Early Stages Of Spermatogenesis Research Articles

Spermatogonial depletion and a spermatogenesis defect in the Dp(16)1Yey mouse model of Down syndrome

In brief Little is known about the mechanism responsible for male infertility in Down syndrome. Duplication of Mmu16 chromosome (an orthologue of Hsa21, bearing Dyrk1A) in Dp(16)1Yey mice is responsible for impaired spermatogenesis through spermatogonial depletion and hypogonadotropic hypogonadism. Abstract Down syndrome (DS), or trisomy 21, is the most common chromosomal disorder in humans. Men with DS are infertile. The DYRK1A gene on Hsa21 is involved in several features of DS. Overexpression of the homolog dyrk1A disrupts primordial germ cell migration in zebrafish, and overexpression of Dyrk1A impairs gonadotropic axis function and the early stages of spermatogenesis in the mouse. Other genes on Hsa21 might be involved in the pathogenesis of infertility in DS. We investigated the Dp(16)1Yey mouse model of DS, which features segmental duplication of chromosome Mmu16 (orthologous to a large part of Hsa21 and carrying Dyrk1A and 112 other genes). Using an immunohistochemical assay for the spermatogonial marker STRA8, we observed spermatogonial depletion in the Dp(16)1Yey mouse. This was correlated with low mRNA expression of GFRα1 (a marker of the self-renewal stem cell pool) in an RT-qPCR assay and low protein expression of PLZF (a marker of differentiating stem cells) in a slot-blot assay. Spermatogenesis was present but impaired, with a low sperm count, low protamine-1 expression, a low testis weight, and a low seminiferous tubule diameter. Low circulating luteinizing hormone and follicle-stimulating hormone levels and an elevated testis anti-Müllerian hormone level (as measured in ELISAs) revealed the presence of hypogonadotropic hypogonadism. The Dp(16)1Yey mouse model of DS recapitulates observations made in zebrafish and mice overexpressing DYRK1A homologs. The presence of an excess of Mmu16 material perturbs spermatogenesis and the gonadotropic axis. More generally, DYRK1A’s role in human infertility (outside DS) remains to be characterized.

Heat wave exposure and semen quality in sperm donation volunteers: A retrospective longitudinal study in south China

BackgroundPrevious studies have suggested an association between non-optimum ambient temperature and decreased semen quality. However, the effect of exposure to heat waves on semen quality remains unclear. MethodsVolunteers who intended to donate sperm in Guangdong provincial human sperm bank enrolled. Heat waves were defined by temperature threshold and duration, with a total of 9 definitions were employed, specifying daily mean temperature exceeding the 85th, 90th, or 95th percentile for at least 2, 3, or 4 consecutive days. Residential exposure to heat waves during 0–90 days before ejaculation was evaluated using a validated gridded dataset on ambient temperature. Association and potential windows of susceptibility were evaluated and identified using linear mixed models and distributed lag non-linear models. ResultsA total of 2183 sperm donation volunteers underwent 8632 semen analyses from 2018 to 2019. Exposure to heat wave defined as daily mean temperature exceeding the 95th percentile for at least 4 consecutive days (P95–D4) was significantly associated with a 0.11 (95% confidence interval [CI]: 0.03, 0.18) ml, 3.36 (1.35, 5.38) × 106/ml, 16.93 (7.95, 25.91) × 106, and 2.11% (1.4%, 2.83%) reduction in semen volume, sperm concentration, total sperm number, and normal forms, respectively; whereas exposure to heat wave defined as P90–D4 was significantly associated with a 1.98% (1.47%, 2.48%) and 2.08% (1.57%, 2.58%) reduction in total motility and progressive motility, respectively. Sperm count and morphology were susceptible to heat wave exposure during the early stage of spermatogenesis, while sperm motility was susceptible to exposure during the late stage. ConclusionHeat wave exposure was significantly associated with a reduction in semen quality. The windows of susceptibility during 0–90 days before ejaculation varied across sperm count, motility, and morphology. Our findings suggest that reducing heat wave exposure before ejaculation may benefit sperm donation volunteers and those attempting to conceive.

