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Besides androgens, estrogen signaling plays a key role in normal development and pathologies of the prostate. Irreversible synthesis of estrogens from androgens is catalyzed by aromatase. Interestingly, animals lacking aromatase do not develop cancer or prostatitis, whereas those with overexpression of aromatase and, consequently, high estrogen levels develop prostatitis and squamous metaplasia via estrogen receptor 1 (ERα). Even with this evidence, the aromatase expression in the prostate is controversial. Moreover, little is known about the occurrence of age-dependent variation of aromatase and its association with histopathological changes commonly found in advanced age, a knowledge gap that is addressed herein. For this purpose, the immunoexpression of aromatase was evaluated in the prostatic complex of young adult to senile Wistar rats. ERα was also investigated, to extend our understanding of estrogen responsiveness in the prostate. Moderate cytoplasmic immunoreactivity for aromatase was detected in the glandular epithelium. Eventually, some basal cells showed intense staining for aromatase. The expression pattern for aromatase appeared similar in the normal epithelium when young and senile rats were compared; this result was corroborated by Western blotting. Conversely, in senile rats, there was an increase in the frequency of basal cells intensely stained for aromatase, which appeared concentrated in areas of intraepithelial proliferation and prostatitis. These punctual areas also presented increased ERα positivity. Together, these findings suggest a plausible source for hormonal imbalance favoring estrogen production, which, by acting through ERα, may favor the development of prostatic lesions commonly found in advanced age.

Estrogens have historically been associated with female reproduction, but work over the last two decades established that estrogens and their main nuclear receptors (ESR1 and ESR2) and G protein-coupled estrogen receptor (GPER) also regulate male reproductive and nonreproductive organs. 17β-Estradiol (E2) is measureable in blood of men and males of other species, but in rete testis fluids, E2 reaches concentrations normally found only in females and in some species nanomolar concentrations of estrone sulfate are found in semen. Aromatase, which converts androgens to estrogens, is expressed in Leydig cells, seminiferous epithelium, and other male organs. Early studies showed E2 binding in numerous male tissues, and ESR1 and ESR2 each show unique distributions and actions in males. Exogenous estrogen treatment produced male reproductive pathologies in laboratory animals and men, especially during development, and studies with transgenic mice with compromised estrogen signaling demonstrated an E2 role in normal male physiology. Efferent ductules and epididymal functions are dependent on estrogen signaling through ESR1, whose loss impaired ion transport and water reabsorption, resulting in abnormal sperm. Loss of ESR1 or aromatase also produces effects on nonreproductive targets such as brain, adipose, skeletal muscle, bone, cardiovascular, and immune tissues. Expression of GPER is extensive in male tracts, suggesting a possible role for E2 signaling through this receptor in male reproduction. Recent evidence also indicates that membrane ESR1 has critical roles in male reproduction. Thus estrogens are important physiological regulators in males, and future studies may reveal additional roles for estrogen signaling in various target tissues.

Studies with 6-n-propyl-2-thiouracil (PTU) in laboratory rodents have shown that transient neonatal hypothyroidism leads to increased Sertoli cell (SC) number, testis size and sperm production. However, scarce and inconclusive data are available for farm animals. In the present study, Piau pigs received PTU in a gel capsule containing 8 mg/kg of body weight for 14 weeks starting from the first week of age, whereas control animals received only the vehicle. Blood samples were collected during the experimental period for hormonal evaluation in the serum. The animals were orchiectomized at adulthood and had their testes used for histomorphometric analysis. Indicating that the PTU concentration used was effective in promoting hypothyroidism, PTU-treated pigs showed a 30% lower body weight and reduced thyroxine levels (p 0.05) during the treatment period. At adulthood, the body weight was similar in both groups but, surprisingly, PTU-treated pigs showed 30% lower testis weight (p 0.05). In general, treated pigs presented increased follicle-stimulating hormone levels, whereas testosterone levels tended to be lower from 9 to 23 weeks of age. No significant differences were observed for estradiol, Leydig cell volume and number, tubular diameter, SC number per gram of testis, SC efficiency and meiotic index. However, seminiferous tubule occupancy, total tubular length, SC number per testis, and daily sperm production per testis and per gram of testis (DSP/g/T) were significantly lower (p 0.05) in PTU-treated pigs. Therefore, in contrast to laboratory rodents, our results showed that SC proliferation and DSP/g/T (spermatogenic efficiency) in Piau pigs is diminished by postnatal PTU treatment.

Sertoli cells (SCs) play a crucial role in testis differentiation, development and function, determining the magnitude of sperm production in sexually mature animals. For over 40 years, it has been considered that these key testis somatic cells stop dividing during early pre-pubertal phase, between around 10 to 20 days after birth respectively in mice and rats, being after that under physiological conditions a stable and terminally differentiated population. However, evidences from the literature are challenging this dogma. In the present study, using several important functional markers (Ki-67, BrdU, p27, GATA-4, Androgen Receptor), we investigated the SC differentiation status in 36 days old and adult Wistar rats, focusing mainly in the transition region (TR) between the seminiferous tubules (ST) and the rete testis. Our results showed that SCs in TR remain undifferentiated for a longer period and, although at a lesser degree, even in adult rats proliferating SCs were observed in this region. Therefore, these findings suggest that, different from the other ST regions investigated, SCs residing in the TR exhibit a distinct functional phenotype. These undifferentiated SCs may compose a subpopulation of SC progenitors that reside in a specific microenvironment capable of growing the ST length if needed from this particular testis region. Moreover, our findings demonstrate an important aspect of testis function in mammals and opens new venues for other experimental approaches to the investigation of SC physiology, spermatogenesis progression and testis growth. Besides that, the TR may represent an important site for pathophysiological investigations and cellular interactions in the testis. © 2016 Taylor & Francis.

