Interstitial Leydig Cells Research Articles

The adiponectin paralog C1q/TNF-related protein 3 (CTRP3) stimulates testosterone production through the cAMP/PKA signaling pathway

CTRP3, a paralog of adiponectin, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. It is expressed at high levels in adipose tissue and has recently emerged as a novel adipokine. In the present study, we provide the first evidence for a physiological role of the new adipokine, CTRP3, in the reproductive system. CTRP3 was specifically expressed in interstitial Leydig cells, where testosterone is produced, in the adult mouse testis. CTRP3 increased testosterone production by TM3 mouse Leydig cells in a dose-dependent manner. The increased testosterone production was linked to upregulation of steroidogenic proteins expression, such as steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage cytochrome P450 (P450scc). Moreover, increases in intracellular cyclic AMP (cAMP) concentrations and the phosphorylation of cAMP-response element binding protein (CREB) in CTRP3-stimulated TM3 Leydig cells were observed. Inhibition of this signaling pathway by a specific protein kinase A (PKA) inhibitor, H89, blocked testosterone production in CTRP3-stimulated Leydig cells, suggesting that the stimulatory effect of CTRP3 on testosterone production is associated with activation of the cAMP/PKA signaling pathway. Thus, our results demonstrate a physiological role for CTRP3 in testicular steroidogenesis and provide novel insights in the intracellular mechanisms activated by this protein.

Cellular Distribution, Regulated Expression, and Functional Role of the Anorexigenic Peptide, NUCB2/Nesfatin-1, in the Testis

Nesfatin-1, product of the precursor NEFA/nucleobindin2 (NUCB2), was initially identified as anorectic hypothalamic neuropeptide, acting in a leptin-independent manner. In addition to its central role in the control of energy homeostasis, evidence has mounted recently that nesfatin-1 is also produced in peripheral metabolic tissues, such as pancreas, adipose, and gut. Moreover, nesfatin-1 has been shown to participate in the control of body functions gated by whole-body energy homeostasis, including puberty onset. Yet, whether, as is the case for other metabolic neuropeptides, NUCB2/nesfatin-1 participates in the direct control of gonadal function remains unexplored. We document here for the first time the expression of NUCB2 mRNA in rat, mouse, and human testes, where NUCB2/nesfatin-1 protein was identified in interstitial mature Leydig cells. Yet in rats, NUCB2/nesfatin-1 became expressed in Sertoli cells upon Leydig cell elimination and was also detected in Leydig cell progenitors. Although NUCB2 mRNA levels did not overtly change in rat testis during pubertal maturation and after short-term fasting, NUCB2/nesfatin-1 content significantly increased along the puberty-to-adult transition and was markedly suppressed after fasting. In addition, testicular NUCB2/nesfatin-1 expression was up-regulated by pituitary LH, because hypophysectomy decreased, whereas human choriogonadotropin (super-agonist of LH receptors) replacement enhanced, NUCB2/nesfatin-1 mRNA and peptide levels. Finally, nesfatin-1 increased human choriogonadotropin-stimulated testosterone secretion by rat testicular explants ex vivo. Our data are the first to disclose the presence and functional role of NUCB2/nesfatin-1 in the testis, where its expression is regulated by developmental, metabolic, and hormonal cues as well as by Leydig cell-derived factors. Our observations expand the reproductive dimension of nesfatin-1, which may operate directly at the testicular level to link energy homeostasis, puberty onset, and gonadal function.

Evidence for Almost Complete Sex-reversal in Bovine Freemartin Gonads: Formation of Seminiferous Tubule-like Structures and Transdifferentiation into Typical Testicular Cell Types

During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubule-like structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3β-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.

