Postnatal testicular development in Vietnamese pot-bellied pigs.

The Vietnamese pot-bellied pig is a breed with high investigation potential. However, at the reproductive level, its testicular characteristics are still unknown, as well as the different stages of its development. Therefore, the objective of this work is to describe the postnatal testicular development of Vietnamese pot-bellied pigs. In this study, we used pigs grouped into the neonatal stage, animals at zero weeks; prepubertal stage, animals at three and eight weeks; pubertal stage, animals at twelve and sixteen weeks; and postpubertal stage animals at twenty, twenty-four, twenty-eight and thirty-two weeks of age. The neonatal stage is characterized by gonocytes at different migration phases. In the prepubertal stage, gonocytes were differentiated into spermatogonia at 3 weeks of age, and the first spermatocytes were observed at 7 weeks of age. Puberty was determined to start at 12 weeks because seminiferous tubules are found with complete spermatogenesis and the highest peaks in positive cell counting of androgen receptors (AR) and proliferating cell nuclear antigen (PCNA) factor that later decreased and further stabilized in the following weeks. In the postpubertal stage, an increase in seminiferous tubule areas was observed. The number of apoptotic cells ranged from low to null at all ages. Testosterone (T) and gonadotropin levels had two important peaks at 3 and 24 weeks. The seminiferous epithelium cycle was found to have 11 stages according to acrosome development. These characteristics of Vietnamese pot-bellied pig testes, which are different from rat testes and more similar to human testicles, make them a viable model to study human male reproductive biology. The postnatal testicular development of pot-bellied pigs is different from the postnatal testicular development of other breeds. Therefore, due to this difference in size and easy manipulation, the Vietnamese pig is an alternative for investigation compared to other pig breeds.

Carnitine Diminishes Etoposide Toxic Action on Spermatogonial Self-renewal and Sperm Production in Adult Rats Treated in the Prepubertal Phase.

The aim of this study was to investigate carnitine action against negative effects of etoposide on stem/progenitor spermatogonia and on sperm production. Carnitine (250 mg/kg body weight/day) and etoposide (5 mg/kg body weight/day) were administered from 25-days postpartum to 32-days postpartum. Testes were collected at 32-days postpartum, 64-days postpartum, and 127-days postpartum, and submitted to the immuno-labeling of UTF1, SOX2, and PLZF proteins to identify undifferentiated spermatogonia populations. At 127-days postpartum, sperm were collected for analysis. Carnitine+etoposide group showed a higher numerical density of spermatogonia labeled for all studied proteins at 64-days postpartum (critical age) compared to the etoposide group. Moreover, there was an improvement of spermatic parameters and sperm DNA integrity in rats of the carnitine+etoposide group in comparison with rats of the etoposide group. The results suggest that carnitine improves the self-renewal of undifferentiated spermatogonia and promotes a partial protection on them, alleviating the etoposide harmful late effects and leading to an enhancement of the sperm parameters in adulthood.

Testin regulates the blood-testis barrier via disturbing occludin/ZO-1 association and actin organization.

The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli-Sertoli junctions.

Expression of transcriptional factor EB (TFEB) in differentiating spermatogonia potentially promotes cell migration in mouse seminiferous epithelium.