Estrogen receptor 1 (ESR1) mediates major reproductive functions of 17β-estradiol (E2). Male Esr1 knockout (Esr1KO) mice are infertile due to efferent ductule and epididymal abnormalities. The majority of ESR1 is nuclear/cytoplasmic; however, a small fraction is palmitoylated at cysteine 451 in mice and localized to cell membranes, in which it mediates rapid E2 actions. This study used an Esr1 knock-in mouse containing an altered palmitoylation site (C451A) in ESR1 that prevented cell membrane localization, although nuclear ESR1 was expressed. These nuclear-only estrogen receptor 1 (NOER) mice were used to determine the roles of membrane ESR1 in males. Epididymal sperm motility was reduced 85% in 8-month-old NOER mice compared with wild-type controls. The NOER mice had decreased epididymal sperm viability and greater than 95% of sperm had abnormalities, including coiled midpieces and tails, absent heads, and folded tails; this was comparable to 4-month Esr1KO males. At 8 months, daily sperm production in NOER males was reduced 62% compared with controls. The NOER mice had histological changes in the rete testes, efferent ductules, and seminiferous tubules that were comparable with those previously observed in Esr1KO males. Serum T was increased in NOER males, but FSH, LH, and E2 were unchanged. Critically, NOER males were initially subfertile, becoming infertile with advancing age. These findings identify a previously unknown role for membrane ESR1 in the development of normal sperm and providing an adequate environment for spermatogenesis.

Ppard(-/-) mice exhibit smaller litter size compared with Ppard(+/+) mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard(-/-) mice compared with Ppard(+/+) mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard(+/+) mice as compared with Ppard(-/-) mice, and these were associated with decreased Sertoli cell number in Ppard(+/+) mice. Cyclin D1 and cyclin D2 expression was lower in Ppard(+/+) as compared with Ppard(-/-) mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard(+/+) mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.

Endocrine regulation of rete testis and the efferent ductules involves a complex interaction of multiple steroid hormone receptors and their ligands, with what is now called an androgen/estrogen balance. Estrogen receptor-α shows constitutive expression and is unaltered by most experimental treatments, including DES. However, DES causes the loss of androgen receptor in the efferent duct epithelium. Pathological changes in this region often appear to be linked to either excessive fluid reabsorption or the opposite, an inhibition of reabsorption. With both deviations, there will be luminal dilation in the proximal region near the rete testis, and if the lesion is found closer to the testis there will be a greater involvement of rete testis and seminiferous tubules. Pathological changes have been associated with several diseases in man and typically involve the development of large cysts, as can be found in proximal tubule diseases of the kidney.

The efferent ducts represent an important site of toxicity in the male reproductive tract but are not routinely examined in toxicity studies. This article describes a primary efferent duct toxicity that resulted in secondary testicular changes in rats. Male rats were administered LTI-1, a leukotriene A4 hydrolase inhibitor, at doses up to 250 mg/kg/d for 3 month or 150 mg/kg/d for 6 month. At the highest dose levels, testicular changes were predominantly unilateral and characterized by diffuse dilation or atrophy of the seminiferous tubules. These testicular changes correlated with granulomatous inflammation in the corresponding efferent ducts, suggesting that the mechanism for the testicular changes involves obstruction and impaired fluid reabsorption in the efferent ducts. Subsequent buildup in fluid volume and back-pressure upstream of the blockage cause dilation of the seminiferous tubules, which, in its late stages, progress to tubular atrophy. There are important differences in efferent duct anatomy between rats and larger mammals, including humans, such that the latter are less susceptible to testicular injury by this mechanism. Because of the limited relevance of this rat-specific finding to humans, it is important to distinguish testicular changes secondary to efferent duct toxicity from primary drug-induced testicular toxicity.

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.

Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

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.

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (−/−), which enabled us to study HUFA deficiency without depleting their precursors. In −/−, no in vivo AA synthesis was detected after administration of [U-13C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the −/− developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The −/− also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male −/− lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in −/− declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in −/−. DPAn-6 was absent in −/− brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (−/−) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.—Stroud, C. K., T. Y. Nara, M. Roqueta-Rivera, E. C. Radlowski, P. Lawrence, Y. Zhang, B. H. Cho, M. Segre, R. A. Hess, J. T. Brenna, W. M. Haschek, and M. T. Nakamura. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration.

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.

Cadmium, a ubiquitous environmental contaminant, damages several major organs in humans and other mammals. The molecular mechanisms for damage are not known. At high doses (5 mg/kg cadmium chloride or higher), testicular damage in mice, rats, and other rodents includes interstitial edema, hemorrhage, and changes in the seminiferous tubules affecting spermatogenesis. Necrosis is evident by 48 h. The goal of this study was to fine map and identify the cdm gene, a gene that when mutated prevents cadmium-induced testicular toxicity in mouse strains with a mutation in this gene. A serine-threonine phosphatase, calcineurin (CN), subunit A, alpha isoform (Ppp3ca), was one of the seven candidates in the cdm region that was narrowed from 5.6 to 2.0 Mb on mouse chromosome 3. An inhibitor of CN, the immunosuppressant, FK506, prevented cadmium-induced testicular damage in five pathological categories, including vascular endothelial and seminiferous epithelial endpoints. Inductively coupled plasma-mass spectrometry revealed that FK506 protected without lowering the amount of cadmium in the testes. Ppp3ca(-/-) mice were investigated but were found to exhibit endogenous testicular abnormalities, making them an inappropriate model for determining whether the inactivation of the Ppp3ca gene would afford protection from cadmium-induced testicular toxicity. The protection afforded by FK506, found by the current study, indicated that CN is likely to be important in the mechanism of cadmium toxicity in the testis and possibly other organs.