Evaluation of CAG repeat length of androgen receptor expressing cells in human testes showing different pictures of spermatogenic impairment

The androgen receptor (AR) is a ligand-activated transcriptional factor with crucial importance for spermatogenesis. Its transactivation domain consists of a polymorphic sequence of 9-36 cytosin-adenin-guanin (CAG) repeats. Within the physiological range an increased CAG repeat length is assumed to correlate with the reduced androgen sensitivity resulting in impaired spermatogenesis. In 33 testes of 32 patients showing different histological pictures ranging from normal spermatogenesis, hypospermatogenesis to severe spermatogenetic impairment such as maturation arrest, Sertoli cell only Syndrome (SCO) and mixed atrophy, CAG repeat length was assessed in lymphocyte DNA, DNA/mRNA from testis homogenate and in mRNA of AR expressing Sertoli cells within the seminiferous tubules, and interstitial Leydig cells collected by the laser-assisted cell picking. The latter examination was performed to detect a possible somatic mosaicism of CAG repeat length in different testicular cell populations. CAG repeat lengths varied from 12 to 27 repeats, i.e., within the physiological range. We found deviating CAG repeat numbers in different fractions of AR expressing Sertoli and Leydig cells indicating tissue heterogeneity. We did not find a correlation of CAG repeat length to testicular histology or AR expression, and testosterone or luteinizing hormone levels even in biopsies showing mixed atrophy. Additionally, we evaluated the expression pattern of the AR-dependent gene androgen binding protein (ABP), and did not find a correlation to CAG repeat, but a significant reduction of ABP mRNA related to severe spermatogenic impairment in the monomorphic histologies. These data suggest other factors than CAG repeat to be responsible for severe spermatogenic impairment including mixed atrophy.

Expression of Cytokeratin 10 Protein in the Human Testis Showing Normal and Abnormal Spermatogenesis

Background: CK10 is a heterotetramer of type I and two type II keratins. Aim: This study examines the expression pattern of cytokeratin 10 (CK10) in human testis. Materials and methods: CK10 protein expression was examined using immunofluorescense staining methods in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) obtained from patients undergoing investigations for infertility. Results: In the testis showing normal spermatogenesis, CK10 was expressed in the interstitium and in the seminiferous tubules. A strong cytoplasmic expression was seen in the Leydig cells, Sertoli cells, and spermatocytes. In testes showing spermatogenic arrest, weak CK10 protein expression was observed both in the interstitium and seminiferous tubules (some primary spermatocytes). In the testes showing Sertoli cell only syndrome, negligible CK10 staining was seen both in the seminiferous tubules and in the interstitial cells of Leydig. Conclusions: To the authors’ knowledge, this is the first study indicating CK10 expression in the human testis during normal and abnormal spermatogenesis. The varied expression of CK10 in testes showing abnormal spermatogenesis suggests its possible involvement in this process.

Atrazine induces transcriptional changes in marker genes associated with steroidogenesis in primary cultures of rat Leydig cells

Reproductive toxicity of atrazine (ATZ) is well reported in mammals. However, the underlying mechanisms are poorly understood and need to be explored. Thus, we investigate ATZ induced transcriptional changes in selected markers of steroidogenesis in primary cultures of rat interstitial Leydig cells (ILCs). Cytotoxic studies were carried out by exposing the cells to ATZ (0.5–50μg/mL or 2.32–232μM) for 24–72h, whereas; the exposure period of expression studies was for 2h. ATZ exposure of (25 and 50μg/ml) for 48h onwards was found to be cytotoxic in MTT (dimethylthiazol-diphenyl tetrazolium bromide salt) assay, while in NRU (neutral red uptake) assay, cytotoxicity could be recorded at 50μg/ml exposure of 72h only. A significant dose dependent induction in the levels of mRNA expression of genes of steroidogenic acute regulatory protein (STAR), cytochrome P45011A1, 3β-hydroxysteroid dehydrogenase (3β-HSD), and other steroidogenic proteins were observed in cells exposed to ATZ. Our data suggest the applicability of these selected marker genes of steroidogenesis as an indicator of short term exposure of ATZ induced testicular toxicity in rats interstitial Leydig cells (ILCs).

Kolaviron Protects Primary Cultures of Rat Leydig Cells from Atrazine Induced Damages

BACKGROUND: Atrazine (ATZ) induced testicular impairment has been reported in vivo in rats. The current study examined the cytotoxic effect of ATZ and the protective potentials of kolaviron (KV) in vitro employing interstitial Leydig cells (ILC) in culture. ILC isolated from juvenile rats were co-cultured with 232μM ATZ in the presence or absence of KV (60μM). Expression of steroidogenic acute regulatory proteins (StAR), cytochrome P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase-1(SOD-1) and superoxide dismutase-2(SOD-2) were monitored by real-time PCR following 6 h of treatment.