Spermiogenesis alterations in the absence of CTCF revealed by single cell RNA sequencing.

CTCF is an architectonic protein that organizes the genome inside the nucleus in almost all eukaryotic cells. There is evidence that CTCF plays a critical role during spermatogenesis as its depletion produces abnormal sperm and infertility. However, defects produced by its depletion throughout spermatogenesis have not been fully characterized. In this work, we performed single cell RNA sequencing in spermatogenic cells with and without CTCF. We uncovered defects in transcriptional programs that explain the severity of the damage in the produced sperm. In the early stages of spermatogenesis, transcriptional alterations are mild. As germ cells go through the specialization stage or spermiogenesis, transcriptional profiles become more altered. We found morphology defects in spermatids that support the alterations in their transcriptional profiles. Altogether, our study sheds light on the contribution of CTCF to the phenotype of male gametes and provides a fundamental description of its role at different stages of spermiogenesis.

Single-cell transcriptome sequencing reveals Wolbachia-mediated modification in early stages of Drosophila spermatogenesis.

Wolbachia are the most widely distributed intracellular bacteria, and their most common effect on host phenotype is cytoplasmic incompatibility (CI). A variety of models have been proposed to decipher the molecular mechanism of CI, among which the host modification (HM) model predicts that Wolbachia effectors play an important role in sperm modification. However, owing to the complexity of spermatogenesis and testicular cell-type heterogeneity, whether Wolbachia have different effects on cells at different stages of spermatogenesis or whether these effects are linked with CI remains unknown. Therefore, we used single-cell RNA sequencing to analyse gene expression profiles in adult male Drosophila testes that were infected or uninfected by Wolbachia. We found that Wolbachia significantly affected the proportion of different types of germ cells and affected multiple metabolic pathways in germ cells. Most importantly, Wolbachia had the greatest impact on germline stem cells, resulting in dysregulated expression of genes related to DNA compaction, and Wolbachia infection also influenced the histone-to-protamine transition in the late stage of sperm development. These results support the HM model and suggest that future studies on Wolbachia-induced CI should focus on cells in the early stages of spermatogenesis.

Down-Regulation of Neogenin Decreases Proliferation and Differentiation of Spermatogonia during the Early Phase of Spermatogenesis

Non-obstructive azoospermia is a major clinical issue associated with male infertility that remains to be addressed. Although neogenin is reportedly abundantly expressed in the testis, its role in mammalian spermatogenesis is unknown. We systematically investigated the role of neogenin during spermatogenesis by performing loss-of-function studies. Testis-specific neogenin conditional knock-out (cKO) mice were generated using CRISPR/Cas9 and neogenin-targeting guide RNAs. We analyzed the expression profiles of germ cell factors by RT-PCR and Western blotting. Neogenin localized mainly to spermatogonia in seminiferous tubules of mouse testes. RT-PCR and Western blot analyses further demonstrated that neogenin expression varied during spermatogenesis and was dramatically increased at postnatal day 12-25 during the pubertal stage. In neogenin-cKO mouse testes, the ratio of primary and secondary spermatocytes was significantly decreased compared with the control, while the number of apoptotic testicular cells was significantly increased. Taken together, these results suggest that neogenin plays a pivotal role in the maintenance and proliferation of spermatogonia during the early stage of spermatogenesis in mice.

Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15–20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine‐treatment groups, T‐57 (castration at 57 days, n = 7) or T‐100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T‐57 and T‐100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra‐scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Lipidomic profile of seminal plasma in non-obstructive azoospermia with sperm maturation arrest

