Arrest Of Spermatogenesis Research Articles

TBHP-induced oxidative stress alters microRNAs expression in mouse testis.

Reactive oxygen species (ROS) and oxidative stress is one of the main reasons of male infertility. MicroRNAs (miRNAs) regulate multiple intracellular processes. Alterations in miRNAs expression may occur in different conditions and diseases. In this study, the effect of oxidative stress induced by tertiary-butyl hydroperoxide (TBHP) on the expression of candidate miRNAs in mouse testis was investigated. After determining median lethal dose (LD50), TBHP was intraperitoneally (ip) injected at the dilution of 1:10 LD50 into the adult male mice for 2 weeks, and then testis tissues were removed in order to assay the ROS level. Total RNA was extracted and the expression of five miRNAs was quantified by reverse transcription-real time polymerase chain reaction (RT-qPCR). The flow cytometry analysis showed a significant increase in ROS level in testis. The expression of three out of five selected miRNAs, including miR-34a, miR-181b and miR-122a, showed some degrees of changes following exposure to oxidative stress. These miRNAs are involved in antioxidant responses, inflammation pathway and spermatogenesis arrest. In conclusion, TBHP alters the miRNA expression profile of testis which might play a potential role in oxidative and antioxidative responses and spermatogenesis.

Ghrelin improves rat sperm kinematic parameters during abdominal position of the testis

BACKGROUND: Disruption of testicular function and arrest ofspermatogenesis are the consequence of cryptorchidism inresponse to elevated temperature. OBJECTIVES:This investigationwas set to clarify the possible ghrelin efficacy in altering somesperm quality parameters upon experimentally-induced cryptorchidism.METHODS: Thirty male adult rats were scheduled for thestudy and were divided into three groups: group 1 was served ascontrol-saline (CS), group 2 was designed as cryptorchidism-saline(CrS), and group 3 was defined as cryptorchidism-ghrelin (CrG).After surgically inducing cryptorchidism in groups 2 and 3, theresearchers gave 10 nmol of ghrelin to CrG rats for 7 consecutivedays. Five animals in each group were equally killed on days 3 and7 after operation and their testes were taken for sperm evaluation.RESULTS: Testicular weight, sperm forward progressive motility(FPM), functional membrane integrity (assessed by HOS-test), andsperm concentration displayed slight changes after heating on day3. However, abdominal position of the testes for 7 days caused asignificant reduction in the percentages of HOS-positive cells(p 0.05).CONCLUSIONS: Indeed, this function of ghrelin could be attributedto its antioxidant properties and it may be implicated as a potentialagent in attenuation of impaired spermatogenesis after cryptorchidism.

Biochemical and histopathological evaluations of ghrelin effects following cadmium toxicity in the rat testis.

Numerous reports demonstrate that cadmium (Cd) induces oxidative stress by increasing lipid peroxidation and altering antioxidative enzymes status. Thirty male rats were subdivided into control-saline, Cd-saline and Cd-ghrelin groups. A single dose of Cd was injected to induce testicular injury and also ghrelin for 10 consecutive days to group 3. SOD activity decreased and lipid peroxidation increased by Cd administration. The mean activities of GPx and CAT as well as GSH content were lower in the Cd-saline rats; however, they did not statistically differ compared with the controls. Exposure to Cd resulted in complete degeneration of seminiferous tubules with severe depletion of germ cells and arrest in spermatogenesis. Notably, ghrelin treatment not only prevented reduction in SOD, GPx, CAT and GSH level, but also increased enzyme activities form their normal values. Moreover, TBARS concentration was significantly reduced by ghrelin administration. Furthermore, ghrelin pre-treatment resulted in partial but not significant prevention in testicular histopathological features damaged by Cd. In conclusion, the obtained results indicate for the first time the novel evidences of ghrelin ability in promotion of antioxidant enzyme activities and reduction of lipid peroxidation following Cd-induced oxidative stress in the rat testis. These observations also demonstrate that ghrelin may be considered as promising antioxidant agent in prevention and attenuation of testicular injury upon Cd toxicity.

The low expression of Dmrt7 is associated with spermatogenic arrest in cattle-yak.