Histological and ultrastructural changes in the adult male albino rat testes following chronic crude garlic consumption

Several studies have presented Garlic (Allium sativum) as a restoring agent for testicular cells after exposure to different types of toxins, however, others have shown that it modified spermatogenesis and might have spermicidal effects. This work has been to assess the positive or negative effect of chronic crude garlic consumption on rat testes using light and transmission electron microscopy with reference to plasma testosterone and LH levels. Forty albino male rats were divided into group A (control) and group B (treated rats). Group B was further subdivided into B1, B2, and B3 subgroups which were administer crude garlic as 20% of their daily food for two, three and four months, respectively. The current study has been the first, to the best of our knowledge, to describe the apoptotic effect of chronic crude garlic consumption targeting Sertoli cells, germ cells and peritubular tissue including interstitial Leydig cells and myoid cells. This might be better explained by inflammatory than degenerative changes in the peritubular tissue and Leydig cells leading to a decrease in testosterone level. Consequently, Sertoli cells degenerate due to a decrease in testosterone and detachment from the basal lamina. Germ cells, which are completely dependent upon Sertoli cells and testosterone to complete there spermatogenesis will be affected. Testicular apoptosis with disruption in spermatogenesis following chronic crude garlic consumption could be correlated with two possible theories; being an anti-hypercholesterolemic agent, it might inhibit steroidogenesis resulting in a decrease in testosterone level and being one of the famous phytoestrogens it possibly has direct estrogen-like actions on adult male rat testes.

Activin A, a product of fetal Leydig cells, is a unique paracrine regulator of Sertoli cell proliferation and fetal testis cord expansion

Formation of tubular structures relies upon complex interactions between adjacent epithelium and mesenchyme. In the embryonic testes, dramatic compartmentalization leads to the formation of testis cords (epithelium) and the surrounding interstitium (mesenchyme). Sertoli cells, the epithelial cell type within testis cords, produce signaling molecules to orchestrate testis cord formation. The interstitial fetal Leydig cells, however, are thought only to masculinize the embryo and are not known to be involved in testis cord morphogenesis. Contrary to this notion, we have identified activin A, a member of the TGF-beta protein superfamily, as a product of the murine fetal Leydig cells that acts directly upon Sertoli cells to promote their proliferation during late embryogenesis. Genetic disruption of activin betaA, the gene encoding activin A, specifically in fetal Leydig cells resulted in a failure of fetal testis cord elongation and expansion due to decreased Sertoli cell proliferation. Conditional inactivation of Smad4, the central component of TGF-beta signaling, in Sertoli cells led to testis cord dysgenesis and proliferative defects similar to those of Leydig cell-specific activin betaA knockout testes. These results indicate that activin A is the major TGF-beta protein that acts directly on Sertoli cells. Testicular dysgenesis in activin betaA and Smad4 conditional knockout embryos persists into adulthood, leading to low sperm production and abnormal testicular histology. Our findings challenge the paradigm that fetal testis development is solely under the control of Sertoli cells, by uncovering an active and essential role of fetal Leydig cells during testis cord morphogenesis.

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.

46,XY DSD due to impaired androgen production

Disorders of androgen production can occur in all steps of testosterone biosynthesis and secretion carried out by the foetal Leydig cells as well as in the conversion of testosterone into dihydrotestosterone (DHT). The differentiation of Leydig cells from mesenchymal cells is the first walk for testosterone production. In 46,XY disorders of sex development (DSDs) due to Leydig cell hypoplasia, there is a failure in intrauterine and postnatal virilisation due to the paucity of interstitial Leydig cells to secrete testosterone. Enzymatic defects which impair the normal synthesis of testosterone from cholesterol and the conversion of testosterone to its active metabolite DHT are other causes of DSD due to impaired androgen production. Mutations in the genes that codify the enzymes acting in the steps from cholesterol to DHT have been identified in affected patients. Patients with 46,XY DSD secondary to defects in androgen production show a variable phenotype, strongly depending of the specific mutated gene. Often, these conditions are detected at birth due to the ambiguity of external genitalia but, in several patients, the extremely undervirilised genitalia postpone the diagnosis until late childhood or even adulthood. These patients should receive long-term care provided by multidisciplinary teams with experience in this clinical management.