Introduction. The difference between obstructive and non-obstructive azoospermia with sperm maturation arrest is important for the choice of treatment tactics and adequate counseling of a married couple.Purpose of the study. The study aimed to assess the semen lipid profile in patients with sperm maturation arrest. Materials and methods. Samples of seminal plasma for lipid composition of 24 men with normozoospermia and 64 men with azoospermia were studied. Patients with azoospermia underwent microdissection testicular biopsy followed by the detection of testicular tissue pathology. Lipid extracts were analyzed by liquid chromatography with mass spectrometry. Lipid data were compared with the results of pathomorphological studies.Results. Comparison of two groups revealed a statistically significant concentration differences for 22 lipids detected in positive-ion mode and 11 lipids detected in negative-ion mode. Those lipids mainly belong to the classes hexosylceramides, sphingomyelins and phosphatidylcholines — simple ethers and oxidized lipids. In multivariate analysis, the following lipids were found to be statistically significant predictors of sperm maturation arrest: PC 16: 0_22: 6 lipid (β-coefficient: -0.73; 95% confidence interval (95% CI): -1.42 to -0.27; odds ratio (OR): 0.48; OR CI: 0.24 to 0.76; Wald's test: -2.58; p = 0.01), SM d20: 1/22:2 lipid (β-coefficient 4.96; 95% CI 2.29 to 9.13; OR: 142.31; OR CI: 9.90 to 9.22^103; Wald's test: 2.93; p = 0.003); PG 20:3_22: 6 lipid (β-coefficient 2.52; 95% CI 1.13 to 4.49; OR: 12.37; OR CI: 3.10 to 89.27; Wald's test: 3.02; p = 0.002); PC O- 16: 1/16:0 lipid (β-coefficient 1.96; 95% CI -4.12 to 0.27; OR: 0.14; OR CI: 0.02 to 0.76; Wald's test: -2.05; p = 0.04). The prediction model characteristics of sperm maturation arrest, obtained during cross-validation in the positiveion mode composed: sensitivity 91%, specificity 85%; in negative-ion mode: sensitivity 75%; specificity 81%.Conclusions. Even though early stages of spermatogenesis are equally preserved in both fertile men and men with homogeneous sperm maturation arrest, the semen in the studied group of patients differed in its lipid profile. Patients with non-obstructive azoospermia, associated with meiosis arrest, may have unique lipidomic characteristics of seminal plasma, which in the future will make it possible to differentiate various variants of severe male infertility using non-invasive methods.

The ultrastructure of spermatogenic cells and morphological evaluation of testicular development in the silver pomfret (Pampus argenteus).

The silver pomfret (Pampus argenteus) is a widely distributed and economically important marine fish in the Indo-Pacific. In this study, we acquired the second generation of wild P.argenteus by artificial breeding and further studied the testicular development and ultrastructure of spermatogenesis. The results of gonadosomatic index (GSI) showed the spawning period of this marine fish was from April to June. Besides, through morphological analysis, we found that P.argenteus had an anastomosing tubular testis surrounded by a layer of tunica albuginea, in which spermatogenesis occurred in cysts where the synchronous germ cells were completely surrounded by the cytoplasmic projection of Sertoli cells. Meanwhile, based on submicroscopic characteristics, the germ cells are classified into nine different types. During the ontogenesis of testis, both the early stage of spermatogenesis and sperm were observed in P.argenteus. At sperm maturation stage, different types of spermatozoa and activation of sperms occurred non-synchronously in the tubules. Cytoplasmic bridges also were observed among synchronous germ cells within the cysts, suggesting an interrelated and differentiated relationship among these germ cells.

Aristolochic acid I induces impairment in spermatogonial stem cell in rodents.

Aristolochic acid I (AAI) is a natural bioactive substance found in plants from the Aristolochiaceae family and impairs spermatogenesis. However, whether AAI-induced spermatogenesis impairment starts at the early stages of spermatogenesis has not yet been determined. Spermatogonial stem cells (SSCs) are undifferentiated spermatogonia that balance self-renewing and differentiating divisions to maintain spermatogenesis throughout adult life and are the only adult stem cells capable of passing genes onto the next generation. The objective of this study was to investigate whether AAI impairs SSCs during the early stages of spermatogenesis. After AAI treatment, we observed looser, smaller and fewer colonies, decreased cell viability, a decreased relative cell proliferation index, and increased apoptosis in SSCs in a concentration- and/or time-dependent manner. Additionally, AAI promoted apoptosis in SSCs, which was accompanied by upregulation of caspase 3, P53 and BAX expression and downregulation of Bcl-2 expression, and suppressed autophagy, which was accompanied by upregulation of P62 expression and downregulation of ATG5 and LC3B expression, in a concentration-dependent manner. Then we found that AAI impaired spermatogenesis in rats, as identified by degeneration of the seminiferous epithelium, and increased apoptosis of testicular cells. Taken together, our findings demonstrate that AAI causes damage to SSCs and implicate apoptosis and autophagy in this process. The impairment of SSCs may contribute to AAI-induced testicular impairment. Our findings provide crucial information for the human application of botanical products containing trace amounts of AAI.

