CXADR: From an Essential Structural Component to a Vital Signaling Mediator in Spermatogenesis.

Canonical coxsackievirus and adenovirus receptor (CXADR) is a transmembrane component of cell junctions that is crucial for cardiac and testicular functions via its homophilic and heterophilic interaction. CXADR is expressed in both Sertoli cells and germ cells and is localized mainly at the interface between Sertoli-Sertoli cells and Sertoli-germ cells. Knockout of CXADR in mouse Sertoli cells specifically impairs male reproductive functions, including a compromised blood-testis barrier, apoptosis of germ cells, and premature loss of spermatids. Apart from serving as an important component for cell junctions, recent progress has showed the potential roles of CXADR as a signaling mediator in spermatogenesis. This review summarizes current research progress related to the regulation and role of CXADR in spermatogenesis as well as in pathological conditions. We hope this review provides some future directions and a blueprint to promote the further study on the roles of CXADR.

Unraveling the effect of the inflammatory microenvironment in spermatogenesis progression.

Experimental autoimmune orchitis (EAO) is a chronic inflammatory disorder that causes progressive spermatogenic impairment. EAO is characterized by high intratesticular levels of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) causing germ cell apoptosis and Sertoli cell dysfunction. However, the impact of this inflammatory milieu on the spermatogenic wave is unknown. Therefore, we studied the effect of inflammation on spermatogonia and preleptotene spermatocyte cell cycle progression in an EAO context and through the intratesticular DETA-NO and TNFα injection in the normal rat testes. In EAO, premeiotic germ cell proliferation is limited as a consequence of the undifferentiated spermatogonia (CD9+) cell cycle arrest in G2/M and the reduced number of differentiated spermatogonia (c-kit+) and preleptotene spermatocytes that enter in the meiotic S-phase. Although inflammation disrupts spermatogenesis in EAO, it is maintained in some seminiferous tubules at XIV and VII-VIII stages of the epithelial cell cycle, thereby guaranteeing sperm production. We found that DETA-NO (2 mM) injected in normal testes arrests spermatogonia and preleptotene spermatocyte cell cycle; this effect reduces the number of proliferative spermatogonia and the number of preleptotene spermatocytes in meiosis S-phase (36 h after). The temporal inhibition of spermatogonia clonal amplification delayed progression of the spermatogenic wave (5 days after) finally altering spermatogenesis. TNFα (0.5 and 1 µg) exposure did not affect premeiotic germ cell cycle or spermatogenic wave. Our results show that in EAO the inflammatory microenvironment altered spermatogenesis kinetics through premeiotic germ cell cycle arrest and that NO is a sufficient factor contributing to this phenomenon.

Changes in histology, protein expression, and autophagy in dairy goat testes during nonbreeding season†.

Seasonal reproduction contributes to increased chances of offspring survival in some animals. Dairy goats are seasonal breeding mammals. In this study, adult male Guanzhong dairy goats (10-12 months old) were used. Testis size, semen quality, hormone level, apoptosis of germ cells, and autophagy of Sertoli cells were analyzed in dairy goats during the breeding (October) and nonbreeding (April) seasons. We found that, during the nonbreeding season for dairy goats, semen quality, follicle-stimulating hormone (FSH) levels, and testosterone levels were reduced, and the number of apoptotic germ cells increased. The proliferation with decrease activity of germ cells in dairy goat during the nonbreeding season was significantly affected. However, the testis size did not change seasonally. Interestingly, Sertoli cell autophagy was more active during the nonbreeding season. The expression levels of FSH receptor, wilms tumor 1, androgen binding protein, glial cell derived neurotrophic factor, and stem cell factor decreased in dairy goats during the nonbreeding season. In summary, our results indicate that spermatogenesis in dairy goats during the nonbreeding season was not completely arrested. In addition, germ cell apoptosis and the morphology of Sertoli cells considerably changed in dairy goats during the nonbreeding season. Sertoli cell autophagy is involved in the seasonal regulation of spermatogenesis in dairy goats. These findings provide key insights into the fertility and spermatogenesis of seasonal breeding animals.

Rubicon prevents autophagic degradation of GATA4 to promote Sertoli cell function.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.

MicroRNA-125a-5p modulates the proliferation and apoptosis of TM4 Sertoli cells by targeting RAB3D and regulating the PI3K/AKT signaling pathway.