Dmrt7 is a member of the DM domain family of genes. Dmrt7 deficiency is also a strong candidate as a cause for male cattle-yak infertility, as it is regarded as essential for male spermatogenesis, between the pachynema and diplonema stages. In our study, the coding region sequence of yak and cattle-yak Dmrt7 was cloned by molecular cloning techniques, and the sequence, conserved domains, functional sites, and secondary and tertiary structures of the Dmrt7-encoded protein were predicted and analyzed using bioinformatics methods. The coding region sequences of the Dmrt7 gene, encoding 370 amino acids, were consistent in yak and cattle-yak. The protein encoded by yak and cattle-yak Dmrt7 contains a DM domain. We detected Dmrt7 mRNA expression in testis, but not in any other tissue. Dmrt7 mRNA and protein expression was significantly higher in testis of cattle and yak than that in cattle-yak (p<0.01). Histological analysis indicated that seminiferous tubules in male cattle-yak were highly vacuolated and contained primarily Sertoli cells and spermatogonia, while those of cattle and yak contained abundant primary spermatocytes. Male cattle-yak testis contained a significantly larger number of apoptotic cells than those in cattle and yak assessed by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) analysis. The accumulation of SCP3-positive spermatocytes indicated the arrest of spermatogenesis at the pachynema stage in the cattle-yak. These results suggest low levels of Dmrt7 expression lead to male sterility in cattle-yak. The molecular function of Dmrt7 and the regulation of its expression warrant need to be examined in future studies.

N-wasp is required for structural integrity of the blood-testis barrier.

During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties. Here we demonstrate that the actin nucleation promoting factor Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) provides an essential function necessary for BTB restructuring, and for maintaining spermatogenesis. Our data suggests that the N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling. These arrays are proposed to mediate the restructuring process through endocytic recycling of BTB components. Disruption of N-WASP in Sertoli cells results in major structural abnormalities to the BTB, including mis-localization of critical junctional and cytoskeletal elements, and leads to disruption of barrier function. These impairments result in a complete arrest of spermatogenesis, underscoring the critical involvement of the somatic compartment of the seminiferous tubules in germ cell maturation.

Association of cellular and molecular alterations in Leydig cells with apoptotic changes in germ cells from testes of Graomys griseoflavus × Graomys centralis male hybrids

Spermatogenesis is disrupted in Graomys griseoflavus×Graomys centralis male hybrids. This study was aimed to determine whether morphological alterations in Leydig cells from hybrids accompany the arrest of spermatogenesis and cell death of germ cells and whether apoptotic pathways are also involved in the response of these interstitial cells. We used three groups of 1-, 2- and 3-month-old male animals: (1) G. centralis, (2) G. griseoflavus and (3) hybrids obtained by crossing G. griseoflavus females with G. centralis males. Testicular ultrastructure was analyzed by transmission electron microscopy. TUNEL was studied using an in situ cell death detection kit and the expression of apoptotic molecules by immunohistochemistry. The data confirmed arrest of spermatogenesis and intense apoptotic processes of germ cells in hybrids. These animals also showed ultrastructural alterations in the Leydig cells. Fas, FasL and calbindin D28k overexpression without an increase in DNA fragmentation was detected in the Leydig cells from hybrids. In conclusion, the sterility of Graomys hybrids occurs with ultrastructural changes in germ and Leydig cells. The enhancement of Fas and FasL is not associated with cell death in the Leydig cells. Probably the apoptosis in these interstitial cells is inhibited by the high expression of the antiapoptotic molecule calbindin D28k.

Suppression of fertility in adult cats.

Cats are animals with highly efficient reproduction, clearly pointing to a need for suppression of fertility. Although surgical contraception is highly effective, it is not always the method of choice. This is predominantly because it is cost-intensive, time-consuming and irreversible, with the latter being of major importance for cat breeders. This article reviews the use of progestins, scleroting agents, immunocontraception, melatonin, GnRH antagonists and finally, GnRH agonists, in adult male and female cats in detail, according to the present state of the art. By now, various scientific and clinical options are available for the suppression of fertility in adult cats and the decision as to which should be chosen - independent of the legal registration of any state - depends on different facts: (i) feral or privately owned animal? (ii) temporary or permanent suppression of fertility wanted/needed? (iii) sex of the animal? New effective and available methods for hormonal contraception include melatonin implants for short-term post ponement of oestrus in adult queens and slow-release GnRH-agonist implants containing deslorelin (Suprelorin(®) ) for short- and long-term contraception in male and female companion and breeding cats.