The present study was undertaken to examine the antispermatogenic effect of l-CDB-4022 in the adult male cynomolgus monkey. Monkeys (four per group) were dosed via nasogastric tube for 7 d with l-CDB-4022 at 12.5 mg/kg.d or vehicle (d 0=first day of dosing). Plasma levels of l-CDB-4022 and its deesterified metabolite were nondetectable prior to treatment and in all vehicle-treated monkeys. Peak levels of l-CDB-4022 and its metabolite were observed at 4 h after dosing with steady-state levels apparent around d 4. Sperm concentration and total sperm per ejaculate were decreased to levels below 1x10(6) sperm/ml or sperm/ejaculate in l-CDB-4022-treated monkeys by d 17 and remained suppressed through wk 6. Sperm motility also declined to 0% for 6 wk. Testicular volume was reduced in l-CDB-4022-treated monkeys through d 21. The left testis and epididymis were removed from all monkeys on d 24. At this time, the most mature germ cells in the seminiferous tubules of testes from l-CDB-4022-treated monkeys were either spermatocytes or round spermatids. Immature germ cells, but not mature sperm, were found in the efferent ducts and collapsed epididymal lumen of l-CDB-4022-treated monkeys. A steady recovery in sperm motility, concentration, and total sperm per ejaculate was observed in l-CDB-4022-treated monkeys such that these parameters were not different from those of vehicle-treated monkeys by wk 16. Volume of the remaining testis increased in vehicle- and l-CDB-4022-treated monkeys after hemicastration; however, the increase in l-CDB-4022-treated monkeys was delayed compared with that observed in the vehicle-treated monkeys. The morphology of the remaining testis and epididymis, which were removed on wk 17, was normal. Serum inhibin B levels were increased in l-CDB-4022-treated monkeys during the dosing interval; thereafter serum inhibin B levels declined such that there was no difference between the groups by wk 3. l-CDB-4022 treatment did not affect circulating levels of testosterone, LH, FSH, or estradiol. In conclusion, these data indicate that in the cynomolgus monkey, a representative higher primate, l-CDB-4022 exerts a selective antispermatogenic action, which was reversible under the conditions of this study and thus has potential as a nonhormonal oral male contraceptive.

Portions of the reproductive tract of the male (Trachemys scripta) turtle were examined by immunocytochemistry for evidence of the capacity to produce and respond to estrogen hormones (via the expression of P450 aromatase and estrogen receptors). Aromatase was detected in both the Sertoli and Leydig cells of the testis and was expressed at different levels during the spermatogenic cycle, being highest in the quiescent testis and lowest during germ cell meiosis. ERα was found in the Leydig cells surrounding the seminiferous tubules as well as in the epithelial cells of the excurrent canals (rete testis, efferent ductule, and epididymis). ERβ immunoreactivity was found in both the spermatogonia and Sertoli cells in the testis, and in the epithelial cells of excurrent canals.

Vitamin D3 is a steroid hormone well known by its role in maintaining calcium homeostasis, however this hormone may also participate in other biological functions, including control of reproductive processes. The vitamin D3 action is mediated by the vitamin D3 receptor (VDR). VDR is widely distributed in the rodent reproductive tract, however the occurrence of VDR and the role of the vitamin D3 in the avian reproductive tract remain unknown. The aim of the present study was to investigate the expression and cellular distribution of VDR in the epididymal region of roosters. VDR expression was investigated by Western blotting analysis and the tissue distribution of the receptor was determined by immunohistochemistry. The Western blotting assay revealed a major VDR protein band of 61kDa in the epididymal region of rooster. Nuclear VDR expression was found in all segments of the epididymal region, namely rete testis, efferent ductules, connecting ducts and epididymal ducts. Nonciliated cells of the distal efferent ductules showed the highest levels of VDR expression, followed by the proximal efferent ductules and rete testis. The connecting and epididymal ducts showed less intense VDR immunostaining. The differential VDR expression in the epididymal region segments reveals that several extratesticular ducts may be target for vitamin D3 action and suggests that vitamin D3 may have a regional-specific function, such as calcium transport, that is modulated through VDR activity.

The E2F transcription factors are primarily implicated in the regulation of entry and exit from the cell cycle. However, in vivo studies have established additional roles for E2Fs during organ development and homeostasis. With the goal of addressing the intestinal requirements of E2f4 and E2f5, we crossed mice carrying Vil-cre, E2f4 conditional and E2f5 germline alleles. E2f4 deletion had no detectable effect on intestinal development. However, E2f4f/f;E2f5+/−;Vil-cre males, but not E2f4f/f;Vil-cre littermates, were unexpectedly sterile. This defect was not due to defective spermatogenesis. Instead, the seminiferous tubules and rete testes showed significant dilation, and spermatozoa accumulated aberrantly in the rete testis and efferent ducts. Our data show that these problems result from defective efferent ducts, a tissue whose primary function is to concentrate sperm through fluid absorption. First, Vil-cre expression, and consequent E2F4 loss, was specific to the efferent ducts and not other reproductive tract tissues. Second, the E2f4f/f;E2f5+/−;Vil-cre efferent ducts had completely lost multiciliated cells and greatly reduced levels of critical absorptive cell proteins: aquaporin1, a water channel protein, and clusterin, an endocytic marker. Collectively, the observed testis phenotypes suggest a fluid flux defect. Remarkably, we observed rete testis dilation prior to the normal time of seminiferous fluid production, arguing that the efferent duct defects promote excessive secretory activity within the reproductive tract. Finally, we also detect key aspects of these testis defects in E2f5−/− mice. Thus, we conclude that E2f4 and E2f5 display overlapping roles in controlling the normal development of the male reproductive system., National Institutes of Health (U.S.) (Grant NIH-P01 CA42063)

