Rat Testis Cells Research Articles

Dose‐dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n‐butyl) phthalate

Adult male rats gestationally exposed to di(n-butyl)phthalate (DBP) have dysgenetic testes characterized by seminiferous epithelial degeneration, clustering of Leydig cells, and decreased spermatogenesis. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. To determine whether gestational exposure to DBP affects cell proliferation and apoptosis in the developing rat testis. Pregnant dams were exposed to different dose levels of DBP in mid-gestation and cellular outcomes in fetal and early postnatal testes were assayed by histological and morphometric approaches. Gestational exposure to high dose DBP inhibited proliferation of fetal testicular somatic cells but did not affect apoptosis. Exposed fetal testes had a smaller volume and decreased cell numbers, with decreases in both the tubular and interstitial cell populations. A reduction was observed in the testis volume and altered seminiferous tubule morphometry at > or =50 mg/kg/d, and a decreased testicular cell number at > or =30 mg/kg/d DBP. The number of multinucleated gonocytes in DBP-exposed fetal testes increased after exposure to > or =100 mg/kg/d. The number of proliferating cells in the DBP-exposed testis rapidly rose after birth (when exposure stopped), and the testis volume and the total cell number was comparable to control by postnatal day 2. DBP reversibly inhibits proliferation of somatic cells in the fetal rat testis. Decreased proliferation, rather than increased apoptosis, is the underlying mechanism of altered fetal development of DBP-exposed seminiferous tubules contributing to testicular dysgenesis.

Regulation of Neuropeptide Y Y1 Receptors by Testosterone in Vascular Smooth Muscle Cells in Rat Testis

It is well established that testosterone and neuropeptide Y (NPY), via its Y1 receptor (Y1R), are involved in the central control of the gonadotrope axis in male rats. Here we examined if a similar interaction also occurs in the male peripheral reproductive target organ, the testes. Expression of the Y1R transcript and protein and changes in testicular microcirculation were studied in normal rats and 12 days following hypophysectomy with and without testosterone substitution (1 or 25 mg s.c.). In situ hybridization and immunohistochemistry showed strong expression of, respectively, Y1R messenger RNA (Y1R mRNA) and Y1R-like immunoreactivity (Y1R-LI) in vascular smooth muscles in the testes of control and hypophysectomized rats treated with testosterone, but was not seen without testosterone substitution. In parallel, control animals and hypophysectomized, testosterone-supplemented rats showed a strong (approximately 40%) decrease in testicular blood flow following intratesticular (i.t.) injection of the Y1-R agonists, [Leu³¹, Pro³⁴]NPY, [D-Arg²⁵]NPY or NPY, an effect which was completely blocked by prior intravenous administration of the Y1R antagonist, BIBP3226. No significant change in testicular blood flow following i.t. injection of NPY was seen in hypophysectomized rats without testosterone substitution. These findings suggest that the high levels of Y1R mRNA and Y1R-LI in the testes reflect expression of functional Y1Rs mediating vasoconstriction, and that testosterone regulates expression of functional Y1Rs.

Regulation of Neurotrophin 3 Expression by SRY During Male Sex Determination.