Comparative Transcriptome Analysis of Spermatogenesis Arrest in Cattle-Yak

The male infertility of cattle-yak, which is the hybrid offspring of cattle and yak, is a difficult problem for cross-breeding and improvement of yak. In this study, we used high-throughput sequencing technology to obtain the transcriptomes of the testicular tissues of both cattle-yak and yak, and investigated the expression profiles of the genes regulating hormones, spermatogenesis and apoptosis. The results showed that, there were 17784 and 18529 genes expressed in cattle-yak and yak testicular tissues respectively, and statistical analysis revealed 5000 genes with increased mRNA levels and 4089 with reduced levels in cattle-yak testicular tissue as compared with those in yak. The genes involved in testosterone synthesis and inhibin alpha chain have a high level of expression in cattle-yak testicular tissue. The former genes with increased expression may induce the secretion of intratesticular testosterone which can be up-regulated expression of the latter. Inhibin alpha chain with increased mRNA levels possible restrain the secretion of follicle-stimulating hormone of the pituitary gland, but not of luteinizing hormone. This may cause imbalance or deficiency of hormones in cattle-yak which lead to male infertility. By comparing the expression of the testicular marker genes between the two species, we showed that expressions of the marker genes of spermatogonial stem cell, sertoli cell, Leydig cell and myoid cell (fibrosis of testis) are increased, whereas those of differentiation of SSCs (especially those expressed at late stage of spermatid formation) are reduced. The abnormality of self-renewal and differentiation of SSCs, which may be tightly associated with the disorder of retinoic acid signaling pathway, might lead to spermatogenesis dysfunction in cattle-yak. Syce3 , Fkbp6 and Dmrt7 are down-regulated significantly in cattle-yak testicular tissue, which may be related with the formation of synaptonemal complex between homologous chromosomes, especially between sex chromosomes. Spo11 and Dmc1 are involved in double strand break and homologous repair process respectively, and are down-regulated in cattle-yak resulting in reduced numbers of synaptonemal complex and impaired homologous repair respectively. Tnp2 , Hmgb4 and H1fnt that are involved in formation of concentrated nucleus are hardly transcriptionally active, which confirmed the finding that germocyte of cattle-yak arrest at round-spermatid stage. This may be caused by suppressed expression of Crem , GRTH/DDX25 in cattle-yak. Proapoptosis genes, such as p53 , TNF- α , Trail , Bmp8b , Bax , Caspase-3 , Caspase-6 and Caspase-7 are up-regulated, and apoptosis genes, such as survivin , Bcl-2 are down-regulated, suggesting increased cell death occurs in cattle-yak testicular tissue. Together, our analysis of transcriptomes of the testicular tissues of cattle-yak and yak moves a step toward revealing the mechanism of cattle-yak male infertility and provides the theoretical basis for cross-breeding and improvement of yak.

Ultrasonographic caput epididymis diameter is reduced in non-obstructive azoospermia compared with normozoospermia but is not predictive for successful sperm retrieval after TESE.

Is the ultrasonographic determination of the caput epididymis diameter predictive for sperm retrieval after testicular sperm extraction (TESE) in non-obstructive azoospermia (NOA)? Ultrasonographic determination of the caput epididymis diameter did not give any relevant clinical information in NOA and was not predictive for positive sperm retrieval after TESE. The diameter of the caput epididymis in ultrasonography (US) has a diagnostic relevance in azoospermic men to correctly identify obstructive azoospermia; however, its clinical value in NOA is not yet determined. We performed a retrospective study of 100 azoospermic and 160 normozoospermic men attending a university infertility clinic. Participants were submitted to scrotal US to determine the mean value of bilateral testicular volumes (ml), the bilateral longitudinal caput diameter (mm) and the antero-posterior diameter of the corpus (mm) epididymis. The number of spermatozoa retrieved after TESE and the testicular histology of azoospermic men was obtained and the percentage of seminiferous tubules with elongated spermatids (%T) was used to classify cases with normal spermatogenesis (obstructive azoospermia) (OA) (n = 20; %T ≥ 80) or with NOA (n = 80; %T < 70). The US testes volumes and caput diameters were reduced (P < 0.05) in NOA compared with OA and with normozoospermia, but the corpus values were not different. The caput diameter in the side submitted to biopsy was significantly reduced when germinal epithelium was absent (Sertoli cell only) (P < 0.05) and the lowest value of caput diameter was observed when the seminiferous epithelium and tubule lumen were absent (testicular hyalinosis). On the contrary, a total arrest of spermatogenesis at the first meiosis level, or a defect of spermiogenesis resulting in scattered elongated spermatids in each tubule, did not show a reduced diameter of caput epididymis compared with normozoospermia. The caput diameter did not show any difference between NOA patients with or without successful sperm retrieval at TESE. On the contrary testicular volume was significantly reduced in NOA patients with no sperm retrieval (P = 0.0037). The caput diameter was not correlated with the number of retrieved sperm, the serum level of follicle stimulating hormone, or with the percentage of tubules with elongated spermatids at histological analysis. The aetiology of NOA was not included in the statistical analysis due to the low rate of cases with a specific aetiology for a testicular failure. Larger studies should exclude the possibility that besides testicular histology, aetiology of NOA might influence the diameter of caput epididymis. Moreover, whether a reduced diameter of caput epididymis is only a result of a testicular pathologic phenotype or whether it may underscore a primitive dysfunction influencing the number of ejaculated spermatozoa is not yet determined. We reported that US diameter of the caput epididymis is reduced in cases of NOA but, in contrast with the testicular volume, it is independent of the completion of spermatogenesis and subsequent presence of spermatozoa in the epididymis. Therefore ultrasonographic determination of caput epididymis diameter is not predictive for positive sperm retrieval after TESE in cases of a primitive testicular failure. Our novel findings may help to define which reproducible parameters of scrotal US should be assessed in the work-up of male infertility. This work was supported by the Ministero dell'Università e Ricerca (I) PRIN 2009. The authors declare no competing interest.

