Sertoli Cell Nuclei Research Articles

Zearalenone impairs the male reproductive system functions via inducing structural and functional alterations of sertoli cells.

The aim of this study was to investigate the effects of ZEA on the cytoskeletal structure, and factors specifically expressed by Sertoli cells. Primary Sertoli cells from rats aged 18-21 days were exposed to increasing ZEA concentrations (0, 5, 10, 20 μg mL(-1)) for 24 h. The results of immunofluorescence showed disruption of α-tubulin filaments and F-actin bundles, and damage to the nucleus of Sertoli cells on exposure to ZEA. In the control group, the protein level expression of androgen-binding protein (ABP), transferrin, vimentin, N-cadherin, and follicle-stimulating hormone receptor (FSHR) were decreased significantly (p<0.05, p<0.01). The mRNA levels of ABP, transferrin, vimentin, N-cadherin, and FSHR varied significantly in the experimental group (p<0.05). The results of enzyme-linked immunosorbent assay indicated a significant decrease in the levels of inhibin-β and transferrin in the cultural supernatants (p<0.05). Additionally, the ultrastructural analysis indicated the absence of mitochondria and Golgi apparatus, and presence of vacuoles in the cytoplasm. These findings showed that ZEA treatment can damage the cytoskeletal structure and affect specific secretory functions of Sertoli cells, which may be an underlying cause of ZEA-induced reproductive toxicity.

The effects of long-term leptin administration on morphometrical changes of mice testicular tissue.

Leptin is a novel and interesting hormone for anyone trying to lose weight, but its effects on male gonad structure in longitudinal study is unknown. The present study was designed to explore morphometrical changes of mouse testicular tissue after long-term administration of leptin. Thirty healthy mature male mice were randomly assigned to either control (n=15) or treatment (n=15) groups. Leptin was intraperitoneally injected to the treatment group (0.1 µg/100 µl of physiological saline) once a day for 30 consecutive days, and control animals received normal saline with the same volume and route. Five mice from each experimental group were sacrificed at 15, 30 and 60 days after the beginning of treatments. Left testes were removed, weighted and then fixed in 10% buffered formalin, and stained with hematoxylin and eosine for morphometrical assays. Except for sertoli cell nucleus diameter, which was affected from 30(th) day, evaluation of other morphometrical parameters such as Johnsen's score, meiotic index, spermatogenesis, epithelial height, seminiferous tubules diameter and spermatogonial nucleus diameter revealed significant decrease from 15(th) day after leptin administration compare to those of the control group (P<0.05). Thus, meiotic index and spermatogonial cell nucleus diameter were two parameters that were further disturbed on 30(th) day compare to the day 15 (3.09±0.03 vs. 3.23±0.03, P=0.006 and 5.50±0.09 vs. 6.08±0.14, P=0.007, respectively). Our results showed that long-term administration of leptin could disturb testicular tissue structure and delay spermatogenesis process.

Expression of CCM2 and CCM3 during mouse gonadogenesis.

Three cerebral cavernous malformation (CCM) proteins, CCM1, CCM2, and CCM3, regulate cell-cell adhesion, cell shape and polarity, and most likely cell adhesion to extracellular matrix. Recently, CCM2 and CCM3 are known to be expressed in control and varicocele-induced rat testes, but little is known about these proteins during gonadogenesis. This led us to study the CCM proteins during the mouse gonadogenesis. Neonatal (PND 0), postnatal, and adult mice testes and ovaries were obtained from mice. CCM2 and CCM3 expression were analyzed during mouse testicular and ovarian development by immunohistochemistry and quantitative real-time PCR. The results showed that in both sexes, Ccm2 and Ccm3 mRNA and protein were first detectable after gonadogenesis when the gonads were well differentiated and remained present until the adult stage. In the testis, CCM2 and CCM3 expression were restricted to the nuclei of Sertoli cells, suggesting a conserved role in testicular differentiation. In the ovary, the CCM2 and CCM3 proteins were localized in the cytoplasm of oocytes, suggesting an unexpected role during oogenesis. Quantitative real-time PCR (qRT-PCR) results showed that expression of Ccm2 and Ccm3 genes could play a role in the regulation of mouse gonadogenesis translational activation upon testicular and ovarian development. The localization of CCM2 and CCM3 proteins show their different functions for CCM2 and CCM3 which may have important roles in testicular and ovarian differentiation. In conclusion, CCM2 and CCM3 may be involved in establishing the differential expression pattern in developing mouse testis and ovary.