BACKGROUND: Spermatogenesis is a complex process involving the self-renewal and differentiation of spermatogonia into mature spermatids in the seminiferous tubules. During spermatogenesis, germ cells migrate from the basement membrane to cross the blood-testis barrier (BTB) and finally reach the luminal side of the seminiferous epithelium. However, the mechanism for regulating the migration of germ cells remains unclear. In this study, we focused on the expression and function of transcriptional factor EB (TFEB), a master regulator of lysosomal biogenesis, autophagy and endocytosis, in spermatogenesis. METHODS: The expression pattern of the TFEB in mouse testes were investigated by Western blotting and immunohistochemistry analyses. Either undifferentiated spermatogonia or differentiating spermatogonia were isolated from testes using magnetic-activated cell sorting based on specific cell surface markers. Differentiation of spermatogonia was induced with 100 nM retinoic acid (RA). shRNA was used to knock down TFEB in cells. TFEB expression was detected by immunofluorescence, qRT-PCR, and Western blotting. Cell migration was determined by both transwell migration assay and wound healing assay applied to a cell line of immortalized spermatogonia, GC-1 cells. RESULTS: During testicular development, TFEB expression was rapidly increased in the testes at the period of 7 days post-partum (dpp) to 14 dpp, whereas in adult testis, it was predominantly localized in the nucleus of spermatogonia at stages VI to VIII of the seminiferous epithelial cycle. Accordingly, TFEB was observed to be mainly expressed in differentiating spermatogonia and was activated for nuclear translocation by RA treatment. Moreover, knockdown of TFEB expression by RNAi did not affect spermatogonial differentiation, but significantly reduced cell migration in GC-1 cells. CONCLUSION: These findings imply that regionally distinct expression and activation of TFEB was strongly associated with RA signaling, and therefore may promote cell migration across the BTB and transport along the seminiferous epithelium.

Revisiting the human seminiferous epithelium cycle.

STUDY QUESTION: Can all types of testicular germ cells be accurately identified by microscopy techniques and unambiguously distributed in stages of the human seminiferous epithelium cycle (SEC)? SUMMARY ANSWER: By using a high-resolution light microscopy (HRLM) method, which enables an improved visualization of germ cell morphological features, we identified all testicular germ cells in the seminiferous epithelium and precisely grouped them in six well-delimitated SEC stages, thus providing a reliable reference source for staging in man. WHAT IS ALREADY KNOWN: Morphological characterization of germ cells in human has been done decades ago with the use of conventional histological methods (formaldehyde-based fixative -Zenker-formal- and paraffin embedding). These early studies proposed a classification of the SEC in six stages. However, the use of stages as baseline for morphofunctional evaluations of testicular parenchyma has been difficult because of incomplete morphological identification of germ cells and their random distribution in the human SEC. STUDY DESIGN, SIZE, DURATION: Testicular tissue from adult and elderly donors with normal spermatogenesis according to Levin's, Johnsen's and Bergmann's scores were used to evaluate germ cell morphology and validate their distribution and frequency in stages throughout human spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular tissue from patients diagnosed with congenital bilateral agenesis of vas deferens (n = 3 adults) or prostate cancer (n = 3 elderly) were fixed in glutaraldehyde and embedded in araldite epoxy resin. Morphological analyses were performed by both light and transmission electron microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: HRLM method enabled a reliable morphological identification of all germ cells (spermatogonia, spermatocytes and spermatids) based on high-resolution aspects of euchromatin, heterochromatin and nucleolus. Moreover, acrosomal development of spermatids was clearly revealed. Altogether, our data redefined the limits of each stage leading to a more reliable determination of the SEC in man. LIMITATIONS, REASONS FOR CAUTION: Occasionally, germ cells can be absent in some tubular sections. In this situation, it has to be taken into account the germ cell association proposed in the present study to classify the stages. WIDER IMPLICATIONS OF THE FINDINGS: Our findings bring a new focus on the morphology and development of germ cells during the SEC in human. Application of HRLM may be a valuable tool for research studies and clinical andrology helping to understand some testicular diseases and infertility conditions which remain unsolved. STUDY FUNDING/COMPETING INTEREST: Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Annexin A2 is critical for blood-testis barrier integrity and spermatid disengagement in the mammalian testis.