Neurotrophin 3 (Nt3) plays a significant role in male gonadal development. It is required for mesonephric cell migration into the testis for testis cord formation. The expression profiles for Nt3 are consistent with those expected for a gene directly regulated by SRY, and potential SRY binding HMG box elements were identified in the Nt3 promoter. Therefore, Nt3 regulation by SRY was investigated. Nt3 expression profiles in rat were verified spatially and temporally by immunohistochemical localization and Nt3 was found to be in the developing testis cords in Sertoli cells of rat gonads. Nt3 promoter constructs with luciferase reporters were made using mouse and rat Nt3 promoters and HMG boxes mutated by site specific mutagenesis. Promoter activity was monitored using transfection assays in primary P20 rat Sertoli cells and in E13 rat testis cell lines. SRY element site specific mutant promoters had reduced luciferase reporter activity compared to wild type promoters. Since SRY and SOX9 are important for sex determination and SOX9 binds to DNA sequences highly similar to those bound by SRY, the potential roles of both SRY and SOX9 in regulation of the Nt3 promoter were investigated. The potential for SRY and SOX9 to affect Nt3 promoter activity was examined in co-transfection assays using SRY and SOX9 expression vectors. Results indicate that both SRY and SOX9 can stimulate the Nt3 promoter in transfection assays. Further analysis is required to reveal the key players in vivo: Knock down of Sry and Sox9 expression is being used to determine if there is a change in Nt3 expression when Sry or Sox9 are silenced at the initiation of gonadal differentiation. Gel shifts and high sensitivity ChIP assays are being utilized to determine the relative binding affinities of SRY and SOX9 to HMG boxes in the Nt3 promoter and investigate binding to the Nt3 promoter in vivo. These studies have helped clarify the regulation of Nt3 in the gonad during sex determination and reveal the potential for Nt3 to be regulated directly by SRY.

Oxidative alterations induced by d-aspartic acid in prepubertal rat testis in vitro: A mechanistic study

The objective of the present study was to investigate the oxidative induction response following in vitro treatment with D-aspartic acid (DA) in prepubertal rat testis (homogenates, explants, and cell suspensions). In all three preparations, DA enhanced (P<0.001) lipid peroxidation, manifest as increased reactive oxygen species (ROS) and malondialdehyde (MDA). Further, DA-induced oxidative induction was potentiated (P<0.001) in the presence of iron (5 microM) and 3-amino triazole and mercaptosuccinate (P<0.001), known inhibitors of the peroxide metabolizing enzymes, catalase and glutathione peroxidase, respectively. Testis homogenates exposed to L-arginine (LA) per se had reduced (P<0.001) endogenous levels of ROS and MDA; furthermore, pre-incubation with L-arginine markedly suppressed (P<0.001) DA-induced oxidative induction, suggesting an antagonistic action, perhaps due to LA-derived nitric oxide. In conclusion, DA caused significant oxidative induction in prepubertal rat testis, but this action was abrogated by L-arginine. The relevance of this phenomenon in vivo merits further study, as both of these molecules have specific physiological functions in the testis.

Effects of hypothyroidism on anti-mullerian hormone expression in the prepubertal rat testis.

Differentiation of adult Leydig cells (ALC) in the prepubertal rat testis is stimulated by thyroid hormone (Thy) and inhibited by the Anti-Mullerian Hormone (AMH) produced by the immature Sertoli cell (SC). As Thy induces SC maturation in the prepubertal rat testis, we hypothesized that Thy stimulation of ALC differentiation is mediated via inhibition of AMH production by the SC with their maturation. If this hypothesis is true, AMH production by the prepubertal Sertoli cells in hypothyroid rats should not decline immediately after birth as in euthyroid rats, but should be maintained throughout the hypothyroid period at a similar or higher level to that of day 1 rats. This concept was tested using control rats of postnatal days (pd) 1, 7 and 14 and hypothyroid (fed 0.1% propyl thiouracil/PTU to lactating mothers) rats of pd7 and pd14. Presence of AMH in SC was examined by immunocytochemistry for AMH. Results demonstrated that testes of pd1 rats had intense AMH positive labeling exclusively in cytoplasm of SC. In testes of pd7 and pd14 control and PTU rats, a positive but weak labeling was also observed in cytoplasm of some SC; Germ cells and testicular interstitial cells were negative for AMH at all tested ages in both experimental groups. These findings suggest that AMH production by the prepubertal SC is independent of Sertoli cell maturation and not regulated by Thy. Therefore, Thy regulation of ALC differentiation in the prepubertal rat testis is unlikely to be mediated via inhibition of AMH produced by the SC with their maturation.