Expression of prothymosin alpha in meiotic and post-meiotic germ cells during the first wave of rat spermatogenesis

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non-mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post-meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27 dpp (completed meiosis), 35 dpp (occurring spermiogenesis), and 60 dpp (first wave of spermatogenesis definitely ended). RT-PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60 dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27 dpp, while at 35 and 60 dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells.

Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia ( Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.

ATROFIA TESTICULAR EM Alouatta caraya MANTIDO EM CATIVEIRO: RELATO DE CASO

This study sought to assess anatomopathological aspects of a case of testicular atrophy in a specimen of Alouatta caraya kept in captivity at the Centro Nacional de Primatas - CENP/SVS/MS. Semen and biometrical exams were performed for the testicles. The results demonstrated that the animal presented azoospermia and in the biometric exam the right testicle presented an average volume of 0.438cm and the left one 0.407cm, values considerably below of those recorded in the literature for normal males of the species. Upon inspection, both testicles presented a firmer consistency that usual and the parenchyma cut was darker. The histopathological exam revealed a marked reduction in the number of seminiferous tubules; some were totally atrophied and hyalinized. In others there was an absence of the germinative epithelium and presence of vacuolized Sertoli cells while the interstitial (Leydig) cells were found proliferated. It is concluded that gonadopathy was the result of a degenerative process, with the lesion at a stage of chronicity. Keywords: Alouatta caraya, testicular atrophy, reproduction.

Posthatching development of Alligator mississippiensis ovary and testis.

We investigated ovary and testis development of Alligator mississippiensis during the first 5 months posthatch. To better describe follicle assembly and seminiferous cord development, we used histochemical techniques to detect carbohydrate-rich extracellular matrix components in 1-week, 1-month, 3-month, and 5-month-old gonads. We found profound morphological changes in both ovary and testis. During this time, oogenesis progressed up to diplotene arrest and meiotic germ cells increasingly interacted with follicular cells. Concomitant with follicles becoming invested with full complements of granulosa cells, a periodic acid Schiff's (PAS)-positive basement membrane formed. As follicles enlarged and thecal layers were observed, basement membranes and thecal compartments gained periodic acid-methionine silver (PAMS)-reactive fibers. The ovarian medulla increased first PAS- and then PAMS reactivity as it fragmented into wide lacunae lined with low cuboidal to squamous epithelia. During this same period, testicular germ cells found along the tubule margins were observed progressing from spermatogonia to round spermatids located within the center of tubules. Accompanying this meiotic development, interstitial Leydig cell clusters become more visible and testicular capsules thickened. During the observed testis development, the thickening tunica albuginea and widening interstitial tissues showed increasing PAS- and PAMS reactivity. We observed putative intersex structures in both ovary and testis. On the coelomic aspect of testes were cell clusters with germ cell morphology and at the posterior end of ovaries, we observed "medullary rests" resembling immature testis cords. We hypothesize laboratory conditions accelerated gonad maturation due to optimum conditions, including nutrients and temperature. Laboratory alligators grew more rapidly and with increased body conditions compared with previous measured, field-caught animals. Additionally, we predict the morphological maturation observed in these gonads is concomitant with increased endocrine activities.