Throughout spermatogenesis, two important processes occur at late stage VIII of the seminiferous epithelial cycle in the rat testis: preleptotene spermatocytes commence entry into the adluminal compartment and step 19 spermatids release from the seminiferous epithelium. Presently, it is not clear how these processes, which involve extensive restructuring of unique Sertoli-Sertoli and Sertoli-germ cell junctions, are mediated. We aimed to determine whether annexin A2 (ANXA2), a Ca2+-dependent and phospholipid-binding protein, participates in cell junction dynamics. To address this, in vitro and in vivo RNA interference studies were performed on prepubertal Sertoli cells and adult rat testes. The endpoints of Anxa2 knockdown were determined by immunoblotting, morphological analyses, fluorescent immunostaining, and barrier integrity assays. In the testis, ANXA2 localized to the Sertoli cell stalk, with specific staining at the blood-testis barrier and the concave (ventral) surface of elongated spermatids. ANXA2 also bound actin when testis lysates were used for immunoprecipitation. Anxa2 knockdown was found to disrupt the Sertoli cell/blood-testis barrier in vitro and in vivo. The disruption in barrier function was substantiated by changes in the localization of claudin-11, zona occludens-1, N-cadherin, and ß-catenin. Furthermore, Anxa2 knockdown resulted in spermiation defects caused by a dysfunction of tubulobulbar complexes, testis-specific actin-rich ultrastructures that internalize remnant cell junction components prior to spermiation. Additionally, there were changes in the localization of several tubulobulbar complex component proteins, including actin-related protein 3, cortactin, and dynamin I/II. Our results indicate that ANXA2 is critical for the integrity of the blood-testis barrier and the timely release of spermatids.

Dramatic changes in 67 miRNAs during initiation of first wave of spermatogenesis in Mus musculus testis: global regulatory insights generated by miRNA-mRNA network analysis.

We mapped global changes in miRNA and mRNA profiles spanning the first wave of spermatogenesis using prepubertal (Postnatal Day 8 [P8]), pubertal (P16), and adolescent (P24) Mus musculus testes and identified the differential expression of 67 miRNAs and 8226 mRNAs. These two data sets were integrated into miRNA-dependent regulatory networks based on miRWalk predictions. In a network representing the P8 to P16 transition, downregulation of four miRNAs and upregulation of 19 miRNAs were linked with 81 upregulated target mRNAs and 228 downregulated target mRNAs, respectively. Furthermore, during the P16 to P24 transition, two miRNAs were downregulated, and eight miRNAs were upregulated, which linked with 64 upregulated mRNAs and 389 downregulated mRNAs, respectively. Only three of the miRNAs present in the network (miR-34b-5p, miR-34c, and miR-449a) showed a progressive increase from P8 through P16 to P24, while the remaining miRNAs in the network showed statistically significant changes in their levels either during the P8 to P16 transition or during the P16 to P24 transition. Analysis of the chromosomal location of these differentially expressed miRNAs showed that 14 out of 25 miRNAs upregulated from P8 to P16, and 18 out of 40 miRNAs upregulated from P8 to P24 were X-linked. This is suggestive of their escape from meiotic sex chromosome inactivation and postmeiotic sex chromatin. This integrated network of miRNA-level and mRNA-level changes in mouse testis during the first wave of spermatogenesis is expected to build a base for evaluating the role of miRNA-mediated gene expression regulation in maturing mammalian testis.

Cell-type-specific regulation of genes involved in testicular lipid metabolism: fatty acid-binding proteins, diacylglycerol acyltransferases, and perilipin 2.

Male germ cell differentiation entails the synthesis and remodeling of membrane polar lipids and the formation of triacylglycerols (TAGs). This requires fatty acid-binding proteins (FABPs) for intracellular fatty acid traffic, a diacylglycerol acyltransferase (DGAT) to catalyze the final step of TAG biosynthesis, and a TAG storage mode. We examined the expression of genes encoding five members of the FABP family and two DGAT proteins, as well as the lipid droplet protein perilipin 2 (PLIN2), during mouse testis development and in specific cells from seminiferous epithelium. Fabp5 expression was distinctive of Sertoli cells and consequently was higher in prepubertal than in adult testis. The expression of Fabp3 increased in testis during postnatal development, associated with the functional differentiation of interstitial cells, but was low in germ cells. Fabp9, together with Fabp12, was prominently expressed in the latter. Their transcripts increased from spermatocytes to spermatids and, interestingly, were highest in spermatid-derived residual bodies (RB). Both Sertoli and germ cells, which produce neutral lipids and store them in lipid droplets, expressed Plin2. Yet, while Dgat1 was detected in Sertoli cells, Dgat2 accumulated in germ cells with a similar pattern of expression as Fabp9. These results correlated with polyunsaturated fatty acid-rich TAG levels also increasing with mouse germ cell differentiation highest in RB, connecting DGAT2 with the biosynthesis of such TAGs. The age- and germ cell type-associated increases in Fabp9, Dgat2, and Plin2 levels are thus functionally related in the last stages of germ cell differentiation.