Androgen receptor binding to an androgen-responsive element in the promoter of the Srsf4 gene inhibits its expression in mouse Sertoli cells.

The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y)) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells.

Musashi-1 maintains blood-testis barrier structure during spermatogenesis and regulates stress granule formation upon heat stress.

In mouse testes, Musashi-1 (Msi-1) was predominantly expressed in the cytoplasm and nuclei of Sertoli cells. Here we demonstrate that knockdown of Msi-1 in Sertoli cells altered the levels and distribution of blood-testis barrier (BTB)-associated proteins. Moreover, Msi-1 knockdown in vivo disrupted BTB functional structure and spermatogenesis. In addition, we report a novel role of Msi-1 in regulating Sertoli cells survival following heat-induced injury. Endogenous Msi-1 protein in heat-treated Sertoli cells was recruited to stress granules. The formation of stress granules was considerably disrupted, and apoptosis was significantly up-regulated in Msi-1-knockdown Sertoli cells after heat treatment. p-ERK1/2 acted downstream of stress granule formation, and inhibition of p-ERK1/2 signaling triggered Sertoli cell apoptosis upon heat stress. In conclusion, we demonstrate that Msi-1 is critical for constructing a functional BTB structure and maintaining spermatogenesis. We also note a role for Msi-1 in regulating Sertoli cell fate following heat-induced injury, likely through the induction of stress granule formation and subsequent activation of p-ERK1/2 signaling.

Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice.

Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.

The immunoexpression of androgen receptor, estrogen receptors alpha and beta, vanilloid type 1 receptor and cytochrome p450 aromatase in rats testis chronically treated with letrozole, an aromatase inhibitor.

The function of testis is under hormonal control and any disturbance of hormonal homeostasis can lead to morphological and physiological changes. Therefore the aim of the study was to investigate the expression of androgen and estrogen receptors (AR, ERs), vanilloid receptor (TRPV1), cytochrome P450 aromatase (P450arom), as well as apoptosis of cells in testis of adult rats chronically treated with letrozole (LT), a non-steroidal aromatase inhibitor, for 6 months. The testicular tissues were fixed in Bouin's fixative and embedded in paraffin. Immunohistochemistry with monoclonal antibodies (abs) against AR, ERa, P450arom, and polyclonalabs against ERβ, TRPV1, caspase-3 was applied. Long-lasting estradiol deficiency, as an effect of LT treatment, produced changes in the morphology of testis and altered the expression of the studied receptors in cells of the seminiferous tubules and rate of cell apoptosis. The immunostaining for AR was found in the nuclei of Sertoli cells and the cytoplasm of spermatogonia and spermatocytes in III-IV stages of the seminiferous epithelium cycle. The intensity of staining for P450arom was lower in the testis of LT-treated rats as compared to control animals. The immunofluorescence of ERα and ERβ was observed exclusively in the nuclei of Leydig cells of LT-treated rats. There were no changes in localization of TRPV1, however, the intensity of reaction was stronger in germ cells of the seminiferous epithelium after LT treatment. The apoptosis in both groups of animals was observed within the population of spermatocytes and spermatids in II and III stages of the seminiferous epithelium cycle. In testis of LT-treated rats the immunoexpression of caspase-3 was additionally found in the germ cells in I and IV stages, and Sertoli, myoid and Leydig cells. In conclusion, our results underline the important role of letrozole treatment in the proper function of male reproductive system, and additionally demonstrate that hormonal imbalance can produce the morphological abnormalities in testis.