In Utero Exposure to Di-(2-Ethylhexyl) Phthalate Decreases Mineralocorticoid Receptor Expression in the Adult Testis

In utero exposure to di-(2-ethylhexyl) phthalate (DEHP) has been shown to result in decreased androgen formation by fetal and adult rat testes. In the fetus, decreased androgen is accompanied by the reduced expression of steroidogenic enzymes. The mechanism by which in utero exposure results in reduced androgen formation in the adult, however, is unknown. We hypothesized that deregulation of the nuclear steroid receptors might explain the effects of in utero DEHP exposure on adult testosterone production. To test this hypothesis, pregnant Sprague Dawley dams were gavaged with 100–950 mg DEHP per kilogram per day from gestational d 14-19, and testes were collected at gestational d 20 and postnatal days (PND) 3, 21, and 60. Among the nuclear receptors studied, the mineralocorticoid receptor (MR) mRNA and protein levels were reduced in PND60 interstitial Leydig cells, accompanied by reduced mRNA expression of MR-regulated genes. Methylation-sensitive PCR showed effects on the nuclear receptor subfamilies NR3A and −3C, but only MR was affected at PND60. Pyrosequencing of two CpG islands within the MR gene promoter revealed a loss of methylation in DEHP-treated animals that was correlated with reduced MR. Because MR activation is known to stimulate Leydig cell testosterone formation, and MR inhibition to be repressive, our results are consistent with the hypothesis that in utero exposure to DEHP leads to MR dysfunction and thus to depressed testosterone production in the adult. We suggest that decreased MR, possibly epigenetically mediated, is a novel mechanism by which phthalates may affect diverse functions later in life.

In UteroExposure to Di-(2-Ethylhexyl) Phthalate Decreases Mineralocorticoid Receptor Expression in the Adult Testis

In utero exposure to di-(2-ethylhexyl) phthalate (DEHP) has been shown to result in decreased androgen formation by fetal and adult rat testes. In the fetus, decreased androgen is accompanied by the reduced expression of steroidogenic enzymes. The mechanism by which in utero exposure results in reduced androgen formation in the adult, however, is unknown. We hypothesized that deregulation of the nuclear steroid receptors might explain the effects of in utero DEHP exposure on adult testosterone production. To test this hypothesis, pregnant Sprague Dawley dams were gavaged with 100-950 mg DEHP per kilogram per day from gestational d 14-19, and testes were collected at gestational d 20 and postnatal days (PND) 3, 21, and 60. Among the nuclear receptors studied, the mineralocorticoid receptor (MR) mRNA and protein levels were reduced in PND60 interstitial Leydig cells, accompanied by reduced mRNA expression of MR-regulated genes. Methylation-sensitive PCR showed effects on the nuclear receptor subfamilies NR3A and -3C, but only MR was affected at PND60. Pyrosequencing of two CpG islands within the MR gene promoter revealed a loss of methylation in DEHP-treated animals that was correlated with reduced MR. Because MR activation is known to stimulate Leydig cell testosterone formation, and MR inhibition to be repressive, our results are consistent with the hypothesis that in utero exposure to DEHP leads to MR dysfunction and thus to depressed testosterone production in the adult. We suggest that decreased MR, possibly epigenetically mediated, is a novel mechanism by which phthalates may affect diverse functions later in life.

Do Microwaves of Mobile Phone Affect the Testicular Tissue Structure? (A histopathological and ultrastructural study)

Background: Some studies have reported that microwave radiation have adverse effects on reproduction. Therefore, the purpose of this study was to investigate the possible effects of mobile phone microwaves on the histological structure and ultrastructure of mice testis. Material: five adult male mice were subjected to the mobile phone microwaves (900- 1800 MHz) for 30 days, one hour / day. Their exposure resulted in many histopathological and ultrastructural changes. Results: The light microscopic examination of testis of irradiated animals revealed: disorganization of the germinal epithelia, the seminiferous tubules taking elliptical aspects with intratubular vacuolization, abnormal morphology of early and late spermatids, rupture of the boundary membrane of the seminiferous tubules, pyknotic germ cells, damaged and ill- defined interstitial cells. The semithin sections demonstrated an increased incidence of Sertoli cells but they are detached from the basal membrane, the pachytene spermatocytes appeared with abnormal faint heterochromatin and abnormal shapes of rounded and elongated spermatids and spermatozoa. The ultrastructural examination illustrated Sertoli cells with lobulated nuclei, ill-defined nucleoli and ruptured cell boundary. Pachytene spermatocytes appeared with low integrity and faint and dispersed chromatin material. Some of them were necrotic and others were apoptotic. Early (rounded) and late (elongated) spermatids appeared with vacuolated cytoplasm and deformed morphology. The interstitial cells of Leydig appeared smaller in size with more electron dense components, vacuolated cytoplasm, ruptured mitochondria and fragmented nuclei. Their neighbouring blood capillaries appeared congested with damaged lateral pericytes . Conclusion: The results of this study showed that 1h / day (30 consecutive days) exposure of 900-1800 MHz microwaves affected the histological structure of mice testis particularly the Leydig cells and recorded an apoptosis-inducing effect on the spermatogenic cells. Further investigations are required to confirm these findings and to clarify the mechanism of action of the applied microwave exposure on male reproductive organs, as well as to establish the biological significance of these observations.