Pathological changes of testicular tissue in normal adult mice: A retrospective analysis

Mouse testicular experimental models are widely used in the study of andrology, reproductive toxicology and pharmacology. Under physiological conditions, a normal adult mouse is usually considered to have normal testes. However, whether normal adult mouse testes exhibit pathological changes has not been evaluated. The objective of this study was to investigate the pathological changes of testicular tissues in normal adult mice. A retrospective analysis of 720 adult male Kunming mice, used in previous studies as controls, was performed. Bilateral testicular tissues were stained with hematoxylin and eosin for pathological examinations. Among the 720 mice, nine had abnormal testes, an incidence of 1.3%. The nine mice with abnormal testes included two with microrchidia (22.2%) while the others had a normal testicular size. The observed pathological changes associated with microrchidia were seminiferous epithelial vacuolation, spermatogenesis arrest at the spermatocyte stage and the absence of sperm in all tubules. In other abnormal testes, pathological alterations included seminiferous epithelial vacuolation, severe hypospermatogenesis and symplasts composed of collapsed spermatids in tubules. The results demonstrate that normal adult male mice exhibit testicular pathological changes. Therefore, the possibility of abnormal testes in normal adult mice must be considered when using mice to establish a testicular experimental model.

Histological and histometric study of testis in albino rats treated with amlodipine

Amlodipine is the most common drug of choice to treat hypertension, one of its side effects is infertility and its effect on the testis of male albino rats is not well documented. Aim: To observe the effect of amlodipine in testis of male albino rats by the histological and histometric method . Materials& Method: we selected 12 adult male albino rats divided into 2 groups, group 1 treated as control group 2 treated as experiment and amlodipine is administered for 30 days. After 30 days testis were removed and analysed histologically and histometrically. Result: Though there are no marked changes, but early degenerative changes and reduction in weight of testis of experimental rats observed. Conclusion: Presence of vacuolated spermatogenic cells in some of the seminiferous tubules indicates early degeneration and ar rest of spermatogenesis.

Effects of Dietary Gossypol on Testicular Histology and Ultrasonograms of Yankasa Rams

This study evaluated the effect of dietary gossypol from whole cottonseed (WCS) and cottonseed cake (CSC) on testicular morphology of Yankasa rams. Fifteen (15) Yankasa rams weighing an average of 20 2 Kg were randomly distributed into three groups (A, B and C) of five rams each and were fed diets containing 48 % WCS, 48 % CSC and control diet (gossypol-free), respectively for 14 weeks. . An ultrasonographic examination of the testes of each ram was done every month for 3 months. At the end of the feeding period, the rams were castrated and histopathogical examination of the testicular tissues (n = 6). Histopathological changes observed in the testes of the rams in the experimental group were depletion of spermatogenic cell layers and arrest of spermatogenesis at the spermatocyte and spermatids levels and an exfoliation of the spermatogonia. There was vacuolation of the germ layers in the seminiferous tubules. These changes were more severe in group B (CSC) than A (WCS) and were indicative of testicular degeneration. Ultrasonograms of the testis of rams from experimental groups showed loss of homogeneity and an increased hyperechoic appearance which were also indicative of testicular degeneration. It was concluded that gossypol from whole cottonseed and cottonseed cake has detrimental effects on testicular histology and the effect on testicular histology was more severe in rams that were fed cottonseed cake.