Characterization of testicular morphology and spermatogenesis in the nurse sharks Ginglymostoma cirratum (Bonnaterre, 1788)

This study reports a novel form of testicular development in nurse sharks (Ginglymostoma cirratum) and provides the first histological description of spermatogenesis in this species. Radially structured testes developed from the caudal to the cranial regions, a pattern that appears to be unique among shark species. Testes from immature males had spermatogonia and Sertoli cells, while in testes of developing and capable to reproduce males, the nuclei of Sertoli cells were evident in basal positions of spermatocyst. The spermatogonia divide to form primary spermatocytes, secondary spermatocytes divide to produce spermatids, and spermatids then move to the periphery of the spermatocyst. Subsequently, the cysts with spermatids were observed at different stages of maturation, and spermatids became spermatozoa. In the final stage of spermatogenesis, spermatozoa had their heads directed towards the basal membrane and flagella directed towards the lumen. Examinations of testes development in a broad range of shark species are now required to determine whether this novel form of testes development observed in nurse sharks is shared by other elasmobranch species.

Actin dynamics regulate subcellular localization of the F-actin-binding protein PALLD in mouse Sertoli cells

Sertoli cells undergo terminal differentiation at puberty to support all phases of germ cell development, which occurs in the mouse beginning in the second week of life. By ∼18 days postpartum (dpp), nearly all Sertoli cells have ceased proliferation. This terminal differentiation is accompanied by the development of unique and regionally concentrated filamentous actin (F-actin) structures at the basal and apical aspects of the seminiferous epithelium, and this reorganization is likely to involve the action of actin-binding proteins. Palladin (PALLD) is a widely expressed F-actin-binding and bundling protein recently shown to regulate these structures, yet it is predominantly nuclear in Sertoli cells at puberty. We found that PALLD localized within nuclei of primary Sertoli cells grown in serum-free media but relocalized to the cytoplasm upon serum stimulation. We utilized this system with in vivo relevance to Sertoli cell development to investigate mechanisms regulating nuclear localization of this F-actin-binding protein. Our results indicate that PALLD can be shuttled from the nucleus to the cytoplasm, and that this relocalization occurred following depolymerization of the F-actin cytoskeleton in response to cAMP signaling. Nuclear localization was reduced in Hpg-mutant testes, suggesting the involvement of gonadotropin signaling. We found that PALLD nuclear localization was unaffected in testis tissues from LH receptor and androgen receptor-mutant mice. However, PALLD nuclear localization was reduced in the testes of FSH receptor-mutant mice, suggesting that FSH signaling during Sertoli cell maturation regulates this subcellular localization.

Expression of testicular androgen receptor in non-obstructive azoospermia and its change after hormonal therapy.

Several trials aimed at improving the sperm retrieval from men with non-obstructive azoospermia (NOA) by optimizing intratesticular testosterone (ITT) have reported partial responses, however, an appropriate level of ITT has not been identified. In this study, we examined the expression of the testicular androgen receptor (AR) in NOA and investigated its correlation with clinical and pathological parameters. Expression of the testicular AR was investigated in 52 men with NOA and 22 men with obstructive azoospermia (OA). Twenty-two patients for whom sperm retrieval failed during microdissection testicular sperm extraction (micro-TESE) were enrolled in hormonal therapy using hCG with or without recombinant human follicle stimulating hormone (rhFSH) prior to a second micro-TESE. Sertoli cells were identified by vimentin immunostaining, and positivity in Sertoli cells was used as the AR index. AR immunostaining was robust in the nuclei of Sertoli cells [Sertoli cell androgen receptor (SCAR)] in both OA and NOA. The mean AR index in NOA was significantly higher than that in OA (p < 0.05). In NOA patients, there was no correlation between the AR index and the clinical parameters, whereas the AR index of early maturation arrest (MA) was significantly lower than that of Sertoli cell only, late MA and hypospermatogenesis (p < 0.05). A significant increase in the AR index after salvage hormonal therapy was shown, particularly when using rhFSH. The AR index in patients from whom spermatozoa could be retrieved at the second micro-TESE increased significantly after hormonal therapy. In human testes, the expression of AR is dominant in Sertoli cells, and the expression of SCAR is upregulated by FSH. Germ cell maturation, especially during spermatogonia to spermatocyte stage, has been shown to be SCAR-dependent. Taken together, the results indicate that SCAR elevation is closely associated with sperm retrieval after hormonal therapy and that FSH-based hormonal therapy is potentially effective in NOA men with MA.