Effect of isoflavone-enriched soybean flour, zinc (Zn) and vitamin E in the ration on testosterone level and total permatogenic cell in seminiferous tubules of rat

The objective of this experiment are to evaluate the effects of isoflavoneen-enriched soybean flour, zinc (Zn) and vitamin E on testosterone level of male rats and total spermatogenic cells in the seminiferous tubules of rat testes as animal model. Diet was given as isonitrogen and isocaloric with 15% of dietary protein. Thirty male Sprague Dawley rats were divided into six groups and treated with isoflavoneen-riched soybean flour, Zn and vitamin E in different combination. Isoflavoneen-riched soybean flour (3mg/day) was given by oral administration, whereas Zn and vitamin E were mixed wih the basic diet. The treatment was done for 2 month. Results indicated that complete treatment of isoflavoneen-riched soybean flour, Zn and vitamin E on male rats increased testosteron level and total spermatogenic cells in comparison with single treatment and the other combination. The best results showed in the group that given isoflavoneen-enriched soybean flour with diet containing both Zn and vitamin E i.e; testosteron level 3.49 ± 0.31 ng/ml; while the number of spermatogonia, spermatocytes, early spermatids, late spermatids, and total spermatogenic cells were 37.56 ± 4.48, 67 ± 4.72, 287.11 ± 31.75, 227.22 ± 29.78, and 618.89 ± 47.38, respectively. It was concluded that synergic interaction between isoflavoneen-enriched soybean flour, Zn and vitamin E increased testosteron level and total spermatogenic cells of rat testes. Key Words : Isoflavone-Riched Soybean Flour, Zn, Vitamin E, Testosterone, Spermatogenic Cells

Androgen-secreting tissue construction with co-cultured rat testes somatic cells by tissue engineering technique

To explore the feasibility of constructing androgen-secreting tissue of a certain size and shape using co-cultured somatic cells of rat testis. Thirty male Wistar rats were castrated. model and implanted rat model were prepared by resecting bilateral testes. The suspension of mixed testes cells was cultured to obtain various somatic cells of testes and Leydig cells were collected by differential anchorage-dependent method. These two kinds of cells were seeded onto biodegradable scaffolds of polyglycolic acid (PGA) fibers and cultured in vitro. The tissue formation of cell-scaffold constructs was observed by optical microscope and electronic microscope and the level of testosterone in the supernatant was detected regularly. After 7-day culture in vitro, the 2 kinds of cell-scaffold constructs, scaffold with purified Leydig cells or co-cultured testis somatic cells (seed cells), were implanted into the gastrocolic omentum or cavity of tunica vaginalis of the castrated rats. The implants were harvested 4, 6, 9, 12, and 24 weeks later to evaluate the tissue formation of cell-scaffold constructs in vivo. The serum testosterone level of the implanted rats was assayed to evaluate the testosterone secreting function of the regenerative tissue. Both the co-cultured testis somatic cells and Leydig cells had fine compatibility with the PGA fibers and adhered to the scaffolds very well. Testosterone was detected at a certain degree in the supernatant of cell-scaffold constructs, indicating the testosterone secreting function of the constructs. Two months after the implantation both kinds of cell-scaffold constructs formed testosterone secreting tissue in both gastrocolic omentum and cavity of tunica vaginalis of the implanted rats. The regenerative tissues were vascularized very well with a certain size and shape. Six weeks after implantation the serum testosterone level of the Leydig cell group was 0.60 ng/ml +/- 0.04 ng/ml, and that of the co-culture group was 0.84 ng/ml +/- 0.03 ng/ml, both significantly higher than that of the control castrated rats (0.56 ng/ml +/- 0.05 ng/ml, both P < 0.01), and the serum testosterone level of the co-cultured testes somatic cell implantation group was significantly higher than that of the Leydig cell implantation group too (P < 0.01). It is completely feasible to construct androgen-secreting tissue in vitro and in vivo using tissue engineering technique. Co-cultured testis somatic cells may serve as the better seed cells for androgen-secreting tissue engineering than purified Leydig cells in terms of the quantity and function of cells.