Association of Wolbachia with Gene Expression in Drosophila Testes.

Wolbachia is a genus of intracellular symbiotic bacteria that are widely distributed in arthropods and nematodes. These maternally inherited bacteria regulate host reproductive systems in various ways to facilitate their vertical transmission. Since the identification of Wolbachia in many insects, the relationship between Wolbachia and the host has attracted great interest. Numerous studies have indicated that Wolbachia modifies a variety of biological processes in the host. Previous studies in Drosophila melanogaster (D. melanogaster) have demonstrated that Wolbachia can affect spermatid differentiation, chromosome deposition, and sperm activity in the early stages of spermatogenesis, leading to sperm dysfunction. Here, we explored the putative effect of Wolbachia in sperm maturation using transcriptomic approaches to compare gene expression in Wolbachia-infected and Wolbachia-free D. melanogaster adult testes. Our findings show that Wolbachia affects many biological processes in D. melanogaster adult testes, and most of the differentially expressed genes involved in carbohydrate metabolism, lysosomal degradation, proteolysis, lipid metabolism, and immune response were upregulated in the presence of Wolbachia. In contrast, some genes that are putatively associated with cutin and wax biosynthesis and peroxisome pathways were downregulated. We did not find any differentially expressed genes that are predicted to be related to spermatogenesis in the datasets. This work provides additional information for understanding the Wolbachia-host intracellular relationships.

Meiotic susceptibility for induction of sperm with chromosomal aberrations in patients receiving combination chemotherapy for Hodgkin lymphoma

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from <0.05 to 0.6 Gy. Late meiosis was found to be the most sensitive to NOVP treatment for the production of sperm with chromosomal abnormalities, both in structure and number. Earlier stages of spermatogenesis were less sensitive and there was no evidence that therapy-exposed stem cells resulted in increased frequencies of sperm with abnormalities in chromosomal structure or number. This indicates that NOVP therapy may increase the risks for paternal transmission of chromosomal structural aberrations for sperm produced 32 to 45 days after a treatment with these drugs and implies that there are no excess risks for pregnancies conceived more than 6 months after this therapy. This clinical evaluation of the AM8 sperm FISH protocol indicates that it is a promising tool for assessing an individual’s burden of sperm carrying chromosomal structural aberrations as well as aneuploidies after cancer therapy, with broad applications in other clinical and environmental situations that may pose aneugenic or clastogenic risks to human spermatogenesis.

Expression and localization of PIWI proteins in testis and ovary of domestic sheep

The piRNA-PIWI protein complex plays crucial roles in safeguarding the genome against inordinate transposon mobilization and regulation of embryonic development. A previous study indicated the presence of piRNA in sheep reproductive organs. However, the tissue distribution and cellular localization of PIWI proteins in sheep remains unclear. Therefore the present study aimed to explore the expression profiles of mRNAs of mammalian PIWI proteins (PIWIL1, PIWIL2, PIWIL4 and AGO3) in 9 tissues derived from adult male and female sheep. Results showed the expression of PIWIL1, PIWIL2, and PIWIL4 was significantly higher in the testis and ovary than in the other tissues. Immunohistochemistry analysis of testes indicated that each of the 4 proteins had specific cellular localizations, and some of the localizations were different from those of other species. All the proteins were mainly localized in the primary spermatocytes, suggesting that they are crucial for silencing of transposon to guarantee the integrity of the gamete genome during early stage of spermatogenesis. In the ovaries, the PIWI proteins were mainly localized in oocytes from antral follicles and leukocytes in ovarian blood. Our results provide insights to better understand the functions of PIWI proteins during spermatogenesis, oogenesis and immune defence in sheep.

