PI3K/AKT/mTOR pathway mediated-cell cycle dysregulation contribute to malignant proliferation of mouse spermatogonia induced by microcystin-leucine arginine.

Environmental cyanotoxin exposure may be a trigger of testicular cancer. Activation of PI3K/AKT/mTOR signaling pathway is the critical molecular event in testicular carcinogenesis. As a widespread cyanotoxin, microcystin-leucine arginine (MC-LR) is known to induce cell malignant transformation and tumorigenesis. However, the effects of MC-LR on the regulatory mechanism of PI3K/AKT/mTOR pathway in seminoma, the most common testicular tumor, are unknown. In this study, mouse spermatogonia cell line (GC-1) and nude mice were used to investigate the effects and mechanisms of MC-LR on the malignant transformation of spermatogonia by nude mouse tumorigenesis assay, cell migration invasion assay, western blot, and cell cycle assay, and so forth. The results showed that, after continuous exposure to environmentally relevant concentrations of MC-LR (20 nM) for 35 generations, the proliferation, migration, and invasion abilities of GC-1 cells were increased by 120%, 340%, and 370%, respectively. In nude mice, MC-LR-treated GC-1 cells formed tumors with significantly greater volume (0.998 ± 0.768 cm3 ) and weight (0.637 ± 0.406 g) than the control group (0.067 ± 0.039 cm3 ; 0.094 ± 0.087 g) (P < .05). Furthermore, PI3K inhibitor Wortmannin inhibited the PI3K/AKT/mTOR pathway and its downstream proteins (c-MYC, CDK4, CCND1, and MMP14) activated by MC-LR. Blocking PI3K alleviated MC-LR-induced cell cycle disorder and malignant proliferation, migration and invasive of GC-1 cells. Altogether, our findings suggest that MC-LR can induce malignant transformation of mouse spermatogonia, and the PI3K/AKT/mTOR pathway-mediated cell cycle dysregulation may be an important target for malignant proliferation. This study provides clues to further reveal the etiology and pathogenesis of seminoma.

Morphometric and immunohistochemical analysis as a method to identify undifferentiated spermatogonial cells in adult subjects with Klinefelter syndrome: a cohort study.

OBJECTIVE: To study the prevalence of spermatogonia in adult subjects with Klinefelter syndrome (KS) using MAGE-A4 and UCHL1 (PGP9.5) immunohistochemistry as markers for undifferentiated spermatogonial cells. We aimed to compare this method to the gold standard of hematoxylin and eosin (H & E) staining with histologic analysis in the largest reported cohort of adult subjects with KS. DESIGN: A retrospective cohort study. SETTING: Infertility Clinic and Institute for Regenerative Medicine. PATIENT(S): This study consisted of 79 adult subjects with KS and 12 adult control subjects. INTERVENTION(S): The subjects with KS (n = 79) underwent bilateral testicular biopsy in an initial effort to recover spermatozoa for in vitro fertilization and intracytoplasmic sperm injection. The institutional review board approved the use of a portion of the archived diagnostic pathology paraffin blocks for the study. The samples were superimposed onto microscopic slides and labeled with the PGP9.5 and MAGE-A4 antibodies. Subjects (n = 12) who had previously consented to be organ donors via the National Disease Research Interchange were selected as controls. Dedicated genitourinary pathologists examined the H & E-, PGP9.5-, and MAGE-A4-stained tissue for presence of undifferentiated spermatogonia and spermatozoa with the use of a virtual microscopy software. MAIN OUTCOME MEASURE(S): The primary outcome was the presence of MAGE-A4-positive or UCHL1-positive tubules that indicate undifferentiated spermatogonia. Supportive outcomes include assessing the biopsy specimen for the following: total surface area; total seminiferous tubule surface area; total interstitium surface area; the total number of seminiferous tubules; and MAGE-A4- negative or UCHL1-negative tubules. Additionally, clinical information, such as age, karyotype, height, weight, mean testicle size, and hormonal panel (luteinizing hormone, follicle-stimulating hormone, and testosterone), was obtained and used in a single and multivariable analysis with linear regression to determine predictive factors for the number of UCHL1-positive tubules. RESULT(S): The mean age of the subjects in the KS group was 32.9 ± 0.7 years (range, 16-48). UCHL1 (PGP9.5) and MAGE-A4 staining showed that 74.7% (n = 59) and 40.5% (n = 32) of the subjects with KS, respectively, were positive for undifferentiated spermatogonia compared with 100% (n = 12) of the control subjects who were positive for both the markers. Hematoxylin and eosin with microscopic analysis showed that only 10.1% (n = 8) of the subjects were positive for spermatogonia. The mean number of positive tubules per subject with KS was 11.8 ± 1.8 for UCHL1 and 3.7 ± 1.0 for MAGE-A4. Secondary analysis showed 7 (8.9%) adult subjects with KS as positive for spermatozoa on biopsy. The population having negative testicular sperm extraction results (n = 72) showed a spermatogonia-positive rate of 1.4%, (n = 1), 72.2% (n = 52), and 34.7% (n = 25) using H & E, UCHL1, and MAGE-A4, respectively. Further analysis showed that 54 (75.0%) subjects were either positive for UCHL1 or MAGE-A4. Twenty (27.8%) subjects were positive for both UCHL1 and MAGE-A4. Multivariate analysis with linear regression showed no significant correlation between clinical variables and the number of UCHL1-positive tubules found on biopsy specimens. CONCLUSION(S): We report a cohort of adult subjects with KS undergoing analysis for the presence of undifferentiated spermatogonia. UCHL1 and MAGE-A4 immunostaining appear to be an effective way of identifying undifferentiated spermatogonia in testicular biopsy specimens of subjects with KS. Despite observing deterioration in the testicular architecture, many patients remain positive for undifferentiated spermatogonia, which could be harvested and potentially used for infertility therapy in a patient with KS who is azoospermic and has negative testicular sperm extraction results.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Dissecting mammalian spermatogenesis using spatial transcriptomics.