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli–Sertoli and Sertoli–germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell–cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood–testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli–germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.

Ethanol Exposure Suppresses Survival Kinases Activation in Adult Rat Testes

The present study was designed to evaluate whether ethanol suppresses survival-signaling pathways in rat testes. Ethanol (1.5 g/kg or 3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Ethanol treatment significantly increased the number of TUNEL-positive cells in rat testes. Potential activation was measured by phosphorylation of Akt and Erk1/2 using Western blot analysis. Ethanol decreased the levels of activated survival kinases, pAkt and pErk1/2. The phosphorylation of Bad at Ser112 and Ser136 was decreased in ethanol-treated animals in comparison to saline-treated animals. Moreover, the interaction of pBad with 14-3-3 was decreased by ethanol exposure. In conclusion, our findings suggest that ethanol induces apoptotic cell death by suppressing the activation of survival kinases and the phosphorylation of their downstream targets in rat testes.

Acrosome‐specific gene AEP1: Identification, characterization and roles in spermatogenesis

Spermatogenesis is a tightly regulated process leading to the development of spermatozoa. To elucidate the molecular spermatogenic mechanisms, we identified an acrosome-specific gene AEP1 in spermatids, which is located in rat chromosome 17p14 with a transcript size of 3,091 bp encoding a signal peptide, zinc finger-like motif, coiled-coil region, several predicted glycosylation and phosphorylation sites. Northern blot and RT-PCR analyses revealed the restricted expression of AEP1 to the testis only. In postnatal rat testes, AEP1 mRNA became detectable from postnatal 25 dpp (round spermatids) and onwards. By using in situ hybridization (ISH) and flow cytometry-fluorescent ISH, only the haploid spermatids yielded the positive AEP1 signal. Immunohistochemistry showed that AEP1 was expressed in the acrosomal cap of late-staged germ cells in rat testis, and co-localized with the acrosomal marker, peanut agglutinin. The spatial expression of AEP1 immunoreactivity in testis was conserved among diverse mammalian species (rat, pig, monkey, human). To further study its roles in spermatogenesis, we showed AEP1 and beta-actin was associated together in complex by co-immunoprecipitation in adult germ cells and by immunofluorescence assay in isolated spermatozoon. In human testes diagnosed with hypospermatogenesis, lower expression of AEP1 was observed, whereas there was no detectable signal in undescended testes. In short, AEP1 is an evolutionary-conserved acrosome-specific gene and likely functions in acrosome-cap formation.

Tumor necrosis factor α reversibly disrupts the blood–testis barrier and impairs Sertoli–germ cell adhesion in the seminiferous epithelium of adult rat testes

The timely restructuring of the blood-testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor alpha (TNFalpha), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFalpha directly to adult rat testes in vivo at 0.5 and 2 mug/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, alpha-, and beta-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFalpha treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFalpha by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring ('opening'?) of the BTB during spermatogenesis to facilitate germ cell migration.

Tissue-cell- and species-specific expression of gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) in gonads, adrenal and brain: Identification of novel forms in the brain

Gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) is a novel hormonally regulated fatty acyl-CoA synthetase (FACS) with activity for long-chain fatty acids. The presence of this enzyme in the Leydig cells of the mature rat testis and its mode of regulation suggest that it participates in testicular steroidogenesis. This study demonstrates that GR-LACS expression is tissue, cell and species-specific. The 79 kDa GR-LACS protein is expressed in rodent gonads and brain, and only in the mouse in the adrenal cortex. In the ovary of both species it is associated with follicles undergoing atresia. It is present in the newborn and immature testis tubules and after puberty only in the Leydig cells. A distinct GR-LACS protein species of 64 kDa that was more abundant than the 79 kDa long form was found in the rat brain. Also, a minor 73 kDa form was observed in the rat brain and mouse ovary. Two novel species resulting from alternatively splicing of the GR-LACS gene were identified in a rat brain cDNA library: a short form 1 (S1) lacking exon 8 and short form 2 (S2) lacking exons 6–8. Expression studies revealed that the sizes of the S1/S2 proteins are comparable to those of the endogenous variant species. Neither S form contains FACSs activity, suggesting that exon 8 is essential for the enzymatic function. GR-LACS variants exhibit small but significant dominant negative effects on the FACS activity of the long form. GR-LACS variants may regulate the long form's activity in the brain.