Ultrastructure of spermatogenesis, spermatozoon and processes of testicular regression and recrudescence in Eptesicus furinalis (Chiroptera: Vespertilionidae)

Studies have shown that the annual reproductive cycle of Eptesicus furinalis includes at least one period of total testicular regression. Thus, this study aimed to evaluate their reproductive cycle ultrastructurally. The annual reproductive cycle was divided into four periods: active, regressing, regressed and recrudescence. The active period was similar to that of other bats, including the completion of spermatogenesis with three main types of spermatogonia (Ad, Ap and B) and 12 steps in the process of spermatid differentiation. However, its spermatozoa differed in that outer dense fibers 1, 5, 6 and 9 are larger than the others and due to the presence of what is likely a probably genera-specific bulging in the anterior portion. In the regressing period, Sertoli cell nuclei migrate to the basal compartment with the nuclei close to the basal lamina. The basal compartment had a more compact appearance than the adluminal compartment, with relaxed cellular connections. In the regressed period, spermatogenesis ceased; the seminiferous epithelium was composed only of Sertoli cells and three types of spermatogonia: types Ad, 1 and 2. In the recrudescence period, spermatogenesis restarted, with the process of reactivation divided into three phases: early, medial and late recrudescence. In conclusion, our study described the process of spermatogenesis and the ultrastructure of the spermatozoa and confirmed the presence of a process of total testicular regression in the annual cycle of E. furinalis. We characterize distinct morphologic variations in the ultrastructure of the testicular cells during the four different periods of the annual reproductive cycle.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas.

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season.

Differential expression of aromatase, estrogen receptor alpha and 17β-HSD associated with the processes of total testicular regression and recrudescence in the bat Myotis nigricans (Chiroptera: Vespertilionidae)

Despite the worldwide distribution and many unique reproductive adaptations that bats present, many aspects of their reproductive hormonal regulation have not been adequately studied, especially in species that presented patterns of total testicular regression. Thus, this study aimed to evaluate the testicular expression of 17β-HSD type 1, aromatase and ERα in the bat Myotis nigricans, during the four periods of its reproductive cycle. Immunoreactivity for ERα was detected only in the cytoplasm of elongated spermatids and in the nuclei of spermatogonia and Sertoli cells. Expression of aromatase was observed in round and elongated spermatids and in Sertoli and Leydig cells. Immunoreactivity for 17β-HSD was restricted to the cytoplasm of Leydig cells. The three expression patterns varied significantly during the four periods of the reproductive cycle. Expression of ERα and aromatase in spermatids was continuous, while expression of ERα in spermatogonia occurred only in initial types (Ap). Expression of ERα and aromatase in Sertoli cells varied, with expression only in periods of spermatogenetic activities; and the same variation was observed for the expression of aromatase and 17β-HSD in Leydig cells. We, therefore, propose that the processes of total testicular regression and posterior recrudescence suffered by M. nigricans from September to January in the northwest of the São Paulo State of Brazil, are directly regulated by testosterone and estrogen. This occurs via the production of testosterone by 17β-HSD, its conversion into estrogen by aromatase, and activation/deactivation of Sertoli cells’ AR and spermatogonia’s ERα.

The Death of Sertoli Cells and the Capacity to Phagocytize Elongated Spermatids During Testicular Regression due to Short Photoperiod in Syrian Hamster (Mesocricetus auratus)1

In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during this process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells, and whether, during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. To this end, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of several morphological techniques using conventional light microscopy (hematoxylin and eosin [H&E], semi-thin section vimentin, immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy, and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells.

Molecular Reproduction &amp; Development Volume 80, Issue 5, May 2013 cover image

The actin cytoskeleton is an integral part of specialized structures in Sertoli cells of the postpubertal mammalian testis, contributing to the blood testis barrier and the apical ectoplasmic specialization. In a study by Niedenberger et al. (this issue), a dominant regulator of the actin cytoskeleton, Palladin, unexpectedly moves into Sertoli cell nuclei as these cytoskeletal structures form at puberty. This image is a cross‐section of an adult stage‐VIII tubule stained for Palladin (brown) and counterstained with hematoxylin (blue).