Sertoli cells provide protection and nutrition for developing sperm. Each stage of sperm development occurs on the surface of Sertoli cells. MicroRNA (MiR)-125a-5p is involved in male reproduction. The current research aimed to probe the role of miR-125a-5p in Sertoli cell function. Functionally, miR-125a-5p knockdown facilitated Sertoli cell proliferation, while miR-125a-5p overexpression suppressed Sertoli cell proliferation, as evidenced by 5-ethynyl-20-deoxyuridine incorporation assay. Additionally, miR-125a-5p knockdown inhibited Sertoli cell apoptosis, while miR-125a-5p upregulation facilitated Sertoli cell apoptosis, as evidenced by flow cytometry analysis. Computationally, we identified four predicted mRNA targets of miR-125a-5p. Based on the results of luciferase reporter assay, miR-125a-5p was confirmed to bind to the predicted sequence in the Ras-related protein Rab-3D (RAB3D) 3'UTR. Rescue experiments showed that miR-125a-5p suppressed the proliferative ability of TM4 Sertoli cells and facilitated their apoptosis by targeting RAB3D. Finally, our data confirmed that miR-125a-5p and RAB3D modulated activation of the PI3K/AKT pathway. In conclusion, our data showed that miR-125a-5p regulated Sertoli cell proliferation and apoptosis by targeting RAB3D and regulating the PI3K/AKT pathway.

Effect of EPA on Neonatal Pig Sertoli Cells "In Vitro": A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies.

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 µM vs SCs and ***p < 0.001 at 3.33µM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 µM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 µM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

Neonatal maternal separation increases the number of p27-positive Sertoli cells in prepuberty.

Neonatal maternal separation (NMS) is an experimental model for early life stress, which affects the growth and development of various organs, resulting in adverse health effects in humans and animals. In our previous study, we demonstrated that NMS [(0.5-, 1-, 2-h/day NMS, from postnatal day (PND) 1-10] induced morphological changes to the male reproductive system, including decreased Sertoli cell numbers in mouse testes at PND 70. To clarify the mechanism by which NMS decreases Sertoli cell numbers, we evaluated the effects of NMS on mouse testes at PNDs 10 and 16. At PND 10, the Sertoli cell number was not significantly different among experimental groups; however, it decreased in 0.5- and 2-h/day NMS mice at PND 16. The termination of Sertoli cell proliferation in prepuberty can be induced by p27, a cyclin-dependent kinase inhibitor. At PND 10, we observed an increase in the number of p27-positive Sertoli cells in 2-h/day NMS mice. The seminiferous tubule diameters decreased significantly in 1- and 2-h/day NMS mice, and the relative interstitial area increased in 2-h/day NMS mice. Serum corticosterone level significantly increased, and serum testosterone level significantly decreased in the 2-h/day NMS mice. At PND 16, the tubule diameters and height of seminiferous epithelium were significantly higher in 0.5- and 2-h/day NMS mice. Our results suggest that NMS disturbs serum corticosterone and testosterone levels and increases the number of p27-positive Sertoli cells at PND 10, resulting in a decrease in the number of Sertoli cells at PND 16.

Di-n-butyl phthalate diminishes testicular steroidogenesis by blocking the hypothalamic-pituitary-testicular axis: relationship with germ cell apoptosis in Japanese quail.

Although di-n-butyl phthalate (DBP) induces germ cell apoptosis, the underlying mechanism is not yet clear in quail. In this study, prepubertal quails were given a single dose of 500mg kg-1 DBP by gavage and were then killed 3, 6 and 24h after treatment. There was a significant reduction in intratesticular testosterone (ITT) concentrations and testicular steroidogenic enzyme mRNA expression and a significant increase in germ cell apoptosis in DBP-treated compared with control quails at all time points. Maximum apoptosis was detected 6h after treatment and the maximum reduction in testosterone concentrations was at 3h. To investigate whether DBP suppressed testicular steroidogenesis by affecting the hypothalamic-pituitary-testicular axis, we analysed pituitary LH subunit ß (Lhb) mRNA expression and serum LH concentrations. At all time points, pituitary Lhb expression and serum LH concentrations were significantly decreased following DBP treatment. The present observations suggest the possibility that DBP blocked LH secretion from the hypothalamus and/or pituitary, thereby decreasing LH stimulation of Leydig cells and reducing ITT concentrations. DBP-induced decreases in ITT concentrations may cause changes to the physical structure of Sertoli cells, which, in turn, may induce germ cell apoptosis.

Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men.

Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as "non-self" by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is "outside" the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses.

Low retinoic acid levels mediate regionalization of the Sertoli valve in the terminal segment of mouse seminiferous tubules.

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator: lesson from the testis.

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood-testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell-cell and also Sertoli-spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

Wild-type p53-induced phosphatase 1 (WIP1) regulates the proliferation of swine Sertoli cells through P53.