In search of rat stem Leydig cells: identification, isolation, and lineage-specific development.

Leydig cells (LCs) are thought to differentiate from spindle-shaped precursor cells that exhibit some aspects of differentiated function, including 3beta-hydroxysteroid dehydrogenase (3betaHSD) activity. The precursor cells ultimately derive from undifferentiated stem LCs (SLCs), which are postulated to be present in testes before the onset of precursor cell differentiation. We searched for cells in the neonatal rat testis with the abilities to: (i) proliferate and expand indefinitely in vitro (self renew); (ii) differentiate (i.e., 3betaHSD and ultimately synthesize testosterone); and (iii) when transplanted into host rat testes, colonize the interstitium and subsequently differentiate in vivo. At 1 week postpartum, spindle-shaped cells were seen in the testicular interstitium that differed from the precursor cells in that they were 3betaHSD-negative, luteinizing hormone (LH) receptor (LHR)-negative, and platelet-derived growth factor receptor alpha (PDGFR alpha)-positive. These cells were purified from the testes of 1-week-old rats. The cells contained proteins known to be involved in LC development, including GATA4, c-kit receptor, and leukemia inhibitory factor receptor. The putative SLCs expanded over the course of 6 months while remaining undifferentiated. When treated in media that contained thyroid hormone, insulin-like growth factor I, and LH, 40% of the putative SLCs came to express 3betaHSD and to synthesize testosterone. When transplanted into host rat testes from which LCs had been eliminated, the putative SLCs colonized the interstitium and subsequently expressed 3betaHSD, demonstrating their ability to differentiate in vivo. We conclude that these cells are likely to be the sought-after SLCs.

Detection of platelet-derived growth factor-alpha (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis.

Platelet-derived growth factor-A (PDGF-A) is a locally produced growth factor in the rat testis secreted by both Sertoli cells and Leydig cells. It has been suggested that PDGF-A may be involved in modulation of testosterone production and may be essential to Leydig cell differentiation, however it is not known at what stage of differentiation PDGF-A begins to be expressed in the cells of Leydig lineage in the postnatal rat testis. Therefore, the objectives of this research were to determine at what postnatal age and in which cell type is PDGF-A first expressed in cells of the adult Leydig cell lineage, and does PDGF-A expression coincide with expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), an indicator of steroid hormone synthesis. Male Sprague Dawley rats of postnatal day 1, 7, 9-14, 21, 28, 40, 60, and 90 were used (n=6). Animals were euthanized and their testicles removed, fixed in Bouin's solution, embedded in paraffin, and 5 micrometers sections were prepared. Immunolocalization of PDGF-A and 3beta-HSD was carried out using a peroxidase-streptavidin-biotin method. PDGF-A was first detected in cells of the Leydig cell lineage at postnatal day 10 in progenitor cells, which were surrounding the seminiferous tubules (peritubular). These cells were confirmed to be the progenitor cells and not the mesenchymal or any other spindle-shaped cells in the testis interstitium by immunolocalization of 3beta-HSD and PDGF-A in the cells in adjacent sections of testis tissue from rats of postnatal days 10-14. After postnatal day 10, PDGF-A was continued to be expressed in subsequent cells of the Leydig lineage through day 90 (adult), however, was not present in peritubular mesenchymal precursor cells of the Leydig cell lineage or any other spindle-shaped cells in the testis interstitium at any tested age. These results revealed that PDGF-A first appears in Leydig progenitor cells in the postnatal rat testis at the onset of mesenchymal cell differentiation into progenitor cells at postnatal day 10 and suggest that a functional role(s) of PDGF-A in postnatally differentiated Leydig cells in the rat testis is established at the time of the onset of postnatal Leydig stem cell differentiation. It is suggested that the significance of the first expression of PDGF-A in the Leydig progenitor cells may be associated with inducing cell proliferation and migration of this cell away from the peritubular region during Leydig cell differentiation.

