Venlafaxine-induced damage to seminiferous epithelium, spermiation, and sperm parameters in rats: A correlation with high estrogen levels.

BACKGROUND: Venlafaxine (selective serotonin and norepinephrine reuptake inhibitor) use has increased worldwide. However, the impact of venlafaxine on testes and sperm parameters has not been investigated. OBJECTIVES: We evaluated venlafaxine impact on testicular and sperm parameters and verified whether the changes are reversible. METHODS: Animals from venlafaxine-35 days and venlafaxine-65 days groups received 30 mg/kg of venlafaxine for 35 days. Control-35 days and control-65 days received distilled water. In control-65 days and venlafaxine-65 days, the treatment was interrupted for 30 days. Sperm concentration, morphology, motility, and mitochondrial activity were analyzed. Number of step 19 spermatids (NLS), frequency of tubules with spermiation failure, Sertoli cells number, and TUNEL-positive germ cells were quantified. Testicular aromatase, connexin 43 (Cx43) immunoexpression, Cx43 protein levels, and Cx43 expression were evaluated. Either intratesticular testosterone or estrogen levels were measured. RESULTS: Venlafaxine impaired sperm morphology, reduced sperm concentration, mitochondrial activity, and sperm motility. The frequency of tubules with spermiation failure and NLS increased in parallel to increased Cx43 immunoexpression; mRNA and protein levels; and aromatase, testosterone, and estrogen levels. An increase in germ cell death and decreased Sertoli cells number were observed. In venlafaxine-65 days, except for sperm motility, mitochondrial activity, Sertoli cells number, and germ cell death, all other parameters were partially or totally recovered. CONCLUSION: Venlafaxine increases testosterone aromatization and Cx43. This drug, via high estrogen levels, disturbs Sertoli cells, induces germ cell death, and impairs spermiation and sperm parameters. The restoration of spermiation associated with the decreased Cx43 and hormonal levels in venlafaxine-65 days reinforces that high estrogen levels are related to venlafaxine-induced changes. The presence of damaged Sertoli cells, germ cell death, and low sperm motility in venlafaxine-65 days indicates that interruption of treatment for 30 days was insufficient for testicular recovery and points to a long-term estrogen impact on the seminiferous epithelium.

Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17ß) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17ß-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell-cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell-expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.-Hakkarainen, J., Zhang, F.-P., Jokela, H., Mayerhofer, A., Behr, R., Cisneros-Montalvo, S., Nurmio, M., Toppari, J., Ohlsson, C., Kotaja, N., Sipilä, P., Poutanen, M. Hydroxysteroid (17ß) dehydrogenase 1 expressed by Sertoli cells contributes to steroid synthesis and is required for male fertility.

CYP26 Enzymes Are Necessary Within the Postnatal Seminiferous Epithelium for Normal Murine Spermatogenesis.

The active metabolite of vitamin A, retinoic acid (RA), is known to be essential for spermatogenesis. Changes to RA levels within the seminiferous epithelium can alter the development of male germ cells, including blocking their differentiation completely. Excess RA has been shown to cause germ cell death in both neonatal and adult animals, yet the cells capable of degrading RA within the testis have yet to be investigated. One previous study alluded to a requirement for one of the RA degrading enzymes, CYP26B1, in Sertoli cells but no data exist to determine whether germ cells possess the ability to degrade RA. To bridge this gap, the roles of CYP26A1 and CYP26B1 within the seminiferous epithelium were investigated by creating single and dual conditional knockouts of these enzymes in either Sertoli or germ cells. Analysis of these knockout models revealed that deletion of both Cyp26a1 and Cyp26b1 in either cell type resulted in increased vacuolization within the seminiferous tubules, delayed spermatid release, and an increase in the number of STRA8-positive spermatogonia, but spermatozoa were still produced and the animals were found to be fertile. However, elimination of CYP26B1 activity within both germ and Sertoli cells resulted in severe male subfertility, with a loss of advanced germ cells from the seminiferous epithelium. These data indicate that CYP26 activity within either Sertoli or germ cells is essential for the normal progression of spermatogenesis and that its loss can result in reduced male fertility.