Identification of key genes associated with spermatogenesis arrest in fox hybrids using weighted gene co-expression network analysis

The silver fox and the blue fox represent different genera, but produce viable offspring. Although these hybrids show obvious heterosis, they are completely sterile due to spermatogenic arrest at the early stages of spermatogenesis, especially mitosis and meiosis I; the hybrids produce few spermatogonia and primary spermatocytes, and no secondary spermatocytes. Although the mechanisms of spermatogenic arrest have been well investigated, transcriptomic differences between hybrid and the pure-species testes have not clarified. In the present study, we used RNA sequencing (RNA-Seq) to generate testicular transcriptomic profiles for silver foxes, blue foxes, and reciprocal hybrids during the pre-breeding period and the breeding season. In total, 1,344,022 transcripts (≥200 bp) were generated; 1,057,724 genes were obtained; and 33,423 genes were shown to have fragments per kilobase of transcript per million mapped reads (FPKM) > 0.3. To identify the hub genes associated with spermatogenesis arrest, weighted gene co-expression network analysis (WGCNA) was used. Nine modules were explored. Genes in only a single module were consistently downregulated in the hybrids as compared to the pure species; these genes were significantly associated with fox hybrid male infertility. Six of the genes in this module (CATSPERD, DMRTC2, RNF17, NME5, SPEF2, SPINK2) also play key roles in mitosis and meiosis during spermatogenesis. Therefore, these six genes might be associated with fox hybrid male infertility.

Deficiency of X‐ray repair cross‐complementing group 1 in primordial germ cells contributes to male infertility

X-ray repair cross-complementing group 1 (Xrcc1), a key DNA repair gene, plays a vital role in maintaining genomic stability and is highly expressed in the early stages of spermatogenesis, but the exact functions remain elusive. Here we generated primordial germ cell-specific Xrcc1 knockout (cXrcc1-/-) mice to elucidate the effects of Xrcc1 on spermatogenesis. We demonstrated that Xrcc1 deficiency results in infertility in male mice due to impaired spermatogenesis. We found that cXrcc1-/- mice exhibited smaller size of testes as well as lower sperm concentration and motility than the wild-type mice. Mechanistically, we demonstrated that Xrcc1 deficiency in primordial germ cells induced elevated levels of reactive oxygen species, mitochondria dysfunction, apoptosis, and loss of stemness of spermatogonial stem cells (SSCs) in testes. In Xrcc1-deficienct SSCs, elevated oxidative stress and mitochondrial dysfunction could be partially reversed by treatment with the antioxidant N-acetylcysteine (NAC), whereas NAC treatment did not restore the fertility or ameliorate the apoptosis caused by loss of Xrcc1. Overall, our findings provided new insights into understanding the crucial role of Xrcc1 during spermatogenesis.-Xu, C., Xu, J., Ji, G., Liu, Q., Shao, W., Chen, Y., Gu, J., Weng, Z., Zhang, X., Wang, Y., Gu, A. Deficiency of X-ray repair cross-complementing group 1 in primordial germ cells contributes to male infertility.

Comparative studies on testis, epididymis and serum hormone concentrations in foxes, and hybrids during the pre-breeding period

Silver fox and blue fox belong to different genera, and the hybrid males are reproductively sterile. In the present study, there was a comparison of testicular and epididymal morphology and serum hormone concentrations among silver foxes, blue foxes, and the hybrids during the pre-breeding period, using 20 male silver foxes, 20 male blue foxes, 15 male HSBs (silver fox male × blue fox female hybrids) and 15 male HBSs (blue fox male × silver fox female hybrids), respectively. Hybrids had a smaller diameter of seminiferous tubules than pure-species males, and testes of hybrid males did not differ in mean size and relative weight from pure-species males. There were many Sertoli cells and spermatogenic cells in silver foxes and blue foxes, while numbers of spermatogonia and primary spermatocytes were less with no secondary spermatocytes in the hybrids. Furthermore, mean serum testosterone and estradiol concentrations in the hybrids were less, and FSH, LH, and PRL were greater than that in silver foxes and blue foxes (P < 0.05), suggesting that lesser concentrations of testosterone and estradiol and greater concentrations of FSH, LH and prolactin can inhibit the completion of spermatogenesis during the pre-breeding period. The results indicate that fox hybrid sterility may result from failures at the early stages of spermatogenesis.