Estrogens play an important role in the male reproductive tract, and this is especially so for the efferent ductules, where alpha-estrogen receptors (ERalpha) have been localized. Mice deficient in ERalpha (alphaERKO mice) are infertile, and the effect appears to be due in part to retention of water at the level of the efferent ductules. In the present study, we examined the consequences of ERalpha deletion on the distribution of certain aquaporins (AQPs), water protein channels, in the efferent ductules and on sperm numbers and motility. In addition, the effects of feeding mice a regular lab chow diet, which contains phytoestrogens, known to affect male reproductive tract functions, and a casein diet, which lacks phytoestrogens, were also assessed. Light microscope immunolocalizations of AQP-1 and AQP-9 revealed dramatic reduction and patchier staining in alphaERKO mice with distal areas of the efferent ductules being more affected than proximal areas. No other changes in immunolocalizations were noted as a consequence of diet. Computer-assisted sperm analyses demonstrated a 62% reduction in cauda epididymal sperm/ml in alphaERKO mice fed lab chow, whereas 87% fewer sperm/ml were observed in alphaERKO mice fed casein, suggesting an enhanced role for sperm production and concentration in a diet containing phytoestrogens. All sperm motility parameters were altered to some degree in alphaERKO mice fed lab chow. Alterations in sperm motility parameters were also detected, but were less dramatic in alphaERKO mice fed casein. These data suggest that the decrease in AQP expression in the efferent ductules of alphaERKO mice contributes in part to water retention in this tissue, eventually leading to backflow of water into the testis, with subsequent decreases in sperm concentration and motility. The data also suggest that phytoestrogens, which are present in regular lab chow, can influence the male reproductive tract with and without the presence of ERalpha, promoting efferent ductule and epididymal functions when ERalpha is expressed, but inhibiting these same functions when ERalpha is missing. Taken together the data underscore the importance of estrogens and ERalpha in maintaining sperm maturation and preventing male infertility.

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.

Background information. Efferent ductules reabsorb more than 90% of the rete testis fluid, a process that involves ion transporters and AQP (aquaporin) water channels. Oestrogen has been shown to modulate the expression of the ion transporters involved in this activity, but reports of AQP regulation in the male tract have been confounding. To understand better the regulation of AQP1 and AQP9, we investigated their expression in rat efferent ductules and initial segment of the epididymis after treatment with the pure antioestrogen ICI 182,780 or bilateral efferent duct ligation, or castration, followed by hormone replacement. Results. Results show that AQP9 is modulated by oestrogen in the efferent ductule epithelium, but not in the initial segment of the epididymis. DHT (5α-dihydrotestosterone) also modulated AQP9 in efferent ductules. AQP9 was down-regulated by the antioestrogen in efferent ductules on day 45 post-treatment, which occurred before the non-ciliated cells had shown significant loss of microvilli. DHT, but not oestradiol, modulated AQP9 expression in the initial segment of the epididymis. In contrast, testosterone, DHT, oestrogen or the antioestrogen did not alter AQP1 staining, indicating constitutive expression of AQP1 in the efferent ductule epithelium. AQP1 expression was induced in peritubular cells of efferent ductules and in the initial segment of the epididymis after castration and long-term treatment with the antioestrogen. Although peritubular AQP1 staining in efferent ductules was partially reversed by the androgens, it was not reversed after any treatment in the initial segment of the epididymis. Conclusions. These results demonstrate that efferent ductules are unique in requiring both oestrogen and androgen to regulate an important mediator of fluid reabsorption, whereas the initial segment is dependent only on androgen stimulation.

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.

Epididymal stones have been reported in roosters in the USA and Japan. The cause of this dysfunction, which is associated with low fertility, is not known. The hypothesis of the present study is that a potential cause is the aggressive selection of birds over many centuries based upon female egg laying traits, without concern for potential effects on the male. If this hypothesis is correct, one potential consequence would be the presence of epididymal stones only in domesticated fowl and this observation would be worldwide in distribution. The present study investigated epididymal lithiasis in Brazilian crossbreed roosters and two other fowl strains, in addition to several domestic and wild bird species. The efferent ductules contained stones in 94.3% of the roosters, but stones were absent in all other domestic and wild birds. The stones were irregular in shape, size and colour and consisted mainly of calcium. In affected roosters, the efferent ductules showed epithelial cell vacuolization and sloughing and peritubular mononuclear cell infiltration, culminating with atrophy. Signs of epithelial re-canalization were seen in ductules occluded by abnormal content, such as stones. In the testis, decrease in mass, sloughing of epithelium, mononuclear cell infiltration and tubular atrophy occurred. No correlation was found between the occurrence of stones and a positive test for ELISA IBV (infectious bronchitis virus), or between the number of stones and calcium concentration in water and food, indicating that IBV infection and calcium in the diet were not related to stones formation. This study confirms and extends information about the epididymal lithiasis, which appears to be unique for roosters but to occur around the world. The severity of the lesion points to potentially severe economical impact in the poultry industry.

Male rats, 30 d old, were treated with the antiestrogen ICI 182,780 (3–150 d) to determine sequences of events leading to testicular atrophy and infertility. Plasma testosterone and LH concentrations were unchanged. ICI 182,780 induced dilation of efferent ductules as early as 3 d post treatment, and the dilation increased over time, resulting in an overall increase of 200% in tubule diameter. A gradual reduction in height of the ductule epithelium was observed; however, the microvilli height increased up to d 73 but subsequently decreased. A transient increase in lysosomes in nonciliated cells was seen from d 15 to d 100. Testicular weight increased by d 45 and seminiferous tubules were dilated by d 52. These effects on testes persisted until d 100, but on d 150 the weight decreased and severe atrophy was observed. These testicular effects were probably owing to accumulation of fluid following inhibition of reabsorption in the efferent ductules, similar to the ER-α knockout mouse. In agreement with this conclusion, there was a decrease in Na+-H+ exchanger-3 mRNA and protein, which is consistent with previous studies showing that ER is required for expression of Na+-H+ exchanger-3 and ultimately fluid reabsorption in the efferent ductules.