Endothelial and inducible nitric oxide synthase (NOS) immunoreactivity and NOS-associated NADPH-diaphorase histochemistry in the domestic cat (Felis catus) testis.

In this study, the cellular localization of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the endothelial (eNOS) and inducible (iNOS) forms of nitric oxide (NO) synthase in the cat testis were studied using enzyme histochemical and immunohistochemical techniques. Stage-dependent nuclear and cytoplasmic eNOS/iNOS immunoreactivity and cytoplasmic NADPH-d reactivity were found in all germ cells, including spermatogonia, primary spermatocytes (preleptotene, zygotene, and pachytene spermatocytes), and round (Sa, Sb1) and elongating spermatids (Sb2, Sc) of the seminiferous epithelium. The pachytene spermatocytes exhibited strong positive reactions at all spermatogenic stage. Interestingly, in elongated spermatids (Sd1) at stages VI to VII, eNOS and iNOS immunostainings was observed only in the cytoplasm but not in the nuclei. eNOS and iNOS immunolabeling was observed in the acrosomal vesicle of some round spermatids (Sb1) at stages I, VII, and VIII, and in the acrosomal cap of elongating spermatids (Sb2) at stage II. Furthermore, eNOS, iNOS, and NADPH-d reactions in elongated spermatids (Sd2) just before spermiation at stage VIII were restricted only to the middle and principal pieces of the tail. Positive reactions were also observed in the Sertoli and Leydig cells as well as in other tissues including vascular endothelial and smooth muscle cells and peritubular myoid cells. These results suggest that NO may play an important role in chromatin condensation, spermatid shaping, and the final release of sperm from the spermatogenic epithelium. Furthermore, NO may also be involved in spermiogenesis, steroidogenesis, and apoptotic cell death.

C-Src and c-Yes are two unlikely partners of spermatogenesis and their roles in blood-testis barrier dynamics.

Src family kinases (SFKs), in particular c-Src and c-Yes, are nonreceptor protein tyrosine kinases that mediate integrin signaling at focal adhesion complex at the cell-extracellular matrix interface to regulate cell adhesion, cell cycle progression, cell survival, proliferation and differentiation, most notably in cancer cells during tumorigenesis and metastasis. Interestingly, recent studies have shown that these two proto-oncogenes are integrated components of the stem cell niche and the cell-cell actin-based anchoring junction known as ectoplasmic specialization (ES) at the: (1) Sertoli cell-spermatid interface known as apical ES and (2) Sertoli-Sertoli cell interface known as basal ES which together with tight junctions (TJ), gap junctions and desmosomes constitute the blood-testis barrier (BTB). At the stem cell niche, these SFKs regulate spermatogonial stem cell (SSC) renewal to maintain the proper population of SSC/spermatogonia for spermatogenesis. At the apical ES and the BTB, c-Src and c-Yes confer cell adhesion either by maintaining the proper phosphorylation status of integral membrane proteins at the site which in turn regulates protein-protein interactions between integral membrane proteins and their adaptors, or by facilitating androgen action on spermatogenesis via a nongenomic pathway which also modulates cell adhesion in the seminiferous epithelium. Herein, we critically evaluate recent findings in the field regarding the roles of these two unlikely partners of spermatogenesis. We also propose a hypothetical model on the mechanistic functions of c-Src and c-Yes in spermatogenesis so that functional experiments can be designed in future studies.

Expression of acid phosphatase in the seminiferous epithelium of vertebrates.