TopBP1 deficiency impairs the localization of proteins involved in early recombination and results in meiotic chromosome defects during spermatogenesis

Topoisomerase IIβ-binding protein 1 (TopBP1) is BRCT domain-containing protein that is required for DNA double-strand break (DSB) repair and DNA damage responses; however, its function during the early stage of spermatogenesis is still unclear. To investigate the physiological role of TopBP1, we have generated germ cell-specific TopBP1-depleted mouse model. TopBP1-deleted mice were infertile, showed a loss of germ cells and had meiotic defects. Conditional TopBP1 deletion resulted in reduced testis size, reduced number of epididymal sperm, increased apoptosis, and severely compromised fertility. TopBP1 deficiency caused defects in DMC1 and Rad51 foci formation, abnormal synaptonemal complexes and meiotic chromosome defects. Collectively, these results suggest that TopBP1 deficiency during spermatogenesis impairs the localization of proteins involved in early recombination at DSBs, results in meiotic chromosome defects and leads to infertility.

Heat Shock Factor 5 Is Essential for Spermatogenesis in Zebrafish

Heat shock factors (Hsfs) are transcription factors that regulate responses to heat shock and other environmental stimuli. Four heat shock factors (Hsf1-4) have been characterized from vertebrates to date. In addition to stress response, they also play important roles in development and gametogenesis. Here, we study the fifth member of heat shock factor family, Hsf5, using zebrafish as a model organism. Mutant hsf5-/- males, generated by CRISPR/Cas9 technique, were infertile with drastically reduced sperm count, increased sperm head size, and abnormal tail architecture, whereas females remained fertile. We show that Hsf5 is required for progression through meiotic prophase 1 during spermatogenesis as suggested by the accumulation of cells in the leptotene andzygotene-pachytene stages and increased apoptosis in post-meiotic cells. hsf5-/- mutants show gonadal misregulation of a substantial number of genes with roles in cell cycle, apoptosis, protein modifications, and signal transduction, indicating an important role of Hsf5 in early stages of spermatogenesis.

MP60-15 TRANSCRIPTOME ANALYSIS TO IDENTIFY HUMAN SPERMATOGONIAL CELLS FROM SERTOLI CELL-ONLY TESTES

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2018MP60-15 TRANSCRIPTOME ANALYSIS TO IDENTIFY HUMAN SPERMATOGONIAL CELLS FROM SERTOLI CELL-ONLY TESTES Koji Shiraishi and Hideyasu Matsuyama Koji ShiraishiKoji Shiraishi More articles by this author and Hideyasu MatsuyamaHideyasu Matsuyama More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1907AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Non-obstructive azoospermia (NOA), particularly Sertoli cell-only (SCO), is the most severe form of male factor infertility and is classically thought to lack germ cells and spermatogenic stem cells (SSCs). However, evidence from microsurgical testicular sperm extraction (micro-TESE) on men with SCO has revealed the presence of focal areas of full spermatogenesis, suggesting the possible presence of SSCs in some patients with SCO lacking mature sperm. To determine whether gene products from SSCs could be detected in SCO, transcriptome analysis using next-generation sequencing (NGS) was performed using human biopsied testicular samples. METHODS Under IRB approval, testicular samples were obtained from 20 men (mean age: 35.7 years) with SCO in which no germ cells were found during laboratory examination and from five men with obstructive azoospermia (mean age: 38.5 years) with normal spermatogenesis. Men with sex chromosome or AZF abnormality were excluded. Transcriptomes on the Illumina platform were expressed as FPKM, and Cuffdiff analysis was performed to compare individual differences among testicular tissues from obstructive azoospermia (OA). Expression of mRNA and localization of targeted genes were confirmed by RT-PCR and immunohistochemical analyses, respectively. RESULTS From the 23,003 genes screened with using NGS with a significant signal after background correction and normalization, a total of 5,666 genes were analyzed. As representative markers of immature germ cells, UTF1, OCT4, DDX4, MMP1, GFRα, GPX1, SCP3, CD14, KLF9, GFRA1 and KIT mRNA were detected in SCO samples by transcriptome analysis. RT-PCR showed significant mRNA expression of UTF1, OCT4, DDX4, SCP3 and KIT from SCO samples. Immunohistochemistry showed the localization of DDX4 and/or KIT-positive cells in the seminiferous tubules and basement membrane in eight (40%) of the SCO samples. All the gene products evaluated in SCO samples were present in testes with obstructive azoospermia. There was no significant difference in the patients' background and hormonal data between the men with and without DDX4 and/or KIT-positive cells. CONCLUSIONS Germ cells are present in 40% of SCO samples as immature forms, namely SSCs. These observations help our understanding of the pathophysiology of spermatogenic failure where germ cell development stops during early stages of spermatogenesis in some men with SCO. The azoospermia seen in men with SCO appears to be, at least in part, secondary to a defect in proliferation and differentiation of SSCs. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e795 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Koji Shiraishi More articles by this author Hideyasu Matsuyama More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The effects of environmentally relevant concentrations of aldrin and methoxychlor on the testes and sperm of male Clarias gariepinus (Burchell, 1822) after short-term exposure.