Paracrystalline and crystalline inclusions of the human testis in cases of subfertility

Testicular crystalline inclusions, namely Charcot-Bottcher and Spangaro crystals of Sertoli cells, Lubarsch crystals of spermatogonia and Reinke crystals of Leydig cells, have been considered normal ultrastructural features of the post-pubertal human testis (Reinke, 1896; Chemes et al., 1977; Kaya et al., 1985). Nevertheless, their significance is not known. We have noted that these structures are dynamic and may disappear and reappear in some cases of subfertility. In particular, patients showing spermatogenesis arrest or germ cell aplasia usually do not show any crystalline inclusions. We study herein using transmission electron microscopy testicular biopsies of young infertile men (idiopathic infertility or varicocele) showing intracytoplasmic crystalline and/or paracrystalline inclusions. Sertoli cells’ paracrystalline inclusions consist of closely packed electron-dense longitudinal fibrils, sometimes including a granular and light core. Coarse ca. 5-25 μm long and 2-3 μm thick bundles composed of 5 or 10 nm thick filaments mainly locate in the basal cytoplasm near the nucleus. Reinke’s crystals, in turn, appear as variable-sized (2-5 μm long) polyhedral crystals with a honeycomb lattice and sharp edges consisting of 5-10 nm filaments filling wide areas of the cytoplasm. Alternatively, they may appear as filamentous/tubular, electron dense (0,5 μm long) units. In both cases they associate to mitochondria and dilated smooth endoplasmic reticulum. Due to their structure, whether these inclusions arise or not within the nucleus and are somehow transported to the cytoplasm is not yet clear. Although their exact molecular composition remains to be discovered, they are not likely to be associated to steroidogenesis but rather they may represent a kind of protein deposit.

Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis

Sulfated steroid hormones are commonly considered to be biologically inactive metabolites, but may be reactivated by the steroid sulfatase into biologically active free steroids, thereby having regulatory function via nuclear androgen and estrogen receptors which are widespread in the testis. However, a prerequisite for this mode of action would be a carrier-mediated import of the hydrophilic steroid sulfate molecules into specific target cells in reproductive tissues such as the testis. In the present study we detected predominant expression of the Sodium-dependent Organic Anion Transporter (SOAT), the Organic Anion Transporting Polypeptide 6A1, and the Organic Solute Carrier Partner 1 in human testis biopsies. All of these showed significantly lower or even absent mRNA expression in severe disorders of spermatogenesis (arrest at the level of spermatocytes or spermatogonia, Sertoli cell only syndrome). Only SOAT was significantly lower expressed in biopsies showing hypospermatogenesis. By use of immunohistochemistry SOAT was localized to germ cells at various stages in human testis biopsies showing normal spermatogenesis. SOAT immunoreactivity was detected in zygotene primary spermatocytes of stage V, pachytene spermatocytes of all stages (I–V), secondary spermatocytes of stage VI, and round spermatids (step 1 and step 2) in stages I and II. Furthermore, SOAT transport function for steroid sulfates was analyzed with a novel liquid chromatography tandem mass spectrometry procedure capable of profiling steroid sulfate molecules from cell lysates. With this technique, the cellular inward-directed SOAT transport was verified for the established substrates dehydroepiandrosterone sulfate and estrone-3-sulfate. Additionally, β-estradiol-3-sulfate and androstenediol-3-sulfate were identified as novel SOAT substrates.

Biosynthesis and biological function of sulfoglycolipids

Sulfation confers negative charge on glycolipids and the attached sulfate group presents a part of determinants for the molecular interactions. Mammalian sulfoglycolipids are comprised of two major members, sulfatide (SO3-3Gal-ceramide) and seminolipid (SO3-3Gal-alkylacylglycerol). Sulfatide is abundant in the myelin sheath and seminolipid is unique to the spermatogenic cells. The carbohydrate moiety of sulfatide and seminolipid is biosynthesized via sequential reactions catalyzed by common enzymes: ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST). To elucidate the biological function of sulfoglycolipids, we have purified CST, cloned the CST gene, and generated CST-knockout mice. CST-null mice completely lack sulfoglycolipids all over the body. CST-null mice manifest some neurological disorders due to myelin dysfunction, an aberrant enhancement of oligodendrocyte terminal differentiation, and an arrest of spermatogenesis. CST-deficiency ameliorates L-selectin-dependent monocyte infiltration in the renal interstitial inflammation, indicating that sulfatide is an endogenous ligand of L-selectin. Studies on the molecular mechanisms underlying the biological events for which sulfoglycolipids are essential are ongoing.