Doxorubicin Induces Apoptosis in Germ Line Stem Cells in the Immature Rat Testis and Amifostine Cannot Protect against This Cytotoxicity

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.

Immunohistochemical localisation of PDE5 in Leydig and myoid cells of prepuberal and adult rat testis

Expression of phosphodiesterase 5 (PDE5) in the rat testis at several pre and postnatal developmental stages was investigated by immunohistochemistry. The enzyme was localised in vascular smooth muscle cells, as well as in Leydig and peritubular cells. The latter were identified as myoid, based on their immunoreactivity to desmin and alpha-smooth muscle actin. The presence of PDE5 in myoid cells was confirmed by Western blot analysis and immunohistochemistry performed on highly purified cell fractions, obtained from 16-day-old rats. The expression of PDE5 in these somatic cells of rat testis is discussed in view of the roles played by cGMP signal transduction pathways in the mammalian male reproductive function.

Testicular expression of small ubiquitin-related modifier-1 (SUMO-1) supports multiple roles in spermatogenesis: Silencing of sex chromosomes in spermatocytes, spermatid microtubule nucleation, and nuclear reshaping

SUMO-1 is a member of a ubiquitin-related family of proteins that mediates important post-translational effects affecting diverse physiological functions. Whereas SUMO-1 is detected in the testis, little is known about its reproductive role in males. Herein, cell-specific SUMO-1 was localized in freshly isolated, purified male germ cells and somatic cells of mouse and rat testes using Western analysis, high-resolution single-cell bioimaging, and in situ confocal microscopy of seminiferous tubules. During germ cell development, SUMO-1 was observed at low but detectable levels in the cytoplasm of spermatogonia and early spermatocytes. SUMO-1 appeared on gonosomal chromatin during zygotene when chromosome homologues pair and sex chromatin condensation is initiated. Striking SUMO-1 increases in the sex body of early-to-mid-pachytene spermatocytes correlated with timing of additional sex chromosome condensation. Before the completion of the first meiotic division, SUMO-1 disappeared from the sex body when X and Y chromosomal activity resumed. Together, these data indicate that sumoylation may be involved in non-homologous chromosomal synapsis, meiotic sex chromosome inactivation, and XY body formation. During spermiogenesis, SUMO-1 localized in chromocenters of certain round spermatids and perinuclear ring and centrosomes of elongating spermatids, data implicating SUMO-1 in the process of microtubule nucleation and nuclear reshaping. STAT-4, one potential target of sumoylation, was located along the spermatid nuclei, adjacent but not co-localized with SUMO-1. Androgen receptor-positive Leydig, Sertoli, and some peritubular myoepithelial cells express SUMO-1, findings suggesting a role in modulating steroid action. Testicular SUMO-1 expression supports its specific functions in inactivation of sex chromosomes during meiosis, spermatid microtubule nucleation, nuclear reshaping, and gene expression.

Stage‐ and Cell‐Specific Expression of Soluble Guanylyl Cyclase Alpha and Beta Subunits, cGMP‐Dependent Protein Kinase I Alpha and Beta, and Cyclic Nucleotide‐Gated Channel Subunit 1 in the Rat Testis