Retinoic acid regulation of male meiosis.

PURPOSE OF REVIEW: Description of new evidence to support the model for how retinoic acid regulates spermatogonial differentiation, male meiosis and the cycle of the seminiferous epithelium. RECENT FINDINGS: It has been known since the 1920s that vitamin A is essential for spermatogenesis. However, only recently has significant progress been made toward understanding how the active metabolite of vitamin A, retinoic acid, regulates spermatogenesis at multiple different differentiation steps, including the onset of meiosis. Current publications suggest that the initiation and maintenance of the cycle of the seminiferous epithelium is linked to retinoic-acid-driving spermatogonial differentiation and meiotic onset. SUMMARY: Retinoic acid appears to act in a pulsatile manner, periodically driving spermatogonial differentiation and meiotic onset at discrete points along testis tubules, and as a result, is likely to be responsible for generating and maintaining the cycle of the seminiferous epithelium.

Spermatogenesis is seasonal in the large hairy armadillo, Chaetophractus villosus (Dasypodidae, Xenarthra, Mammalia).

Very little is known about the distinct reproductive biology of armadillos. Very few studies have investigated armadillo spermatogenesis, with data available only for Euphractus sexcinctus and Dasypus novemcinctus. In the present study, we analysed male germ cell differentiation in the large hairy armadillo Chaetophractus villosus throughout the year, describing a cycle of the seminiferous epithelium made of eight different stages. Evaluation of the testis/body mass ratio, analysis of the architecture of the seminiferous epithelium and the frequency of defective seminiferous tubules allowed identification of a temporal interruption of spermatogenesis during the period between mid-May to July (mid-end autumn) in correlation with very low testosterone levels. Overall, these results suggest that spermatogenesis is seasonal in C. villosus.

Transcriptome profiling of testis during sexual maturation stages in Eriocheir sinensis using Illumina sequencing.

The testis is a highly specialized tissue that plays dual roles in ensuring fertility by producing spermatozoa and hormones. Spermatogenesis is a complex process, resulting in the production of mature sperm from primordial germ cells. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. The gene expression pattern of testis in Chinese mitten crab (Eriocheir sinensis) has not been extensively studied, and limited genetic research has been performed on this species. The advent of high-throughput sequencing technologies enables the generation of genomic resources within a short period of time and at minimal cost. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for testis of E. sinensis. In two runs, we produced 25,698,778 sequencing reads corresponding with 2.31 Gb total nucleotides. These reads were assembled into 342,753 contigs or 141,861 scaffold sequences, which identified 96,311 unigenes. Based on similarity searches with known proteins, 39,995 unigenes were annotated based on having a Blast hit in the non-redundant database or ESTscan results with a cut-off E-value above 10(-5). This is the first report of a mitten crab transcriptome using high-throughput sequencing technology, and all these testes transcripts can help us understand the molecular mechanisms involved in spermatogenesis and testis maturation.

Juvenile rats do not exhibit elevated sensitivity to acrylamide toxicity after oral administration for 12 weeks.