Nuclear localization of the actin regulatory protein palladin in sertoli cells

In the testis, F-actin structures are involved in spermatid nuclear remodeling and cytoplasm reduction, maintenance of the blood-testis barrier, support of the spermatogonial stem cell niche, and release of spermatids into the tubular lumen. To gain a better understanding of actin regulation in Sertoli-germ cell interactions, we investigated the expression of the Palladin (Palld) gene, which encodes a widely expressed phosphoprotein that localizes to actin-rich cytoplasmic structures, including focal adhesions, cell-cell junctions, podosomes, and stress fibers, and serves as a molecular scaffold to bundle actin fibers. In germ cells, PALLD was concentrated along the tubulin- and F-actin-containing cytoplasmic manchette that forms adjacent to the elongating spermatid nucleus during spermiogenesis. To our surprise, PALLD relocated from the cytoplasm to the nucleus of Sertoli cells in the juvenile testis, coincident with the onset of puberty, and this localization was maintained in the adult. We provide evidence that the 140 kDa isoform of PALLD predominates in Sertoli cells, and that it is apparently cleaved, with the C-terminus localizing to the nucleus while the N-terminus remains cytoplasmic. We investigated the nuclear localization of the C-terminus of PALLD and found that it is regulated by a putative nuclear export signal. These results provide the foundation for future work employing Sertoli cell- and spermatid-specific Palld-knockout mice to study diverse roles of PALLD as both a nuclear-actin regulatory protein and as a potential regulator of manchette formation during spermatogenesis.

Chicken hemogen homolog is involved in the chicken-specific sex-determining mechanism

Using a comprehensive transcriptome analysis, a Z chromosome-linked chicken homolog of hemogen (cHEMGN) was identified and shown to be specifically involved in testis differentiation in early chicken embryos. Hemogen [Hemgn in mice, EDAG (erythroid differentiation-associated gene protein) in humans] was recently characterized as a hematopoietic tissue-specific gene encoding a transcription factor that regulates the proliferation and differentiation of hematopoietic cells in mammals. In chicken, cHEMGN was expressed not only in hematopoietic tissues but also in the early embryonic gonad of male chickens. The male-specific expression was identified in the nucleus of (pre)Sertoli cells after the sex determination period and before the expression of SOX9 (SRY-box 9). The expression of cHEMGN was induced in ZW embryonic gonads that were masculinized by aromatase inhibitor treatment. ZW embryos overexpressing cHEMGN, generated by infection with retrovirus carrying cHEMGN, showed masculinized gonads. These findings suggest that cHEMGN is a transcription factor specifically involved in chicken sex determination.

The seminiferous epithelial cycle and microanatomy of the koala (Phascolarctos cinereus) and southern hairy‐nosed wombat (Lasiorhinus latifrons) testis

The koala (Phascolarctos cinereus) and southern hairy-nosed wombat (Lasiorhinus latifrons) are iconic Australian fauna that share a close phylogenetic relationship but there are currently no comparative studies of the seminiferous epithelial cell or testicular microanatomy of either species. Koala and wombat spermatozoa are unusual for marsupials as they possess a curved stream-lined head and lateral neck insertion that superficially is similar to murid spermatozoa; the koala also contains Sertoli cells with crystalloid inclusions that closely resemble the Charcot-Bottcher crystalloids described in human Sertoli cells. Eighteen sexually mature koalas and four sexually mature southern hairy-nosed (SHN) wombats were examined to establish base-line data on quantitative testicular histology. Dynamics of the seminiferous epithelial cycle in the both species consisted of eight stages of cellular association similar to that described in other marsupials. Both species possessed a high proportion of the pre-meiotic (stages VIII, I - III; koala - 62.2±1.7% and SHN wombat - 66.6±2.4%) when compared with post-meiotic stages of the seminiferous cycle. The mean diameters of the seminiferous tubules found in the koalas and the SHN wombats were 227.8±6.1 and 243.5±3.9μm, respectively. There were differences in testicular histology between the species including the koala possessing (i) a greater proportion of Leydig cells, (ii) larger Sertoli cell nuclei, (iii) crystalloids in the Sertoli cell cytoplasm, (iv) a distinctive acrosomal granule during spermiogenesis and (v) a highly eosinophilic acrosome. An understanding of the seminiferous epithelial cycle and microanatomy of testis is fundamental for documenting normal spermatogenesis and testicular architecture; recent evidence of orchitis and epididymitis associated with natural chlamydial infection in the koala suggest that this species might be useful as an experimental model for understanding Chlamydia induced testicular pathology in humans. Comparative spermatogenic data of closely related species can also potentially reflect evolutionary divergence and differences in reproductive strategies.