The autonomous innervation of the buffalo (Bubalus bubalis) testis. An immunohistochemical study.

The innervation pattern in the buffalo testis was determined by using histochemical and immunohistochemical methods. Nerves were concentrated in the tunica albuginea and septula testis, and did not show an uniform distribution. The tunica albuginea at the lateral and medial sides and at the free border of the testis is most densely innervated than at the epididymal border. At the cranial pole thick nerve bundles were observed between albugineal vessels and muscle bundles. Rare parenchymal nerves were found in perivascular position between seminiferous tubules and their occurrence is confined to lobules at the cranial and caudal testicular poles. An intense NPY immunoreactivity occurred in nerve bundles and in solitary varicose fibres. Nerves were concentrated in the tunica albuginea at the lateral and medial side and at the free border of the testis, and in the lobules at the cranial and caudal testicular poles. Sub P immunoreactivity was occasionally detected in some thicker nerve bundles and solitary fibers, in the tunica albuginea and in the wall of blood vessels, showing a similar distribution but less intensity and density than NPY immunoreactivity. TH immunoreactivity stained nerve fibers in the buffalo testis with a distribution pattern similar to that obtained with general neuronal markers. The histochemical reaction for AchE was negative, so cholinergic fibers cannot be detected in the buffalo testis. The histochemical NADPHd reaction stained rare nitrergic nerve bundles and solitary fibers. The majority of NADPHd activity was confined to the vascular endothelium, and rarely to the interstitial Leydig cells, whereas the Sertoli and germ cells did not show any reaction.

Distribution of Lectin‐Bindings in the Testis of the Lesser Mouse Deer, Tragulus javanicus

The distribution of lectin bindings in the testis of the smallest ruminant, lesser mouse deer (Tragulus javanicus), was studied using 12 biotinylated lectins specific for d-galactose (peanut agglutinin PNA, Ricinus communis agglutinin RCA I), N-acetyl-d-galactosamine (Dolichos biflorus agglutinin DBA, Vicia villosa agglutinin VVA, Soybean agglutinin SBA), N-acetyl-D-glucosamine and sialic acid (wheat germ agglutinin WGA, s-WGA), D-mannose and d-glucose (Lens culinaris agglutinin LCA, Pisum sativum agglutinin PSA, Concanavalin A Con A), L-fucose (Ulex europaeus agglutinin UEA I), and oligosaccharide (Phaseolus vulgaris agglutinin PHA-E) sugar residues. In Golgi-, cap-, and acrosome-phase spermatids, lectin-bindings were found in the acrosome (PNA, RCA I, VVA, SBA, WGA and s-WGA), and in the cytoplasm (PNA, RCA I, VVA, SBA, WGA, LCA, PSA, Con A and PHA-E). s-WGA binding was confined to the spermatid acrosome, but other lectins were also observed in spermatocytes. In spermatogonia, VVA, WGA, Con A, and PHA-E bindings were observed. Sertoli cells were intensely stained with DBA and Con A, and weakly with PHA-E. In interstitial Leydig cells, RCA I, DBA, VVA, Con A, PSA, LCA, WGA and PHA-E were positive. UEA I was negative in all cell types including spermatogenic cells. Unusual distribution of lectin-bindings noted in the testis of lesser mouse deer included the limited distribution of s-WGA only in the spermatid acrosome, the distribution of DBA in Sertoli cells, Leydig cells and lamina propria, and the absence of UEA I in all type cells. The present results were discussed in comparison with those of other animals and their possible functional implications.