The organochlorine pesticides aldrin (0.14μg/L) and methoxychlor (0.23μg/L) were both present in the Albasini Dam, Limpopo Province, South Africa, during a field survey in 2014. The use of aldrin has been banned in the USA since 1987 and restricted in South Africa since 1992. The use of methoxychlor, however, remains undefined with little information available about its registration in South Africa despite being banned in Europe (2002) and USA (2003). The aim of this study was to determine the potential effects of environmentally relevant concentrations of aldrin and methoxychlor on the reproductive system of male catfish, Clarias gariepinus. Males were exposed for 96h to the two pesticides under controlled laboratory conditions. Following exposure, each fish was weighed and measured, and a necropsy performed to determine any macroscopic abnormalities and the general health of the fish. The fish were killed and dissected and the testes removed, weighed and measured to determine the gonadosomatic index (GSI). The right testis of each fish was sectioned for histopathological assessment and to calculate the testes index (IT). The left testis was used for computer-assisted sperm analysis (CASA). The histopathological assessment of the testes showed histopathological changes such as of melano-macrophage centres (MMCs) and vacuolation of spermatogonia and spermatocytes. However, the classification of these changes indicated that the testes tissue structure was normal with slight histological changes. No statistically significant differences (p > 0.05) were found in the CASA parameters between exposure groups. The results of this study showed that the environmentally relevant concentrations of aldrin and methoxychlor did not have a negative effect on the motility of the mature sperm, but adverse effects were noted in the early stages of spermatogenesis, indicating possible effects over longer exposure periods.

Histological evidence that diploid hybrids of Cobitis taenia and C. elongatoides (Teleostei, Cobitidae) develop into fertile females and sterile males

In Europe, two bisexual fish species, Cobitis taenia (TT) and Cobitis elongatoides (EE), hybridized, producing diploid and polyploid clonal lineages. This study compared, for the first time, embryonic development, hatching success, sex ratio, body size and as well as reproductive ability based on the gonad histology of F1 reciprocal diploid hybrids (TE, ET) of both species. Hybrid F1 progeny showed the same proper pattern of embryonic and larval development. Among TE and ET offspring, slightly more females and males, respectively, occurred, but sex parity among 18-month-old hybrids was observed. Two- and three-year-old F1 hybrid females were mature, possessing all stages of oogenesis in their ovaries. Females (TE) back-crossed with C. taenia males had properly developing progeny. In testes of two- and three-year-old F1 hybrids, only early stages of spermatogenesis and pyknotic cells indicating the degeneration process were observed, but they exhibited an external feature (lamina circularis) of maturation. The results were confirmed by the structure of gonads that two sexual species, C. taenia and C. elongatoides, hybridize, producing F1 progeny of sterile males and fertile females. Hybrid females may participate in subsequent steps of speciation via hybridization and polyploidy or, on the contrary, represent an element of new species isolation.