derivatization of sperm protein carbonyls with dinitrophenyl hydrazine. Immunochemical staining of whole, intact sperm showed the presence of halos of oxidized proteins around the head and midpiece of sperm from TCE-treated animals. The presence of oxidized sperm proteins was confirmed by Western blotting using in vitro-oxidized sperm as a positive control. Thiobarbituric acid reactive substances analyses showed a dose-dependent increase in the level of lipid peroxidation in sperm from treated animals, as well. Oxidative damage to sperm may explain the diminished fertilizing capacity of exposed animals and provide another mechanism by which TCE can adversely affect reproductive capabilities in the male.

Expression of estrogen receptors (ERs) in the reproductive tracts of adult male dogs and cats has not been reported. In the present study, ERalpha and ERbeta were localized by immunohistochemistry using ER-specific antibodies. ERalpha was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ERalpha staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ERalpha. In the cat, except for the initial region of caput epididymis, ERalpha staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERbeta. In rete testis and efferent ductules, epithelial cells were weakly positive for ERbeta. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ERalpha and ERbeta in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ERalpha and ERbeta in male dog and cat reproductive tracts, demonstrating more intensive expression of ERbeta than ERalpha. However, as in other species, the efferent ductules remained the region of highest concentration of ERalpha.

Turkeys which produce yellow semen have abnormal ductuli efferentes’ epithelial morphology, with b!ebbing of cytoplasmic material into the ductal lumen. This could possibly increase the activity or concentration of seminal plasma components. In the present study, seminal plasma from 270 Large White breeder turkeys was evaluated for protein and cholesterol concentrations and the activities of acid phosphatase and asparate aminotransaminase. In a separate experiment, protein concentrations of turkey seminal plasma were estimated by biuret or Bradford methods. Bradford estimates were 46.6% less than those obtained with the biuret assay, using bovine serum albumin as the standard. Estimates of seminal plasma protein concentration in the main study were obtained using the Bradford method, and should be adjusted accordingly when compared with other studies using the biuret technique. Abnormal yellow seminal plasma, compared to normal white seminal plasma, had elevated levels of total protein and cholesterol and increased activities of acid phosphatase and aspartate aminotransasninase. Overall means were: 14.3 mg/mI, 38.9 mg/dl, 232.6 lU/mI, 81.0 lU/mI, respectively. Correlation coefficients for cholesterol concentration, acid phosphatase and aminotransaminase activity with protein concentration were +0.65, 0.70 and 0.50 (P

The objective of this research were twofold: 1) to determine if decreased spermatozoal longevity, a previously reported heritable trait in chickens, was attributable to spermatozoal passage through the excurrent ducts, and 2) to document the morphology of the testicular excurrent ducts from affected roosters. Though spermatozoa were viable at ejaculation, as evidenced by their exclusion of ethidium bromide, fertility after intravaginal insemination of spermatozoa from affected roosters was less (p less than 0.001) than that observed with spermatozoa from nonaffected controls, 37 +/- 2.3 versus 58 +/- 1.5%, respectively, over a 21-day egg-collection interval. In contrast, fertility after intramagnal insemination of testicular spermatozoa from affected roosters was equivalent (p greater than 0.05) to that of nonaffected controls, 47 +/- 2.2 versus 41 +/- 3.6%, respectively. After intravaginal insemination, neither type of testicular spermatozoa fertilized oocytes. The ductuli efferentes proximales from affected roosters were characterized by a greater luminal cross-sectional area as well as a diminished height and number of longitudinal epithelial folds (p less than 0.005). It was concluded that heritable decreased spermatozoal longevity in the chicken is not attributable to an inherent spermatozoal defect. Rather, the defect is acquired during passage of spermatozoa through the extragonadal ducts of the rooster.

A serial breeding technique was used to evaluate the fertility of male Sprague-Dawley rats after exposure to the fungicide carbendazim (methyl 2-benzimidazole carbamate). Proven-fertile male rats (90 days old) received 10 daily doses of corn oil or carbendazim (400 mg/kg/day) peroral. Each male was bred with a new female each week; breeding began on the third day of treatment and continued for 32 wk after the last day of chemical exposure. Twelve days after each breeding period, the females were killed, their uteri were examined for resorptions, and the number of dead and viable fetuses was determined. All males were killed 35 wk post exposure, and testicular tissue was prepared for histopathological examination by vascular perfusion. Fertility (percent fertile as indicated by pregnant females) of males in the carbendazim-treated group was depressed the first post-exposure week; 10 of the 24 treated males failed to produce a pregnant female as compared with no failures in the control group. By the fifth post-exposure week, 16 of the 24 carbendazim-treated males were infertile. Of these 16 males, 4 recovered fertility after a failure to produce a pregnant female for 5-11 consecutive breeding periods. However, 12 of the males did not recover fertility during the remainder of the 32-wk post-exposure period. Histological examinations of testicular sections 245 days post exposure revealed that exposure to carbendazim caused severe seminiferous tubular atrophy (greater than 85% of tubules were atrophic) in those carbendazim-treated males that failed to recover fertility.(ABSTRACT TRUNCATED AT 250 WORDS)