Acid phosphatases (AcPs) are known to provide phosphate to tissues that have high energy requirements, especially during development, growth and maturation. During spermatogenesis AcP activity is manifested in heterophagous lysosomes of Sertoli cells. This phagocytic function appears to be hormone-independent. We examined the expression pattern of AcP during the reproductive period of four species belonging to different vertebrate groups: Tilapia rendalli (Teleostei, Cichlidae), Dendropsophus minutus (Amphibia, Anura), Meriones unguiculatus (Mammalia, Rodentia), and Oryctolagus cuniculus (Mammalia, Lagomorpha). To demonstrate AcP activity, cryosections were processed for enzyme histochemistry by a modification of the method of Gömöri. AcP activity was similar in the testes of these four species. Testes of T. rendalli, D. minutus and M. unguiculatus showed an intense reaction in the Sertoli cell region. AcP activity was detected in the testes of D. minutus and O. cuniculus in seminiferous epithelium regions, where cells are found in more advanced stages of development. The seminiferous epithelium of all four species exhibited AcP activity, mainly in the cytoplasm of either Sertoli cells or germ cells. These findings reinforce the importance of AcP activity during the spermatogenesis process in vertebrates.

Regulation of blood-testis barrier dynamics by focal adhesion kinase (FAK): an unexpected turn of events.

The blood-testis barrier (BTB) is conferred by co-existing tight junctions (TJs), basal ectoplasmic specializations (basal ES), desmosome-like junctions and gap junctions (GJs) between adjacent Sertoli cells near the basement membrane in the seminiferous epithelium. While the concept of the BTB has been known for more than a century and its significance to spermatogenesis discerned for more than five decades, its regulation has remained largely unknown. Recent studies, however, have demonstrated that focal adhesion kinase (FAK), a modulator of the integrin-based signaling that plays a crucial role in cell movement, apoptosis, cell survival and gene expression at the focal adhesion complex (FAC, also known as focal contact, a cell-matrix anchoring junction type), is an integrated component of the BTB, associated with the TJ-integral membrane protein occludin and its adaptor zonula occludens-1 (ZO-1). Herein, we summarize recent findings in the field regarding the significance of FAK in conferring BTB integrity based on some unexpected observations. We also critically discuss the role of FAK in regulating the timely "opening" and "closing" of the BTB to facilitate the transit of primary preleptotene spermatocytes across the BTB at stage VIII of the seminiferous epithelial cycle of spermatogenesis. Lastly, we describe a working model, which can be used to design future functional experiments to explore the involvement of FAK in BTB dynamics by investigators in the field.

Expression of testicular angiotensin-converting enzyme in adult spontaneously hypertensive rats.

Recent studies demonstrated that one isoform of angiotensin-converting enzyme named testicular or germinal (tACE) is localized in postmeiotic male germ cells and is essential for fertilizing ability of spermatozoa. Hypertension in spontaneously hypertensive rats (SHR) is androgen-dependent and reduction in male gametes is reported in this experimental conditions. Expression of tACE was not studied under conditions of spontaneous hypertension. The aim of this work is to characterize immuno-expression of tACE in the testis of adult (16-week-old) SHR rats in relation to the changes in blood pressure and serum testosterone level. In 82% of adult SHR, the immuno-expression of tACE followed the normal stage-specific pattern. Destructive testicular changes, germ cells depletion have been observed in 18% of 16-week-old SHR and stronger expression of tACE in stages 8-11 compared to controls was detected. As a result stage specificity in SHR was not as evident as in control. No reaction was found in germ cell depleted tubules in which elongated spermatids were absent. Degenerating germ cells exhibited strong immunostaining comparable to that in residual bodies. The blood pressure was significantly higher in SHR and testosterone levels were more than twice but non-significantly elevated. There was no clear correlation between testicular structural changes, blood pressure level values or serum testosterone levels. Expression of tACE in postmeiotic germ cells, specifically altered by SHR, suggested possible involvement of components of renin-angiotensin system in the process of spermiogenesis. Loss of enzyme expression we found in germ cell depleted tubules in SHR is due to absence of corresponding stages of spermatid differentiation. Therefore, tACE can be used as a marker for germ cell depletion due to hypertension and other pathological conditions.

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis.

Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.

Interactions of proteases, protease inhibitors, and the beta1 integrin/laminin gamma3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis.