Single-cell RNA sequencing has revealed extensive molecular diversity in gene programs governing mammalian spermatogenesis but fails to delineate their dynamics in the native context of seminiferous tubules, the spatially confined functional units of spermatogenesis. Here, we use Slide-seq, a spatial transcriptomics technology, to generate an atlas that captures the spatial gene expression patterns at near-single-cell resolution in the mouse and human testis. Using Slide-seq data, we devise a computational framework that accurately localizes testicular cell types in individual seminiferous tubules. Unbiased analysis systematically identifies spatially patterned genes and gene programs. Combining Slide-seq with targeted in situ RNA sequencing, we demonstrate significant differences in the cellular compositions of spermatogonial microenvironment between mouse and human testes. Finally, a comparison of the spatial atlas generated from the wild-type and diabetic mouse testis reveals a disruption in the spatial cellular organization of seminiferous tubules as a potential mechanism of diabetes-induced male infertility.

Single-cell RNA-seq unravels alterations of the human spermatogonial stem cell compartment in patients with impaired spermatogenesis.

Despite the high incidence of male infertility, only 30% of infertile men receive a causative diagnosis. To explore the regulatory mechanisms governing human germ cell function in normal and impaired spermatogenesis (crypto), we performed single-cell RNA sequencing (>30,000 cells). We find major alterations in the crypto spermatogonial compartment with increased numbers of the most undifferentiated spermatogonia (PIWIL4+). We also observe a transcriptional switch within the spermatogonial compartment driven by increased and prolonged expression of the transcription factor EGR4. Intriguingly, the EGR4-regulated chromatin-associated transcriptional repressor UTF1 is downregulated at transcriptional and protein levels. This is associated with changes in spermatogonial chromatin structure and fewer Adark spermatogonia, characterized by tightly compacted chromatin and serving as reserve stem cells. These findings suggest that crypto patients are disadvantaged, as fewer cells safeguard their germline's genetic integrity. These identified spermatogonial regulators will be highly interesting targets to uncover genetic causes of male infertility.

Curcumin nanocrystals attenuate cyclophosphamide-induced testicular toxicity in mice.