Seminiferous tubular atrophy may involve indirectly the disruption of estrogen receptor-α (ESR1) function in efferent ductules of the testis. ESR1 helps to maintain fluid resorption by the ductal epithelium and the inhibition or stimulation of this activity in rodent species will lead to fluid accumulation in the lumen. If not resolved, the abnormal buildup of fluid in the head of the epididymis and efferent ductules becomes a serious problem for the testis, as it leads to an increase in testis weight, tubular dilation and seminiferous epithelial degeneration, as well as testicular atrophy. The same sequence of pathogenesis occurs if the efferent ductule lumen becomes occluded. This review provides an introduction to the role of estrogen in the male reproductive tract but focuses on the various overlapping mechanisms that could induce efferent ductule dysfunction and fluid backpressure histopathology. Although efferent ductules are difficult to find, their inclusion in routine histological evaluations is recommended, as morphological images of these delicate tubules may be essential for understanding the mechanism of testicular injury, especially if dilations are observed in the rete testis and/or seminiferous tubules. Signature Lesion: The rete testis and efferent ductules can appear dilated, as if the lumens were greatly expanded with excess fluid or the accumulation of sperm. Because the efferent ductules resorb most of the fluid arriving from the rete testis lumen, one of two mechanisms is likely to be involved: a) reduced fluid uptake, which has been caused by the disruption in estrogen receptor signaling or associated pathways; or b) an increased rate of fluid resorption, which results in luminal occlusion. Both mechanisms can lead to a temporary increase in testicular weight, tubular dilation and atrophy of the seminiferous tubules.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The role of oestrogens in epididymal function is still unclear. Knockout of the oestrogen receptor ESR1 (Esr1-/-) or treatment with the anti-oestrogen Fulvestrant affect epididymal milieu and sperm motility. We investigated the effect of in vivo treatment of rats with Fulvestrant on: (i) expression of genes that may be important for the architecture and function of the epididymal epithelium: prominins 1 and 2, metalloproteinase 7, claudin 7, beta-catenin and cadherin 13, and (ii) levels of oestradiol and testosterone, and expression of oestrogen and androgen receptors, in the initial segment (IS), caput, corpus and cauda epididymis. Fulvestrant (i) reduced gene expression of prominin 1 (variant 1) in the caput, reduced prominin 1 protein content in the caput epididymis and in the efferent ductules, and increased the localization of prominin 1 in microvilli of the caput and corpus; (ii) reduced gene expression of prominin 2 in the corpus and cauda epididymis; (iii) increased the metalloproteinase 7 content in the apical region of principal cells from IS/caput; (iv) reduced in the corpus epididymis, but increased in the efferent ductules, the cadherin 13 mRNA level; (v) reduced testosterone but increased oestradiol levels in the corpus and cauda; (vi) increased the androgen receptor protein content in all regions of the epididymis, and the oestrogen receptor GPER in the corpus and cauda epididymis. in conclusion, treatment with Fulvestrant induced regional-specific changes in hormonal and steroid receptor content, and affected expression of proteins important for epithelial organization and absorption/secretion. the mechanisms of oestrogen action may differ among epididymal regions, which may contribute to determine region-specific sperm functions. Universidade Federal de São Paulo, Dept Pharmacol, Sect Expt Endocrinol, BR-04044020 São Paulo, Brazil Univ Illinois, Dept Comparat Biosci, Urbana, IL 61801 USA Univ Fed Minas Gerais, Dept Morphol, Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, Dept Pharmacol, Sect Expt Endocrinol, BR-04044020 São Paulo, Brazil FAPESP: 2008/56564-1 Web of Science

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.

Human sperm count studies, historic dietary iodination, and an animal model where neonatal goitrogen administration causes unprecedented testis enlargement, together suggest an hypothesis relevant to the postulated fall in human sperm counts. We present the hypothesis with a supporting study extending the model to include iodine deficiency. In a one-generation rat reproduction study, dams were fed an iodine sufficient (control, 200 ppb I) or deficient (low iodine diet [LID],20 ppb I) diet from prebreeding through weaning, when male offspring were divided into three groups: 1) controls from iodine sufficient dams, 2) neonatal LID (NLID) from the LID dams, fed control diet postweaning, and 3) chronic LID (CLID) from LID dams, fed a moderate LID (40 ppb I) postweaning. F1 males were euthanized on postnatal day (PND) 133+/-1. Each of the three diet groups comprised two subgroups in which testicular parameters were evaluated: 1) daily sperm production (DSP), sperm motility, morphology, and histopathology, and 2) Sertoli cell and round spermatid morphometry. In the first subgroup, NLID and CLID testes weights were 8.5% and 14.0% heavier than their unusually heavy controls (3.921 g; historical control mean approximately 3.5 g), with proportional DSP increases. Sperm motility, morphology, and testis histopathology were unaffected. In the morphometry subgroup, respective increases in NLID and CLID rats included testes weights (+28.6% and +20.3%), Sertoli cells (+24.8% and +23.9%), and round spermatids (+20.4% and +15.8%). The results indicate that neonatal iodine deficiency can significantly increase spermatogenic function in rats, and support our hypothesis concerning human sperm counts.

Regulation of the excurrent ducts of the testis is not well understood, particularly in avian species. To investigate the role of steroid hormones in the male reproductive tract, we developed a primary cell culture of epithelia isolated from rooster ductuli efferentes (efferent ductules). Efferent ductules of the avian testis comprise 77% of the epididymal region and form a mass of tubules containing a heavily folded epithelium enmeshed in connective tissue. The epididymal region was separated by microdissection and small epithelial plaques isolated by serial digestion with collagenase, elastase and repeated pipetting. Isolated cell plaques were cultured in a bicameral chamber on Millicell-CM inserts coated with two layers of basement membrane matrix, consisting primarily of laminin and Types I and IV collagen. Active ciliary beat was observed before plating and this activity was maintained for 14 days in culture. Cell plaques attached within 24 h and outgrowths formed a confluent monolayer by 5–6 days. The epithelial nature of cultured cells was demonstrated by immunocytochemical staining for cytokeratin. Light and electron microscopy confirmed that morphology and polarity of the original epithelial cells were maintained in culture. Cultured efferent ductal epithelium was cuboidal in shape and maintained many of the cytoplasmic organelles typical of these cells in vivo. The uptake of cationic ferritin indicated the endocytotic activity of these cultured cells was maintained. Estrogen receptor mRNA expression was maintained in cultured cells. These data demonstrate avian efferent ductal epithelium can be isolated and grown in defined culture medium for the purpose of determining the role of hormones and other factors in regulating the function of the epididymal region in the bird.