During spermatogenesis, developing germ cells migrate progressively across the seminiferous epithelium. This event requires extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as the ectoplasmic specialization (ES, a testis-specific AJ type), between Sertoli cells and elongating/elongate spermatids. It was postulated that proteases and protease inhibitors worked in a yin-yang relationship to regulate these events. If this is true, then it is anticipated that both proteases and protease inhibitors are found at the ES. Indeed, matrix metalloprotease (MMP)-2, membrane-type 1 (MT1)-MMP and their inhibitor, tissue-inhibitor of metalloproteases (TIMP)-2, were shown to localize at the apical ES. In order to identify the putative MMP substrate as well as the unknown binding ligand for alpha6beta1 integrin in the ES, immunofluorescent microscopy coupled with immunoprecipitation techniques were used to demonstrate that laminin gamma3, largely a germ cell product, was present at the apical ES and could form a bona fide complex with beta1-integrin. Furthermore, the structural interactions of MMP-2 and MT1-MMP with laminin gamma3 and beta1-integrin, but not with N-cadherin or nectin-3, have implicated the crucial role of MMP-2/MT1-MMP in the regulation of integrin/laminin-based ES dynamics. Using an in vivo model to study AJ dynamics where adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to disrupt Sertoli-germ cell adhesive function, an induction of active MMP-2, active MT1-MMP and TIMP-2 but not active MMP-9 was detected between 0.5 and 8 h after AF-2364 treatment. This time frame coincided with the depletion of elongating/elongate spermatids from the epithelium, illustrating the synergistic relationships between MMP-2, MT1-MMP, and TIMP-2 in AJ disassembly. Perhaps the most important of all, the use of a specific MMP-2 and MMP-9 inhibitor, (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid, could effectively delay the AF-2364-induced elongating/elongate spermatid loss from the epithelium, demonstrating the pivotal role of MMP-2 activation in ES disassembly. Collectively, these studies illustrate that the beta1-integrin/laminin gamma3 complex is a putative ES-structural protein complex, which is regulated, at least in part, by the activation of MMP-2 involving MT1-MMP and TIMP-2 at the apical ES. The net result of this interaction likely regulates germ cell movement in the seminiferous epithelium.

Localization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the male reproductive tract.

The action of glucocorticoids in target tissues is dependent on the local expression of glucocorticoid receptors and two 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes, 11beta-HSD1 and 11beta-HSD2, which interconvert active and inactive glucocorticoids. This study examined expression of the 11beta-HSD enzymes in the male reproductive tract of the adult rat. 11beta-HSD1 was immunolocalized to the apical region of principal epithelial cells of the caput epididymis, with the less numerous clear cells devoid of signal. Epididymal 11beta-HSD1 expression was confirmed by Western blot analysis, with immunoreactive species identified at 34 kDa (the expected size for 11beta-HSD1) and at approximately 48 kDa. 11beta-HSD bioactivity was readily detectable in the epididymis, with 11-oxoreductase activity clearly the favored reaction (as observed in liver), consistent with 11beta-HSD1 expression. The epithelium of the vas deferens, seminal vesicle, and penile urethra were also immunopositive for 11beta-HSD1, as were smooth muscle cells of the vas deferens and penile blood vessels. 11beta-HSD2 was also immunolocalized to the epididymal epithelium, but its distribution was complementary to that of 11beta-HSD1 (i.e. clear cells showing intense 11beta-HSD2 staining but principal cells devoid of signal). 11beta-HSD2 was also present in the corpora cavernosa of the penis but not in other tissues. In conclusion, the differential expression of 11beta-HSD1 and 11beta-HSD2 throughout the male reproductive tract suggests that these enzymes locally modulate glucocorticoid and mineralocorticoid actions, particularly in the epididymis and penile vasculature.

Function of DNA-protein kinase catalytic subunit during the early meiotic prophase without Ku70 and Ku86.

All components of the double-stranded DNA break (DSB) repair complex DNA-dependent protein kinase (DNA-PK), including Ku70, Ku86, and DNA-PK catalytic subunit (DNA-PKcs), were found in the radiosensitive spermatogonia. Although p53 induction was unaffected, spermatogonial apoptosis occurred faster in the irradiated DNA-PKcs-deficient scid testis. This finding suggests that spermatogonial DNA-PK functions in DNA damage repair rather than p53 induction. Despite the fact that early spermatocytes lack the Ku proteins, spontaneous apoptosis of these cells occurred in the scid testis. The majority of these apoptotic spermatocytes were found at stage IV of the cycle of the seminiferous epithelium where a meiotic checkpoint has been suggested to exist. Meiotic synapsis and recombination during the early meiotic prophase induce DSBs, which are apparently less accurately repaired in scid spermatocytes that then fail to pass the meiotic checkpoint. The role for DNA-PKcs during the meiotic prophase differs from that in mitotic cells; it is not influenced by ionizing radiation and is independent of the Ku heterodimer.