The cancer therapy using cyclophosphamide (CP) has been associated with adverse effects on the testicular function that raises concerns about the future fertility potential among cancer survivors. Curcumin, a polyphenol, has shown to possess a plethora of biological functions including tissue protective effects. In the present study, we investigated the protective effects of curcumin nanocrystals (NC) in mitigation of CP-induced testicular toxicity. Healthy adult (8-10 week) and prepubertal (2 week) male Swiss albino mice were injected with a single dose of CP (200 mg/kg) intraperitoneally (i.p). NC (4 mg/kg, i.p.) was administered every alternate day, for 35 days in adult mice while, a single dose of NC was injected intraperitoneally to prepubertal mice 1 h prior to CP. Administration of multiple doses of NC ameliorated CP-induced testicular toxicity in adult mice, which was evident from the improved sperm functional competence, sperm chromatin condensation, seminiferous tubule architecture and decreased apoptosis in testicular cells. Further, administration of NC 1 h prior to CP in prepubertal mice modulated the expression of genes pertaining to proliferation, pluripotency, DNA damage and DNA repair in spermatogonial cells at 24 h after the treatment. Overall, these results suggest that NC could be a promising chemoprotective agent, which can have potential application in male fertility preservation.

Dietary biotin supplementation increases proliferation pathways in mice testes without affecting serum follicle-stimulating hormone levels and stem cell factor expression.

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1-/- mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1-/- mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1-/- compared to wild-type mice. Furthermore, cultured Uch-l1-/- SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

Formation of spermatogonia and fertile oocytes in golden hamsters requires piRNAs.

PIWI-interacting RNAs (piRNAs) support the germline by suppressing retrotransposons. Studies of the pathway in mice have strongly shaped the view that mammalian piRNAs are essential for male but not for female fertility. Here, we report that the role of the piRNA pathway substantially differs in golden hamsters (Mesocricetus auratus), the piRNA pathway setup of which more closely resembles that of other mammals, including humans. The loss of the Mov10l1 RNA helicase-an essential piRNA biogenesis factor-leads to striking phenotypes in both sexes. In contrast to mice, female Mov10l1-/- hamsters are sterile because their oocytes do not sustain zygotic development. Furthermore, Mov10l1-/- male hamsters have impaired establishment of spermatogonia accompanied by transcriptome dysregulation and an expression surge of a young retrotransposon subfamily. Our results show that the mammalian piRNA pathway has essential roles in both sexes and its adaptive nature allows it to manage emerging genomic threats and acquire new critical roles in the germline.

Responses and coping methods of different testicular cell types to heat stress: overview and perspectives.

To facilitate temperature adjustments, the testicles are located outside the body cavity. In most mammals, the temperature of the testes is lower than the body temperature to ensure the normal progression of spermatogenesis. Rising temperatures affect spermatogenesis and eventually lead to a decline in male fertility or even infertility. However, the testes are composed of different cell types, including spermatogonial stem cells (SSCs), spermatocytes, spermatozoa, Leydig cells, and Sertoli cells, which have different cellular responses to heat stress. Recent studies have shown that using different drugs can relieve heat stress-induced reproductive damage by regulating different signaling pathways. Here, we review the mechanisms by which heat stress damages different cells in testes and possible treatments.

Expression of the C-terminal region of the SSX protein is a useful diagnostic biomarker for spermatocytic tumour.

AIMS: Spermatocytic tumour (ST) is a rare testicular germ cell neoplasm with few confirmatory biomarkers that can be challenging to diagnose. Like normal spermatogonia, STs are known to express SSX proteins. Recently, a novel SSX antibody directed against a conserved C-terminal region of SSX1, SSX2 and SSX4 (SSX_CT) has emerged as a reliable biomarker for these SSX proteins and synovial sarcoma. However, SSX_CT immunostaining has not been demonstrated in ST. The aim of this study was to assess the diagnostic utility of SSX_CT immunohistochemistry in ST and other tumours in the differential diagnosis with ST. METHODS AND RESULTS: SSX_CT, OCT3/4 and c-KIT immunohistochemistry was performed on 15 STs, 38 seminomas, 13 embryonal carcinomas, 12 yolk sac tumours, six choriocarcinomas, four teratomas, seven Sertoli cell tumours, and six lymphomas. Staining was scored as negative, rare, focal, or diffuse. SSX_CT was positive in all (15/15) STs, and diffusely positive in 14 of 15 (93%). SSX_CT was positive in 22 of 38 (58%) seminomas; however, only two cases showed diffuse expression. SSX_CT was negative in all other tumours. OCT3/4 was negative in all STs, but positive in all seminomas and embryonal carcinomas. c-KIT was frequently positive in both STs (12/15; 80%) and seminomas (33/38; 87%). OCT3/4 and c-KIT were negative in all other tumours. CONCLUSIONS: SSX_CT is a valuable and highly sensitive biomarker that supports the diagnosis of ST. Diffuse expression of SSX-CT in STs is also highly specific for ST. Nevertheless, SSX_CT is best used in combination with OCT3/4 when ST is in the differential diagnosis.