Feline Gonads Exhibit Tissue Specific Alternative Splicing of Oestrogen Receptor Alpha (ESR1)

Male felids frequently show teratospermia. At least in the domestic cat model, teratospermia is accompanied by impaired regulation of testicular apoptosis. We hypothesize that this phenomenon is caused by dysregulations in oestrogen signalling pathways. Both classical oestrogen receptors (ESR1 and 2) are expressed in species and/or tissue-specific manners and display different variants, inter alia, caused by alternative splicing. In vitro studies showed that exon deleted transcripts are translated into proteins and that some of the variants modify the effects of the full-length ERs. It has been proposed that some of the functional and morphological dysregulations, for example, during spermatogenesis, could directly derive from this phenomenon. In the present basic study, we investigated the expression pattern of ESR1 splicing variants in the gonads of domestic cats. Testicular, epididymal as well as ovarian tissue samples were collected from routine castrations. ESR1 variants were detected by means of RT-PCR using primers spanning one to three exons. We detected the variants Δ4 and Δ7 in all tissue samples investigated. Additionally, the testicular parenchyma expressed the variant Δ6 and double exon deletions of ESR1 (Δ4/6 and Δ6/7). Using an antiserum recognizing all previously identified ESR1 splicing variants, we revealed ESR1 proteins being expressed in nearly all cells of the testicular and ovarian parenchyma. ESR1 Δ6 protein, however, detected by an antiserum specifically raised against the Δ6 variant, was predominantly located in Sertoli cells. As the exon deletion variants are significantly expressed and show a distinct expression pattern, they could specifically modulate the cellular responsiveness to hormonal stimuli within the gonads.

Expression of the androgen receptor in the testis of mice with a Sertoli cell specific knock-out of the connexin 43 gene (SCCx43KO−/−)

Gap junctions are intercellular channels that connect the cytoplasm of adjacent cells, allowing the passage of small molecules (<1kDa) and thereby the regulation of many different processes. In the male gonad, the most abundant protein that builds gap junctions is connexin 43 (Cx43, GJA1). Specific knock-out of Sertoli cells (SCCx43KO−/−) results in an impaired spermatogenesis up to the Sertoli cell only syndrome. The aim of this study was to compare the testicular expression pattern of the androgen receptor (AR) in wild type (WT) and SCCx43KO−/− mice. In both WT and SCCx43KO−/− testes, the AR staining was restricted to the nuclei of Sertoli, Leydig, and peritubular cells. However, the staining intensity varied between control and mutant mice. In the latter, the AR expression depended on the level of the seminiferous tubule impairment. In tubules with qualitatively normal spermatogenesis, the AR protein expression was similar to that observed in the testes of WT mice. Conversely, seminiferous tubules with an arrest of spermatogenesis at the level of spermatogonial or spermatocyte phase expressed the AR at a lower intensity. In Sertoli cell only tubules (no germ cells in the tubules), the AR immunoreaction was mainly weak or undetectable. Moreover, AR staining was lower in Sertoli and Leydig cells (p<0.001 and p<0.05, respectively) of SCCx43KO−/− mice compared to WT mice, as revealed by a semiquantitative analysis. In conclusion, the deletion of Cx43 leads to a partial disruption of the AR signaling pathway, indicating a possible reason for the observed impaired spermatogenesis.