Inmunorreactividad de enolasa en el epitelio seminífero senil humano / Immunoreactivity to enolase in the human senil seminiferous epithelium

El metabolismo energético intracelular compartimentalizado, activa la enzimo enolasa para formar ácido pirúvico. Este, substrato energético, debe ingresar a la mitocondria para continuar hacia el ciclo de losácidos tricarboxílicos. Sin embargo, durante la proliferación del epitelio seminífero ocurre una distribución y pérdida citoplasmática progresiva y disposición de las mitocondrias a nivel del flagelo inicial. en el presente estudio se describe la inmunoreactividad de la enzimo enolasa en las diferentes poblaciones celulares del epitelio seminífero, en testiculo humano senil. Se trabajo con un paciente de 70 años, sometido a orquiectomía subcapsular terapéutica. El tejido testicular fue fijado inmediatamente en formalina taponada al 10 por ciento y mantenido por 12 horas. Luego se procesó por técnicas histológicas corrientes e incluyo en parafina para obtener secciones de 5 um. Posteriormente se procedió a la reacción inmunohistoquímica para enolasa y revelación con complejo avidina-biotina. Finalmente se cuantificaron las distintas poblaciones celulares del epitelio seminífero con reacción positivo o negativa. Los resultados preliminares se expresan en porcentajes de células positivas respecto del total de células contadas (40x. se observó que la totalidad de las células de Sértoli presentaron reacción negativa a la enolasa. En el epitelio seminífero se encontró que el 76 por ciento de las gonias (gonias tipo A y B) mostraron reacción negativa a la enolasa, mientras que en citos 1 se redujo al 10 por ciento y ausencia total en espermátidas y espermatozoides. Por lo tanto, las célula somáticas (de origen mesodérmico), del epitelio seminífero presentarían isoenzimas enolasas de reactividad diferente en la relación con la línea germinal (espermatogonias). Adicionalmente, en la línea germinal la inmunoreactividad a enolasa disminuye mientras progresa la espermatogénesis

Stage and cell-specific expression of calmodulin-dependent phosphodiesterases in mouse testis.

Calcium and cyclic nucleotides are second messengers that regulate the development and functional activity of spermatozoa. Calcium/calmodulin-dependent phosphodiesterases (CaM-PDEs) are abundant in testicular cells and in mature spermatozoa and provide one means by which calcium regulates cellular cyclic nucleotide content. We examined the spatial and temporal expression profiles of three knownCaM-PDE genes, PDE1A, PDE1B, and PDE1C, in the testis. In situ hybridization and immunofluorescent staining showed that both PDE1A and PDE1C are highly expressed but at different stages in developing germ cells. However, a very low hybridization signal of PDE1B exists uniformly throughout the seminiferous epithelium and the interstitium. More specifically, PDE1A mRNA is found in round to elongated spermatids, with protein expression in the tails of elongated and maturing spermatids. In contrast, PDE1C mRNA accumulates during early meiotic prophase and throughout meiotic and postmeiotic stages. Immunocytochemistry showed a diffuse, presumably cytosolic distribution of the expressed protein. The distinct spatial and temporal expression patterns of CaM-PDEs suggest important but different physiological roles for these CaM-PDEs in developing and mature spermatozoa.

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents.

The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.

Elements of the kallikrein-kinin system are present in rat seminiferous epithelium.

Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein-kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein-kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.

Rat prostaglandin D2 synthetase: its tissue distribution, changes during maturation, and regulation in the testis and epididymis.