A method for utilizing automated machine learning for histopathological classification of testis based on Johnsen scores.

We examined whether a tool for determining Johnsen scores automatically using artificial intelligence (AI) could be used in place of traditional Johnsen scoring to support pathologists' evaluations. Average precision, precision, and recall were assessed by the Google Cloud AutoML Vision platform. We obtained testicular tissues for 275 patients and were able to use haematoxylin and eosin (H&E)-stained glass microscope slides from 264 patients. In addition, we cut out of parts of the histopathology images (5.0 × 5.0 cm) for expansion of Johnsen's characteristic areas with seminiferous tubules. We defined four labels: Johnsen score 1-3, 4-5, 6-7, and 8-10 to distinguish Johnsen scores in clinical practice. All images were uploaded to the Google Cloud AutoML Vision platform. We obtained a dataset of 7155 images at magnification 400× and a dataset of 9822 expansion images for the 5.0 × 5.0 cm cutouts. For the 400× magnification image dataset, the average precision (positive predictive value) of the algorithm was 82.6%, precision was 80.31%, and recall was 60.96%. For the expansion image dataset (5.0 × 5.0 cm), the average precision was 99.5%, precision was 96.29%, and recall was 96.23%. This is the first report of an AI-based algorithm for predicting Johnsen scores.

Z-scores for comparative analyses of spermatogonial numbers throughout human development.

OBJECTIVE: To normalize age-dependent effects on standardized measures of spermatogonial quantity such as the number of spermatogonia per tubular cross-section (S/T) or fertility index. DESIGN: Published quantitative histologic data on human spermatogonial numbers were used to create Z-scores for reference means and tested on archived testicular tissue samples. SETTING: Retrospective cohort study. PATIENT(S): The sample cohort comprised testicular samples from 24 boys with cancer diagnosis and 10 with Klinefelter syndrome, as part of the fertility preservation programs NORDFERTIL and Androprotect, as well as archived histologic samples from 35 prepubertal boys with acute lymphoblastic leukemia and 20 testicular biobank samples. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Z-score values for S/T and fertility index on the basis of morphology and germ cell-specific markers (MAGEA4 and/or DDX4) were calculated, and the impact of cancer therapy exposure and genetic disorders on Z-score values was evaluated. RESULT(S): The Z-scores for S/T values in the nontreated samples (-2.08 ± 2.20, n = 28) and samples treated with nonalkylating agents (-1.90 ± 2.60, n = 25) were comparable within ±3 standard deviations of the reference mean value but differed significantly from samples exposed to alkylating agents (-12.14 ± 9.20, n = 22) and from patients with Klinefelter syndrome (-11.56 ± 4.89, n = 8). The Z-scores for S/T were correlated with increasing cumulative exposure to alkylating agents (r = -0.7020). CONCLUSION(S): The Z-score values for S/T allow for the quantification of genetic and cancer treatment-related effects on testicular tissue stored for fertility preservation, facilitating their use for patient counseling.

Constitutive activation of CTNNB1 results in a loss of spermatogonial stem cell activity in mice.

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal.

Evaluation of multi-epitope recombinant protein as a candidate for a contraceptive vaccine.

Contraceptive vaccine (CV) is a valuable, non-invasive, and alternative method for purposeful contraception. Sperm antigens are useful targets for producing CVs due to their specialized expression in sperm. In this study, a recombinant protein containing three main sperm epitopes (IZUMO1, SACA3, and PH-20) was designed and evaluated as CV to control fertility in male mice. The chimeric recombinant protein was expressed and purified in E. coli. Male mice were immunized by 100 µg purified protein and sera were collected to assess IgG antibodies. Evaluating the reproductive performance, immunized male mice mated with normal-fertile female mice and mating rate and the number of newborns was studied. Immunized mice were sacrificed and necropsy and histopathology studies were conducted. The results revealed that the designed chimeric protein stimulated the immune system of the mice effectively. The level of IgG antibody was significantly higher in vaccinated mouse rather than control mouse. Eighty percent of the vaccinated mice became infertile and in the remaining ones, the number of children decreased to 4-6 offspring instead of 10-12 in normal mice. Histopathological studies showed that no organs including heart, brain, lung, liver, kidney and intestine were damaged. However, Normal spermatogenesis has been disrupted and necrotic spermatogonia cells were reported in Seminiferous tubules. We concluded that the designed chimeric protein containing IZUMO1, SACA3, and PH-20 epitopes can stimulate the immune system and cause male contraception without any side effects.

Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice.

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions.

Insulin-like 3 affects zebrafish spermatogenic cells directly and via Sertoli cells.

Pituitary hormones can use local signaling molecules to regulate target tissue functions. In adult zebrafish testes, follicle-stimulating hormone (Fsh) strongly increases the production of insulin-like 3 (Insl3), a Leydig cell-derived growth factor found in all vertebrates. Little information is available regarding Insl3 function in adult spermatogenesis. The Insl3 receptors Rxfp2a and 2b were expressed by type A spermatogonia and Sertoli and myoid cells, respectively, in zebrafish testis tissue. Loss of insl3 increased germ cell apoptosis in males starting at 9 months of age, but spermatogenesis appeared normal in fully fertile, younger adults. Insl3 changed the expression of 409 testicular genes. Among others, retinoic acid (RA) signaling was up- and peroxisome proliferator-activated receptor gamma (Pparg) signaling was down-regulated. Follow-up studies showed that RA and Pparg signaling mediated Insl3 effects, resulting in the increased production of differentiating spermatogonia. This suggests that Insl3 recruits two locally active nuclear receptor pathways to implement pituitary (Fsh) stimulation of spermatogenesis.

Human X chromosome exome sequencing identifies BCORL1 as contributor to spermatogenesis.

BACKGROUND: Infertility affects approximately 15% of couples worldwide with male infertility being responsible for approximately 50% of cases. Although accumulating evidence demonstrates the critical role of the X chromosome in spermatogenesis during the last few decades, the expression patterns and potential impact of the X chromosome, together with X linked genes, on male infertility are less well understood. METHODS: We performed X chromosome exome sequencing followed by a two-stage independent population validation in 1333 non-obstructive azoospermia cases and 1141 healthy controls to identify variant classes with high likelihood of pathogenicity. To explore the functions of these candidate genes in spermatogenesis, we first knocked down these candidate genes individually in mouse spermatogonial stem cells (SSCs) using short interfering RNA oligonucleotides and then generated candidate genes knockout mice by CRISPR-Cas9 system. RESULTS: Four low-frequency variants were identified in four genes (BCORL1, MAP7D3, ARMCX4 and H2BFWT) associated with male infertility. Functional studies of the mouse SSCs revealed that knocking down Bcorl1 or Mtap7d3 could inhibit SSCs self-renewal and knocking down Armcx4 could repress SSCs differentiation in vitro. Using CRISPR-Cas9 system, Bcorl1 and Mtap7d3 knockout mice were generated. Excitingly, Bcorl1 knockout mice were infertile with impaired spermatogenesis. Moreover, Bcorl1 knockout mice exhibited impaired sperm motility and sperm cells displayed abnormal mitochondrial structure. CONCLUSION: Our data indicate that the X-linked genes are associated with male infertility and involved in regulating SSCs, which provides a new insight into the role of X-linked genes in spermatogenesis.

MicroRNA-449a Suppresses Mouse Spermatogonia Proliferation via Inhibition of CEP55.

At present, infertile patients with maturation arrest (MA) are difficult to obtain mature sperm. Spermatogenesis and its molecular mechanism are still not clear. Patients with MA and normal spermatogenesis (NS) were collected. iTRAQ-based proteomic approach was performed to reveal the different proteins between them. To validate the confidence of proteome data, the individual samples were analyzed by Western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence. The miR-449a and CEP55 were determined by Luciferase assay. Mouse GC-1 cells were transfected with CEP55 siRNAs, miR-449a mimic, or inhibitor, and cell proliferation was determined. Compared with NS, 27 proteins were differentially expressed in MA, and CEP55 protein was the most significant difference. WB and qPCR showed that CEP55 levels were significantly elevated in NS than MA. In transfected cells, overexpression of miR-449a and knockdown of CEP55 both downregulated CEP55 expression and decreased cell proliferation. miR-449a suppresses mouse spermatogonia proliferation via inhibition of CEP55.