Molinate is a thiocarbamate herbicide widely used in rice culture. Studies conducted for regulatory purposes have indicated that molinate exposure causes male reproductive damage in rats. The present study describes the testicular lesion after administration of single doses of molinate. The hypothesis that a metabolite of molinate is responsible for testicular toxicity was also investigated. Testicular damage was evaluated histopathologically in Sprague-Dawley rats 48 h and 1, 2, and 3 weeks after administration of molinate (100-400 mg/kg i.p.). No testicular damage was seen at any time point at the 100 mg/kg dose level. Damage was first seen 1 week after 200 mg/kg and 48 h after 400 mg/kg. The lesion was characterized by Sertoli cell vacuolation, failed spermiation, and phagocytosis of spermatids particularly evident at Stages X and XI. With increasing time, damage progressed until disorganization of the seminiferous epithelium was extensive, multinucleated giant cells were numerous, and neither spermatozoa nor late step spermatids were present. At 3 weeks after administration of the two higher-dose levels, germ cells in the seminiferous tubules were almost completely absent. Administration of the sulfoxide metabolite of molinate (200 mg/kg i.p.) caused testicular damage similar in severity to that seen at the 400 mg/kg dose level for the parent compound, indicating that it was more potent as a testicular toxicant. In vitro metabolism studies using liver and testis microsomes found that the major metabolite in both preparations was molinate sulfoxide. Testis microsomes produced only slightly less sulfoxide when compared with liver microsomes. Molinate was also metabolized via ring hydroxylation to form small amounts of hydroxymolinate. The amount of hydroxymolinate was substantially less in testis microsomes. Overall, these data indicate that sulfoxidation of molinate plays a role in molinat-induced testicular toxicity. Moreover, molinate is metabolized readily by both liver and testis microsomal enzymes, suggesting that the molinate toxic metabolite could be formed in the testis in close proximity to its site of action.

The fungicide carbendazim, a male reproductive system toxicant, affects the adult testis, while prepubertal animals are assumed to be unsusceptible to the chemical. The present study was conducted to re-evaluate the susceptibility of rat prepubertal testis (25-30 days old) to carbendazim based on the incidence of spermatid abnormalities, including nuclear enlargement (megaspermatids) and binucleate round spermatids. In control prepubertal rats treated orally with corn oil vehicle alone, seminiferous tubules containing magno- and/or binucleate round spermatids were often observed in the groups at stages II-III, IV-V and VI-VII. At 3.0 days after treatment with carbendazim (100 mg/kg), seminiferous tubules containing spermatids with identical abnormalities were significantly increased, including groups at all stages. Significant increases were also observed at stages IV-V and VI-VII at day 4.5, and VI-VII at day 7.5. In contrast, the frequency of spermatids with these abnormalities was reduced in the groups at stages II-III and IV-V at day 7.5. These findings confirm that the prepubertal rat testis is susceptible to carbendazim during early spermiogenesis.

Background Benzimidazole fungicide, carbendazim, is known to adversely affect Sertoli cells by disrupting microtubules, which induces sloughing of elongate spermatids in a stage-specific manner. This study determines the direct effects on dividing germ cells and the subsequent effects on spermiogenesis. Methods Carbendazim was administered orally to male rats (100 mg/kg), and their testes were processed for histological evaluation at various post-treatment intervals up to day 20.0. Results The sloughing of elongate spermatids was observed as reported previously. In addition to this Sertoli cell lesion, necrosis of dividing spermatocytes in stage XIV was observed at 8 hours post-treatment. At day 1.5, empty spaces of missing step 1 spermatids were seen in stage I. At days 4.5 and 7.5, normal round spermatids were missing, but large round spermatids (megaspermatids) and binucleate spermatids were common. The megaspermatid nucleus was approximately 33% larger in diameter than normal round spermatids. At day l0.5, megasteps 10–12 spermatids, binucleate spermatids, and three to four different steps of spermatids coexisting in the same tubule section were present in stages X–XII. In addition, abnormally shaped elongating spermatids were observed having distorted heads and nuclear invagination containing microtubules. At day 20.0, empty spaces of missing diplotene spermatocytes were seen in stage XIII. Conclusions The present observations show that carbendazim has rapid direct effects on meiotic spermatocytes and latent effects on spermatids, leading to morphological abnormalities and failure of spermiogenesis. These effects are found independent of occlusions in the efferent ductules. Anat. Rec. 247:379–387, 1997. © 1997 Wiley-Liss, Inc.

Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used phthalate, and it is an endocrine-disrupting chemical. This study tested a hypothesis that prenatal exposure to DEHP lays the foundation for premature gonadal dysfunction and subsequent reproductive senescence in male mice. Pregnant female CD-1 mice were orally dosed with vehicle control (tocopherol-stripped corn oil) or with 20 μg/kg/day, 200 μg/kg/day, 500 mg/kg/day, or 750 mg/kg/day of DEHP from gestational day 11 to birth. Overall, the prenatal DEHP exposure did not cause any overt physical health problems in male offspring, as no significant differences in their body nor gonadal weight were seen up to the age of 23 months. However, an age- and dose-dependent gonadal dysfunction was observed. As early as 7 months of age, the 750 mg/kg/day group of mice exhibited significantly reduced fertility. At 19 months of age, 86% of the 750 mg/kg/day mice became infertile, whereas only 25% of the control mice were infertile. At this age, all of the DEHP-exposed mice had lower serum testosterone levels, higher serum estradiol levels, and higher LH levels compared with control mice. Histological evaluations showed that mice prenatally exposed to DEHP displayed a wide array of gonadal and epididymal abnormalities such as increased germ cell apoptosis, degenerative seminiferous tubules, oligozoospermia, asthenozoospermia, and teratozoospermia in comparison to age-matching control mice. In summary, this study shows that prenatal exposure to DEHP induces premature reproductive senescence in male mice.