Several studies suggest that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) modulate testicular function. In this study, we examined the expression of cGMP-dependent protein kinase G-I (PKG-I), and cyclic nucleotide-gated channel 1 (CNG-1), 2 known mediators of cGMP action, and the expression of soluble guanylyl cyclase (sGC) subunits in the rat testis. Immunohistochemical analysis revealed that the alpha subunit of sGC was expressed in the blood vessels and Leydig cells of adult rat testes. In addition, the sGC alpha subunit was observed in the acrosomal structures of spermatids undergoing the middle and later stages of spermiogenesis, but not in mature spermatozoa. Similar localization and expression patterns were seen for the sGC beta subunit, indicating coexpression of the sGC subunits. PKG-I was expressed in blood vessels and in the acrosomal region of spermatids during the early and middle stages of spermiogenesis but was not observed in Leydig cells or in mature spermatozoa. In contrast to sGC and PKG-I, CNG-1 was expressed only in cytoplasm and the residual bodies of late-stage (17-19) spermatids, with no staining observed in blood vessels and Leydig cells. These results demonstrate that sGC, PKG-I, and CNG-1 are expressed in a stage- and cell-specific manner in the rat testis. The distinct temporal patterns of expression of these components of cGMP signaling pathways suggest different physiological roles for sGC, PKG-I, and CNG-1 in spermiogenesis and steroidogenesis.

289. Comparison of lymphocyte subsets and function in the rat and mouse testis

Testicular leukocytes are assumed to be involved in immunological surveillance against infection and tumours as well as regulation of local immune responses. They are implicated in mechanisms that make the testis a successful site for tissue transplantation in both rats and mice. Our previous studies using multi-colour fluorescence flow cytometric analysis to examine isolated testicular leukocytes in the rat testis have established the existence of a significant population of predominantly CD8+ T cells and a comparable number of lymphocytes expressing natural killer (NK) cell markers (NK and NKT cells). The functional activity of these testicular NK and NKT cells subsequently has been confirmed by a standard flow cytometric cytotoxicity assay using an NK-sensitive tumour cell line (YAC-1) and an NKT-sensitive tumour cell line (U937). Similar analyses of mouse testicular leukocytes have shown a slightly different pattern. The data indicate that mouse testicular lymphocytes comprise T cells, NK cells, and NKT cells, similar to the rat testis. However, while the apparent numerical densities of T cells in rat and mouse testes were similar, the numbers of NK and NKT cells were considerably lower in the mouse. Mouse testicular NKT cells were positive for staining with the tetramer CD1d/αGC, which is used to identify classical NKT cells, whereas rat NKT cells did not stain for this marker. Moreover, the CD8/CD4 T cell ratio in the mouse testis displayed a skewing towards the CD4+ subset. These data highlight the possibility that the immunological environment, and hence the course of immunological events, might be quite different in the testes of the two species. The reasons for these differences are not clear, however they should be taken into account when considering studies of testicular immune processes. Finally, comparative studies of immunological process in the testes of rats and mice may be very informative.

Enhanced Expression of Tyrosine Kinase Receptor A in Germ Cells of Rat Testis with Acute Experimental Testicular Torsion

Objective and Methods: To examine the involvement of neurotrophic factor receptors in the testis with acute experimental testicular torsion, the expression of tyrosine kinase receptors (trk) A and B, and p75 nerve growth factor receptor (NGFR) were studied in the rat testis with ischemia/reperfusion (I/R) injury, using Western blotting and immunohistochemistry. Apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Results: There was a significant increase in TUNEL-positive reaction in spermatogonia in the seminiferous tubules in rat testes after testicular torsion. Western blot analysis showed that trk A expression reached a significant peak at 12 h after reperfusion (p < 0.01), as compared to sham-operated controls, whereas trk B was not increased in the testis after I/R. Constitutive expression of p75 NGFR was at or below the level detectable by Western blot analysis, and it remained unchanged in the testis after I/R. Immunohistochemistry demonstrated that after I/R trk A expression was increased in spermatocytes and spermatids in the seminiferous tubules, in contrast to the basal location of the TUNEL-positive reaction. Immunoreactivity of trk B was seen mainly in the interstitial cells in the sham-operated testis, and its localization was not changed after I/R. Conclusion: It is postulated that trk A and B, but not p75 NGFR, are involved differently in the survival of testicular cells during acute experimental testicular torsion. In particular, increased trk A seems to be related to germ cell survival following I/R.