Histopathological alterations in testicular tissue of male rats exposed to arsenic

The present study was designed to investigate the adverse effect of arsenic on testicular tissue of Swiss albino male rats. Sodium arsenite was administered to adult male rats by gavage at the doses 1, 2 and 3 mg/kg body weight for 30 days. After the treatment, the testis were processed for histopathological observations. Sodium arsenite caused remarkable reduction in testicular weight (P&lt;0.05), while the body weight of experimental animals were reduced but not significantly (P&lt;0.05). Histological evaluation revealed dose-dependent, gradual destruction in histoarchitecture of testicular tissue. Sodium arsenite exposure caused complete arrest of spermatogenesis with disfigured seminiferous tubules in the testes .The lumens of the tubules were devoid of spermatids and were in places filled with cellular debris. The germinal epithelium was distorted. At places interstitial odema was also evident. Sertoli and Leydig cells were damaged. Along with structural alterations, fertility rate in experimental animals was significantly decreased at higher doses i.e. 2 and 3 mg/kg, as 100% infertility was observed. After withdrawal of the treatment over a period of 30 days, recovery was observed in low dose groups as few female rats became pregnant. The study concluded that exposure of arsenic causes testicular toxicity in male albino rat.

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

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

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice

SummaryIt is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty.

Circadian Proteins CLOCK and BMAL1 in the Chromatoid Body, a RNA Processing Granule of Male Germ Cells

Spermatogenesis is a complex differentiation process that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells. RNA nuclear and cytoplasmic bodies appear to be critical for the progress of spermatogenesis. The chromatoid body (CB) is a cytoplasmic organelle playing an important role in RNA post-transcriptional and translation regulation during the late steps of germ cell differentiation. The CB is also important for fertility determination since mutations of genes encoding its components cause infertility by spermatogenesis arrest. Targeted ablation of the Bmal1 and Clock genes, which encode central regulators of the circadian clock also result in fertility defects caused by problems other than spermatogenesis alterations. We show that the circadian proteins CLOCK and BMAL1 are localized in the CB in a stage-specific manner of germ cells. Both BMAL1 and CLOCK proteins physically interact with the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog), a hallmark component of the CB. BMAL1 is differentially expressed during the spermatogenic cycle of seminiferous tubules, and Bmal1 and Clock deficient mice display significant CB morphological alterations due to BMAL1 ablation or low expression. These findings suggest that both BMAL1 and CLOCK contribute to CB assembly and physiology, raising questions on the role of the circadian clock in reproduction and on the molecular function that CLOCK and BMAL1 could potentially have in the CB assembly and physiology.

Abnormalities of meiotic recombination in Han Chinese azoospermic patients

To analyze defective homologous chromosomal recombination in Han Chinese azoospermic patients. Testicular biopsy samples from 7 healthy controls and 7 Han Chinese azoospermic patients including 2 obstructive azoospermia (OA group) and 5 non-obstructive azoospermia (NOA group) were analyzed. Immunofluorescence staining was performed to categorize early stage cells at meiosis prophase and to analyze chromosome pairing and recombination of pachytene spermatocyte. Newly developed meiotic proteins antibodies (anti-SCP3, anti-synaptonemal complex proteins 3, anti-MLH1, anti-Mut-L Homolog 1, anti-CREST, chromosome centromere antibody) were used to identify synaptonemal complex (anti-SCP3), recombination sites (anti-MLH1) and centromere (anti-CREST), respectively. Staging of spermatocyte was determined according to SCP3 formation progression. Qualitative data were compared by a Chi-square test, and ANOVA was used to analyze quantitative data. Respectively, 2346 and 2932 spermatocytes were categorized in the controls and azoospermic patients. The proportions of zygotene cells in both OA group and NOA group were significantly higher than that of the control group. Investigation of 1967 pachytene cells from the controls and 354 pachytene cells from azoospermic patients indicated that the mean MLH1 foci per pachytene cell of NOA group was statistically lower than that of the controls. Compared with the controls, incomplete synaptonemal complexes cells (containing gap and/or split) were significantly increased in the NOA group. Delayed meiosis prophase is relatively common in azoospermic patients, and changes in quantity and distribution of recombination foci may be the cause for spermatogenesis arrest in Han Chinese population.