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.

This review article discusses a novel nontraditional site of estrogen synthesis and the potential targets of estrogen action within the male reproductive system. Our laboratories have recently demonstrated that developing spermatids in several species contain aromatase, the cytochrome P450 enzyme responsible for converting androgens into estrogens. The enzyme was localized by immunocytochemistry and the protein's presence was confirmed by Western blot analysis. Northern blot analysis and in situ hybridization were used to corroborate the presence of mRNA for aromatase. It appears that the aromatase message precedes the synthesis of the protein, and the protein remains in the spermatids several days after the message disappears. The enzyme is located along the tail of newly released sperm and is active in the epididymal sperm as well as in the developing germ cells of the testis. This unique discovery is the basis for our overall hypothesis that estrogen, synthesized by sperm, plays a role in the regulation of epididymal function proportional to the number of sperm being transported. The presence of an estrogen source within the ductal lumen is of special importance to the study of epididymal function because the regulatory mechanisms in this region remain unclear, particularly for the efferent ductules and initial segment regions, although estrogen receptors have been identified in the ductal epithelium. An understanding of the role that estrogen plays in the function of the epididymis may provide benefits in several areas including the treatment of abnormalities in epididymal function, the potential development of a male contraceptive, and insight into the causes of adult epididymal lesions induced by neonatal exposure to estrogenic compounds such as diethylstilbestrol.

The effects of exposure in utero to 1 MHz, continuous-wave ultrasound on adult growth and testicular development in the mouse was investigated. The spatial peak temporal average intensity ( I spta ) employed ranged from 1 to 10 W/cm 2 , with exposure durations ( t ) of 200 s to 20 s. Exposures were made on days 9, 12 or 15 of gestation. Results showed an increase in postpartum deaths, an increase in the number of stillbirths, and a decrease in litter size when I 2 t ≥ 1125 W 2 s/cm 4 , such that there was significant loss of pups. Birthweights of pups from nearly all dosage groups was significantly lower than that of the sham or cage control groups. Results also showed that males exposed to ultrasound in utero had decreased testis size and decreased daily sperm production ranging from 9% to 30%. This study showed that ultrasound exposure in utero is capable of disrupting fetal development and having potential subsequent effects on fertility in the adult male.

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.

Estrogens play key roles in the development and maintenance of male reproductive function and fertility. in this review, we briefly describe the localization and function of estrogen receptors ESR1 and ESR2 (also known as ER alpha and ER beta, respectively) and the expression of G protein-coupled estrogen receptor-1 (GPER, formerly known as GPR30) in efferent ductules and epididymis. the efferent ductules present the highest levels of ESR1 and ESR2 in the male reproductive system, and represent a major target of estrogen action. in efferent ductules, ESR1 has a crucial role in the regulation of fluid reabsorption, and in the epididymis the receptor helps to maintain fluid osmolality and pH. ESR1 expression in the epididymal epithelium shows considerable variation among species, but differences in laboratory techniques may also contribute to this variation. Here we report that Esr1 mRNA and protein are higher in corpus than in other regions of the rat epididymis. the mRNA level for Gper was also higher in corpus. Although ESR1 is expressed constitutively in efferent ductules and down-regulated by estrogen, in the epididymis, both testosterone (T) and estradiol (E2) may regulate its expression. T and E2 are, respectively, higher and lower in the corpus than in the initial segment/caput and cauda regions. It is important to determine the expression of GPER, ESR1, androgen receptor, and their respective cofactors in specific cell types of this tissue, as well as the intracellular signaling pathways involved in efferent ductules and epididymis. These studies will help to explain the consequences of exposures to environmental endocrine disruptors and provide potential targets for the development of a male contraceptive. Univ Illinois, Dept Comparat Biosci, Urbana, IL 61802 USA Universidade Federal de São Paulo, Sect Expt Endocrinol, Dept Pharmacol, Escola Paulista Med, São Paulo, Brazil Univ Fed Minas Gerais, Dept Morphol, Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, Sect Expt Endocrinol, Dept Pharmacol, Escola Paulista Med, São Paulo, Brazil Web of Science

Epididymal lithiasis is a reproductive dysfunction of roosters that is associated with loss of fertility and is characterized by the formation of calcium stones in the lumen of the efferent ductules of the epididymal region. The efferent ductules of birds are responsible for the reabsorption of the fluid coming from the testis as well as luminal calcium. It has been hypothesized that the epididymal stone formation may be related to the impairment of local fluid or calcium homeostasis, which depends on hormones such as estradiol (E2). Therefore, this study aimed to investigate possible alterations in the expression of ERα (ESR1) and ERβ (ESR2) in the epididymal region of roosters affected by epididymal lithiasis. The study was performed by immunohistochemistry and western blotting assays. In addition, the concentrations of E2, vitamin D3, and testosterone, which are also key hormones in maintenance of calcium homeostasis, were determined in the plasma and epididymal region, by ELISA. It was observed that ESR2 expression is increased in all segments of the epididymal region of affected roosters, whereas ESR1 levels are not altered. Moreover, the hormone concentration profiles were changed, as in the epididymal region of roosters with lithiasis the E2levels were increased and vitamin D3 as well as testosterone concentrations were significantly decreased. These results suggest that a hormonal imbalance may be involved with the origin and progression of the epididymal lithiasis, possibly by affecting the local fluid or calcium homeostasis.