The changes in glutathione-independent prostaglandin D2 synthetase (PGD-S) during maturation in the rat were determined in selected organs by an RIA using PGD-S purified from rat cerebrospinal fluid and a monospecific anti-rat PGD-S polyclonal antibody. In a survey of its tissue distribution in various organ extracts and biological fluids, it was found that the concentration of PGD-S was highest in the epididymis-about 6- and 80-fold greater than that in the brain and testis, respectively. During maturation, PGD-S concentration increased steadily in the testis and epididymis; this is in contrast to the pattern of changes in the brain and liver, which showed a general trend of decline. Reverse transcription-polymerase chain reaction and Southern blotting were used to demonstrate the presence of PGD-S mRNA transcript in the testis and in Sertoli and germ cells. In the epididymis, the steady-state PGD-S mRNA level was highest in the caput, followed by the cauda and corpus. Orchiectomy induced a drastic reduction of PGD-S concentration in all three epididymal compartments. Administration of dihydrotestosterone (DHT) failed to restore the reduced epididymal PGD-S level except in the caput epididymis, where 4 days after DHT treatment the level of PGD-S was restored to about 50% of the pre-orchiectomized level; this suggests that the epididymal PGD-S level is not entirely regulated by androgen and that another yet to be identified testicular factor(s) is likely to be involved in its regulation. Germ cell-conditioned medium was also shown to stimulate PGD-S expression in the Sertoli cell. These results illustrate that PGD-S is an important molecule in testicular and epididymal function and that it is likely involved in spermatogenesis and sperm maturation.

Changes in protein prenylation and prenyltransferase activity in the rat seminiferous epithelium during early stages of spermatogenesis.

Changes in protein prenyltransferase activity, levels of prenylated protein, and the type of isoprenoid modification was described in cells of rat seminifereous epithelium and correlated with differentiative events of spermatogenesis. The activity of protein farnesyltransferase (PFT) was at least 10-fold higher than that for protein geranylgeranyltransferase-I (PGGT-I) in seminiferous epithelium and spermatogenic cells of prepubertal rats of different ages. Both activities increased during the meiotic stages of differentiation and peaked at 23 days of age. The activity of farnesyltransferase in seminiferous epithelium was the same as that in mixed spermatogenic cell populations from animals aged 9 and 23 days, indicating that the activity of this enzyme in somatic cells and germ cells was similar at these ages. Farnesyltransferase activities were similar and low in both pachytene spermatocytes and round spermatids from adult rats; however, the activity in pachytene spermatocytes from 23-day old animals was 2-fold higher than in adults. The highest activity was associated with intermediate-sized spermatocytes appearing late during meiosis. PGGT-I activity was at least 10-fold lower than farnesyltransferase activity and was not significantly different among all cell populations. Differentiation-dependent in vivo protein prenylation was demonstrated by labeling of seminiferous epithelium with [3H]mevalonic acid at different prepubertal ages. Total protein prenylation and the ratio of geranylgeranylated to farnesylated protein, in contrast to prenyltransferase activity, decreased with increasing age. Although 20-30-kDa proteins were the most highly labeled at all ages, [3H]-proteins from different-aged prepubertal rats showed age-dependent changes in the level of prenylation of at least 14 proteins as determined by two-dimensional (2D) electrophoresis. Prenylated proteins of round spermatids were distinguished from those of the spermatocytes by the lack of many 20-30-kDa proteins and by low geranylgeranyl/farnesyl (GG/F) ratios. These results show that independent changes in prenyltransferase activity and protein prenylation accompany the differentiation events during the premeiotic and meiotic stages of spermatogenesis. This suggests that prenylation in the seminiferous epithelium may be more dependent on available protein substrate than on protein prenyltransferase activity.

Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium.