Acrylamide (AA), a neurotoxic, testicular toxic, genotoxic and carcinogenic chemical, has been reported to be formed in processed food, and sensitivity to AA intoxication in childhood is a concern. In the present study, to clarify the general toxicological profile of AA in juvenile rats, subchronic toxicity was evaluated in F344 rats administered AA in the drinking water at 0 (control), 10, 20 and 40 ppm, presented to the dams (three per group) immediately after the birth of their litters, through lactation (3 weeks), and directly to the offspring in their drinking water after weaning for a further 9 weeks (12 weeks total). Treatment with AA caused a decrease in body weights in 20 and 40 ppm F(1) females, compared with the controls. Average AA intake throughout the treatment period for the 10, 20 and 40 ppm groups after weaning was equivalent to 1.0, 2.1 and 4.4 mg kg(-1) body weight per day, respectively, in males and 1.2, 2.5 and 4.9 mg kg(-1) body weight per day, respectively, in females. No toxicologically significant organ weight changes were observed. AA-induced histopathological changes were limited to focal degeneration and necrosis of the seminiferous epithelium in the testes and desquamated epithelium in the ducts of epididymides, noted only in 40 ppm males. Taken together with previous reports, juvenile rats are not necessarily more susceptible to AA-induced toxicity as compared with young adults.

Early prepubertal testis criteria, seminiferous epithelium and hormone concentrations as related to testicular development in beef bulls.

The present study was conducted to evaluate testis size, spermatogenesis and hormone concentrations before and when peripheral testosterone reached 1 ng/ml as related to further gonad development of beef bulls (n=28). Blood samples were taken weekly starting at 10 weeks (wk) and when testosterone reached 1 ng/ml (AGE1), the left testis was surgically excised. From AGE1 until 54 wk, blood samples were collected to follow basal and GnRH-stimulated hormone profiles. At 54 wk, the second testis was removed. Testosterone reached 1 ng/ml at 20±0.6 wk and, at this developmental state, the seminiferous tubules occupied 57±1.1% of the testis parenchyma. At this phase, 79.3±1.4% of tubule sections had no germ cells and only 2.4±0.3% of the remaining tubules had spermatocytes as the most advanced germ cell type. Also at AGE1, testis size was correlated with the number of Sertoli cells per testis (r=0.67; P<0.05), but not (P>0.05) with the percentage of tubules with germ cells. There was a consistent increase in body weight and testis size throughout the study showing that hemicastration did not impair the development of the bulls. At 54 wk, seminiferous tubules represented 76±0.7% of the testis parenchyma and 72.3±1.7% of tubule sections were found with either round or elongated spermatids. Quantitative criteria of spermatogenesis in the second testis (excised at 54 wk) were not correlated (P>0.05) with the percentage of seminiferous tubules with germ cells in the first testis (excised at AGE1). As determined by regression analysis, testis diameter measured between 30 and 44 wk (AVTD) was associated with AGE1 and testis diameter averaged at 12 wk and AGE1 (R(2)=0.77; P<0.01). Also, AVTD was related to AGE1, testis diameter at 12 wk and concentrations of 17ß-estradiol (estradiol; basal+GnRH-stimulated) averaged between 10 wk and AGE1 (R(2)=0.79; P<0.01). Yearling testis weight, in turn, was linked to AGE1 and testis weight at AGE1 (R(2)=0.49, P<0.01). In conclusion, early detection of 1 ng of testosterone/ml, larger testis size and greater estradiol before and at that developmental period positively relate to future testis attributes. When testosterone reached 1 ng/ml, the seminiferous tubules had Sertoli cells, spermatogonia and a few spermatocytes and events occurring before and at that phase are potential markers of testis growth and sperm-producing capacity of sires.

Xenografting of human fetal testis tissue: a new approach to study fetal testis development and germ cell differentiation.

BACKGROUND: Abnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development. METHODS: Human fetal testes (at 9 weeks, n = 4 and 14-18 weeks gestation, n = 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls. RESULTS: Xenografts showed >75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P = 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4(+)) into pre-spermatogonia (VASA(+)) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls. CONCLUSIONS: Human fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.

Krüppel-like factor 4 is involved in functional differentiation of testicular Sertoli cells.