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.

Occlusions of the rat efferent ductules were induced by an oral gavage of carbendazim and the capacity for regeneration or recanali- zation of the ductules was histologically evaluated between 2 and 70 days post-treatment. At 2 days post-treatment, the efferent ductules were oc- cluded by sloughed materials and showed various degrees of inflammation. Severely damaged epithelium showed few regenerative features at later intervals. On the other hand, epithelia with medium inflammation often exhibited irregular epithelial growth along the luminal contents or the for- mation of multiple abnormal ductules. These abnormal ductules were formed by migrated and original epithelia at the periphery of the occluded lumen at 16 days post-treatment, indicating attempted recanalization. At later time periods, 32 and 70 days post-treatment, the occluded original lumen was filled in by fibrotic connective tissue and surrounded by a series of abnormal ductules. These abnormal ductules were characterized by cuboidal epithelia, a small luminal diameter, fewer cilitated cells than nor- mal, less developed organelles in the epithelial cells, and basal laminae of irregular thickness. However, there was no evidence that occluded ductules formed patent re-connections via abnormal ductules. The results suggest that occluded efferent ductules have the ability to initiate epithelial regrowth and to form new ductules, but the newly formed ductules are abnormal and are not adequate to recover from azoospermia at least at 70 days post-treatment. Key words: Abnormal epithelial growth, Efferent ductules, Microcanals

Estrogen receptor-alpha (ESR1) is highly expressed in the efferent ductules of all species studied as well as in the epididymal epithelium in mice and other select species. Male mice lacking ESR1 (Esr1KO) are infertile, but transplantation studies demonstrated that Esr1KO germ cells are capable of fertilization when placed in a wild-type reproductive tract. These results suggest that extratesticular regions, such as the efferent ductules and epididymis, are the major source of pathological changes in Esr1KO males. Previous studies have shown alterations in ion and fluid transporters in the efferent duct and epididymal epithelia of Esr1KO males, leading to misregulation of luminal fluid pH. To determine the effect of an altered epididymal milieu on Esr1KO sperm, we assayed sperm morphology in the different regions of the epididymis. Sperm recovered from the epididymis exhibited abnormal flagellar coiling and increased incidence of spontaneous acrosome reactions, both of which are consistent with exposure to abnormal epididymal fluid. Analysis of the epididymal fluid revealed that the osmolality of the Esr1KO fluid was reduced relative to wild type, consistent with prior reports of inappropriate fluid absorption from the efferent ductules. This, along with the finding that morphological defects increased with transit through the epididymal duct, suggests that the anomalies in sperm are a consequence of the abnormal luminal environment. Consistent with this, incubating Esr1KO sperm in a more wild-type-like osmotic environment significantly rescued the abnormal flagellar coiling. This work demonstrates that Esr1KO mice exhibit an abnormal fluid environment in the lumen of the efferent ducts and epididymis, precluding normal sperm maturation and instead resulting in progressive deterioration of sperm that contributes to infertility.

International audience; The role of oestrogens in male reproductive tract physiology has for a long time been a subject of debate. The testis produces significant amounts of oestrogenic hormones, via aromatase, and oestrogen receptors (ERs)alpha (ESR1) and ER beta (ESR2) are selectively expressed in cells of the testis as well as the epididymal epithelium, depending upon species. This review summarizes the current knowledge concerning the presence and activity of aromatase and ERs in testis and sperm and the potential roles that oestrogens may have in mammalian spermatogenesis. Data show that physiology of the male gonad is in part under the control of a balance of androgens and oestrogens, with aromatase serving as a modulator.

Two flocks of yearling Large White turkeys representing high (61 toms; 123 hens) and low (63 toms; 111 hens) hatchability lines were pedigree mated: 1 tom/1-3 hens. In the fifth month of a six month breeding season, infertility (of unknown origin) and poor hatchability were experienced in both flocks. At this time, semen was collected once from the males, and seminal plasma protein concentrations were measured and correlated with fertility and hatchability for a 20 week breeding period. Males with high seminal plasma protein (equal to or greater than 6 g/100mL) were afflicted severely with yellow semen syndrome (YSS). Hens with which these males were mated produced eggs with significantly reduced fertility and hatchability. When the protein concentration was less that 6 g/100mL, no significant relationship between seminal plasma protein concentration and fertility or hatchability of total or fertile eggs occurred. These responses held true for both the high and low hatchability lines. We conclude that a single determination of seminal plasma protein concentration can be used to detect breeder males of poor reproductive potential.

The ductuli efferentes of Sprague-Dawley rats were studied by microdissection and microscopic evaluation to document the presence of blind-ending tubules (ductuli aberrantes) and to describe morphological and ultrastructural differences between normally open ductules and blind-ending tubules. The branching patterns of the ductuli efferentes varied considerably between animals. A majority of the animals studied had either six or seven ductuli connected to the rete testis, with some animals having as few as four or as many as eight. Pairs of ductules began merging in the conus vasculosa, ultimately forming a single terminal duct within the capsule of the initial segment epididymidis. In a majority of animals, the junctions were unequally matched and located at various positions within the conus. Blind-ending tubules, found in 60% of the animals, were surrounded by thick connective tissue, and had a smaller diameter (78.7 +/- 1.4 microns) than normal ductules in the conus vasculosa (119.5 +/- 2.1 microns) or the terminus (102.2 +/- 1.5 microns). The lumina of blind-ending tubules were contracted and did not contain sperm. Nonciliated cells in the epithelium of blind-ending tubules contained fewer PAS-positive granules and electron-dense bodies (lysosomes) than nonciliated cells in normal ductules. Consideration of these characteristics will prevent blind-ending tubules from being mistaken for pathological changes in efferent ductules.