Four genes (ratPDE1/IVc, ratPDE2/IVa, ratPDE3/IVd, and ratPDE4/IVb) encoding different isoforms of phosphodiesterase that specifically hydrolyze the second messenger cAMP (cAMP-PDEs) are present in the rat. Previous data from our laboratory indicated that these genes are differentially expressed in the somatic and germ cells of the seminiferous epithelium of the testis. To further characterize their spatial and temporal expression in the seminiferous tubules, in situ hybridization was used to monitor the expression of the four cAMP-PDE messenger RNAs (mRNAs). The signals corresponding to ratPDE1/IVc and ratPDE2IVa mRNAs were localized in two restricted layers of the seminiferous epithelium. The ratPDE1/IVc mRNA was present in a region of the epithelium corresponding to the location of middle-late pachytene spermatocytes. Conversely, the ratPDE2/IVa signal was confined to a more adluminal area corresponding to the location of maturing round spermatids. The ratPDE3/IVd and ratPDE4/IVb mRNAs were distributed throughout the span of the seminiferous epithelium, indicating a localization in the Sertoli cell cytoplasm. Although the intensity of the signal corresponding to ratPDE4/IVb was similar in all seminiferous tubule stages, the ratPDE3/IVd signal varied in intensity in tubules at different stages of the seminiferous cycle. Maximal expression was present in tubules at stages I-V and XI-XIII of the cycle and minimal at stages VIII-IX of the cycle. The expression of the ratPDE3/IVd mRNA positively correlated with the ability of specific inhibitors of the cAMP-PDEs to potentiate the FSH-dependent cAMP accumulation in tubules at different stages of the seminiferous cycle, with maximal potentiation observed at stages II-VI of the cycle. These data demonstrate that different cAMP-PDE genes are active in different cells of the seminiferous tubules and that the ratPDE3/IVd gene is expressed in the Sertoli cell in a cyclical fashion during the seminiferous cycle.

Developmental expression of the glutathione S-transferase Yo subunit in the rat testis and epididymis using light microscope immunocytochemistry.

BACKGROUND: Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione with various toxic electrophilic compounds. GSTs are composed of several classes based on the degree of sequence homology of their subunits. The Yo subunit, a member of the mu class, is expressed at high levels in the testis and epididymis. The purpose of this study was to immunolocalize the GST-Yo in these tissues during development. METHODS: The testes and epididymides of rats aged 7, 15, 21, 28, 39, 42, 45, 49, and 56 days were fixed in Bouin's fixative, and immunostained for light microscopic analysis. RESULTS: In the testis the cytoplasm of all germ cells was unreactive until day 39. At that time, step 18 spermatids appeared moderately reactive, while the few observed step 19 spermatids were intensely reactive as were their residual bodies. The presence of residual bodies indicates that spermiation takes place as early as day 39; however, the number of step 19 spermatids is low at this age. A progressive increase in the size of the tubule and number of elongating spermatids was seen between days 42 and 49. In addition, by day 49, a weak staining was observed in steps 12-15, moderate in steps 16-17, and intense in steps 18-19 spermatids. In terms of the intensity of staining, cell types stained, size of the tubules, and number of elongating spermatids, no difference was noted between day 49, 56, and adult animals. Thus Yo protein expression in germ cells reached maturity by day 49. The epithelial cells of the rete testis were intensely reactive at day 7 and remained so throughout development. In contrast, while the epithelial cells of the efferent ducts at day 7 were intensely reactive, they were weakly reactive by day 39 and remained so at later ages. Along the entire epididymis, the columnar epithelial cells showed a moderate apical/supranuclear reaction from day 7 to 28. By day 39 principal cells of the initial segment became weakly reactive, while those in the caput and corpus were moderately stained, a situation seen at later ages including adults. Only by day 49 did principal cells of the proximal cauda become moderately stained as seen in adult animals. Thus the expression of the Yo protein in the principal cells of the proximal cauda may be regulated by different factors than those of the caput and corpus epididymidis. Alternatively, the expression of the Yo subunit in principal cells of the proximal cauda may develop later since this region would be the last to receive luminally derived testicular products. In the initial segment, the decrease in staining of principal cells at day 39 may be due to an inhibiting factor emanating from the testis. Spermatozoa appeared in the lumen of each epididymal region well after the expression of Yo had reached its adult staining pattern indicating that they are not a factor. CONCLUSIONS: Overall these results suggest that the expression of GST-Yo in the various cells of the testis and epididymis are controlled by different factors during postnatal development.