Krüppel-like factor 4 (KLF4) is a pleiotropic zinc finger transcription factor that regulates genes being involved in differentiation and cell-cycle control. Knockout studies revealed a critical function for KLF4 in the terminal differentiation of many epithelial cells. In testicular Sertoli cells, Klf4 is strongly inducible by the glycoprotein follicle stimulating hormone (FSH). Because KLF4 is essential for postnatal survival in mice, we deleted Klf4 specifically in Sertoli cells using the Cre/loxP system. Importantly, around postnatal day 18, a critical period of terminal Sertoli cell differentiation, mutant seminiferous tubules exhibited a disorganized germinal epithelium and delayed lumen formation. The ultrastructural finding of highly vacuolized Sertoli cell cytoplasm and the identification of differentially expressed genes, which are known to play roles during vesicle transport and fusion or for maintenance of the differentiated cell state, suggest impaired apical secretion of the Sertoli cell. Interestingly, a high proportion of all identified genes was localized in a small subregion of chromosome 7, suggesting coordinated regulation. Intriguingly, adult mutant mice are fertile and show normal testicular morphology, although the testosterone levels are decreased. In summary, KLF4 plays a significant role for proper and timely Sertoli cell differentiation in pubertal mice.

Spermatogenesis and cycle of the seminiferous epithelium.

Spermatogenesis is a complex biological process of cellular transformation that produces male haploid germ cells from diploid spermatogonial stem cells. This process has been simplified morphologically by recognizing cellular associations or 'stages' and 'phases' of spermatogenesis, which progress through precisely timed and highly organized cycles. These cycles of spermatogenesis are essential for continuous sperm production, which is dependent upon numerous factors, both intrinsic (Sertoli and germ cells) and extrinsic (androgens, retinoic acids), as well as being species-specific.

Ontogeny of leptin and its receptor expression in mouse testis during the postnatal period.

The mechanism by which leptin regulate male reproductive development during postnatal periods remain to be determined. Using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), we established that leptin is expressed in mouse testis with a cell-type and stage-dependent manner during the postnatal period. In testes of 5-day-old mice, leptin expression was mainly restricted to gonocytes, whereas the immunostaining of leptin was confined to spermatogonia in 10-day-old testes. From Day 20 and onwards, stage-specific expression of leptin was evident in spermatocytes at stages VII-XII of the cycle in the seminiferous epithelium. RT-PCR showed that leptin and its receptor isoforms, Ob-Ra, Ob-Rb, and Ob-Re were all expressed in testes from 5- to 60-day-old mice. The mRNA for Ob-Ra and Ob-Re, but not Ob-Rb or leptin were identified in both immature (14-day-old) and adult (60-day-old) isolated Leydig cells. These results suggest that besides its primary actions at the hypothalamic-pituitary level, leptin has direct effects in the proliferation, differentiation of germ cells and the modulation of testicular steroidogenesis, using both autocrine and paracrine mechanisms.

Maternal vitamin B12 deficiency affects spermatogenesis at the embryonic and immature stages in rats.

To evaluate the role of cobalamin (Cbl) on spermatogenesis, the effect of dietary vitamin B(12) deficiency on early spermatogenesis was histologically investigated in male fetuses and newborns in the first filial generation (F(1) males) of rats. There was no difference in the number of gonocytes and supporting cells of Sertoli in the gonad in male fetuses on day 16 of gestation and in the testes in F(1) males at 0 days of age between vitamin B(12)-deficient (VB12-D) and vitamin B(12)-supplemented (VB12-S) groups. However, at 21 days of age, a decreased number of spermatogonia and no spermatocytes were observed in the VB12-D group. Numerous TUNEL positive cells were located among spermatocytes of the spermatogenic epithelium. The ultrastructural features examined using transmission electron microscopy were considered to be indicative of apoptosis. The incidence of seminiferous tubules having apoptotic cells was 51.5% in the VB12-D group. At 60 days of age, aplasia of the spermatids and spermatozoa was detected in the VB12-D group. In the connective tissue between the seminiferous tubules, many interstitial Leydig cells and blood vessels were observed in the VB12-D group, as compared with the VB12-S group. These changes produced by vitamin B(12) deficiency can be reversed by providing a VB12-S diet after weaning at 21 days of age. From these findings, such a vitamin B(12) deficiency during gestation and lactation could affect the germ cells and especially damage spermatocytes in F(1) male rats, which indicates that Cbl may be an essential constituent in the meiosis of spermatogenesis.

Effects of chronic simulated hypobaric hypoxia on mouse spermatogenesis / Efectos de la Hipoxia Hipobárica Simulada Crónica sobre la Espermatogénesis en el Ratón

La disminución del aporte de O2 a los tejidos provoca daños de éstos, incluido el epitelio seminífero. Últimamente, se ha incrementado la población que trabaja a gran altura, interesando así el estudio de la hipoxia hipobárica sobre la espermatogénesis. Para este estudio se utilizaron dos grupos de ratones machos sexualmente maduros: Control (540 metros sobre el nivel del mar (msnm)) y grupo con hipoxia hipobárica simulada crónica (HHSC) (4.600 msnm) expuestos por 8, 16, 24 ó 33 días. Fueron evaluados hematocrito, reticulocitosis, peso de testículos, epidídimos y vesícula seminal; altura del epitelio seminífero, diámetro tubular, recuento y morfología espermática y lipoperoxidación de membranas de espermatozoides y parénquima testicular. El peso de testículos, epidídimos y vesícula seminal se redujo para empezar a recuperarse a los 33 días. El diámetro tubular y la altura del epitelio se redujeron y luego tendieron a aumentar sin normalizarse. El recuento y la morfología espermáticos fluctaron en el tiempo. Se puede concluir que la exposición a HHSC induce daño del epitelio seminífero, disminución de la lipoperoxidación en espermatozoides y tejido testicular, y altera la morfología testicular y espermática.

Reduction of O2 delivery to tissues damage them, including the seminiferous epithelium. Recently, population working in high altitude has increased, so that the study of hypobaric hypoxia on spermatogenesis becomes of interest. In this study we used two groups of male, sexually mature mice Control (C) (540 meters above sea level (masl)) and chronic simulated hypobaric hypoxia (CSHH) (4,600 masl) exposed during 8, 16, 24 or 33 days. Hematocrit; reticulocytosis; testicular, epididymal and seminal vesicle weight; seminiferous epithelium height, tubular diameter, sperm count and morphology and testicular parenchyme and spermatozoa membranes lipoperoxidation were measured. Weight of testis, epididymis and seminal vesicle were reduced but they recuperate at 33 days. Tubular diameter and epithelial height are reduced, subsequently they tend to increase without returning to normal values. The count and sperm morphology fluctuate along the exposure time. Lipoperoxidation levels of spermatozoa and testicular parenchyme are reduced. Therefore, we can conclude that exposure to CSHH induce damage in the seminiferous epithelium, decrease of lipoperoxidation in spermatozoa and testicular tissue, and damages the testicular and sperm morphology.

Spermatogenic cells distal to the blood-testis barrier in rats lack C3 convertase regulators and may be at risk of complement-mediated injury.

On most tissues, multiple membrane complement regulators (CReg) protect self-cells from damage by complement. An exception is the brain, where the blood-brain barrier provides a protected environment within which cells survive with little or no protection from complement. The testis has a functionally similar structure, the blood-testis barrier (BTB). Here, we have investigated the expression of C3/C5 convertase CReg and C3 in the normal rat testis at different ages and different spermatogenetic stages, as well as in rats in which spermatogenesis and the BTB were impaired due to a developmental deficit. Immature testis, prior to BTB formation at puberty, displayed broad expression of the ubiquitous rodent CReg Crry on all elements and no expression of CD46 or CD55. Within days of BTB formation, CReg expression was dramatically altered; Crry was expressed only in the spermatogenetic cells external to the BTB in basal layers of adult seminal epithelium. Spermatogenic cells immediately distal to the BTB at first expressed no C3/C5 convertase regulators but later acquired expression of CD46 and CD55. Staining for C3 was widespread pre-puberty, but absent distal to the BTB in mature rats. In rats with defects in spermatogenesis and BTB integrity, expression patterns of CReg and C3 resembled those in pre-pubertal normals. The relative paucity of CReg and absence of C3 synthesis distal to the BTB suggest the presence of a complement-protected environment analogous to that described in the brain, and suggest also that cells enclosed by the BTB may be susceptible to complement damage when the barrier is breached.