Wild-type p53-induced phosphatase 1 (WIP1) plays an oncogenic function by increasing cell proliferation in various cancer types. Deficiency in WIP1 expression leads to male infertility, possibly by impairing the blood-testis barrier and spermatogenesis. However, how WIP1 functions in the Sertoli cells to affect male reproduction remains unclear. Thus, in the present study we used a swine Sertoli cell line to investigate whether WIP1 regulated the proliferation of Sertoli cells to participate in male reproduction. The WIP1 inhibitor GSK2830371, WIP1-short interference (si) RNAs and an upstream microRNA (miR-16) were used to inhibit the expression of WIP1, after which the proliferation of swine Sertoli cells, P53 expression and the levels of P53 phosphorylation were determined. Inhibiting WIP1 expression suppressed swine Sertoli cell proliferation, increased P53 expression and increased levels of P53 phosphorylation. In addition, overexpression of miR-16 in swine Sertoli cells resulted in a decrease in WIP1 expression and increases in both P53 expression and P53 phosphorylation. Together, these findings suggest that WIP1 positively regulates the proliferation of swine Sertoli cells by inhibiting P53 phosphorylation, and the miR-16 is likely also involved by targeting WIP1.

Role of Akt and mammalian target of rapamycin signalling in insulin-like growth factor 1-mediated cell proliferation in porcine Sertoli cells.

The critical role of insulin-like growth factor (IGF) 1 in promoting Sertoli cell proliferation invivo and invitro has been established, but its downstream signalling mechanisms remain unknown. In addition to mitogenic effects, a role for IGF1 in mediating cholesterol biosynthesis within testes has been implied. The aims of this study were to investigate the roles of: (1) phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signalling in IGF1-mediated Sertoli cell proliferation; and (2) IGF1 in mediating cholesterol biosynthesis in Sertoli cells. Primary cultures of Sertoli cells were prepared from 1-week-old porcine testes. On Day 3 of culture, Sertoli cells were treated with 300ng mL-1 IGF1, alone or in combination with inhibitors of IGF1 receptor (2µM picropodophyllotoxin), Akt (1µM wortmannin) or mTOR (200nM rapamycin). Cells were cultured for 30min and phosphorylation levels of Akt, mTOR and p70 ribosomal protein S6 kinase (p70S6K) were determined by immunoblotting. Cell proliferation and quantitative polymerase chain reaction assays were conducted using cells cultured for 24h. IGF1 increased phosphorylation of Akt, mTOR and p70S6K and cell proliferation, and these effects were inhibited by inhibitors of IGF1R, Akt and mTOR. Furthermore, IGF1 upregulated the expression of cholesterol biosynthetic genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1) and cytochrome P450, family 5, subfamily A, polypeptide 1 (CYP5A1)), but not sterol regulatory element-binding transcription factor 1 (SREBF1). Increased phosphorylation of p70S6K, a major downstream target of mTOR, and upregulated expression of genes involved in cholesterol biosynthesis are indicative of the key role played by IGF1 in regulating the synthesis of cholesterol, the precursor for steroid hormones.

Molecular insights into hormone regulation via signaling pathways in Sertoli cells: With discussion on infertility and testicular tumor.

Spermatogenesis is a complex and elaborate differentiation process and is critical for male fertility. The hypothalamic-pituitary-gonadal axis serves as a significant neuroendocrine system to regulate spermatogenesis. As a constitute of the hypothalamic-pituitary-gonadal axis, Sertoli cells promote spermatogenesis via protecting, nourishing, and supporting germ cells upon hormone determination. Here we clarified how the hormones in the hypothalamic-pituitary-gonadal axis, including FSH, testosterone and LH, regulate spermatogenesis via the androgen receptor, cAMP/PKA, PI3k/Akt signaling pathways in Sertoli cells. Other endogenous hormones in higher vertebrates, including ouabain, estradiol, leptin, MIS, PGD2, and thyroid hormone, also regulate spermatogenesis via the AR or cAMP/PKA signaling pathway. Among them, the dynamics of adherens junctions, gap junctions, and blood-testis barrier, glucose uptake, lactate supply and differentiation of Sertoli cells are regulated by more comprehensive hormones and signaling pathways in Sertoli cells. In infertile patients or patients with blocked spermatogenesis, the AR, cAMP/PKA and PI3k/Akt signaling pathways and related components exhibit abnormal activity or disordered content. The clinical specimens from patients with testicular cancer show similar mutated AR genes. According to the existing clinical evidence, it is valuable to study the deep mechanism of male infertility and testicular tumors from the perspective of hormones and signaling pathways in Sertoli cells.

Oxidative stress mediates heat-induced changes of tight junction proteins in porcine sertoli cells via inhibiting CaMKKß-AMPK pathway.

Heat stress causes reversible changes in tight junction proteins in immature Sertoli cells via inhibition of the AMPK signaling pathway; these effects are accompanied by an increase in the early apoptotic rate and decrease in the cell viability of Sertoli cells. Since heat stress is known to also cause oxidative damage, in the present study, we investigated whether the earlier mentioned effects of heat stress were brought about via the induction of oxidative stress in boar Sertoli cells. Immature Sertoli cells obtained from 3-week-old piglets were subjected to heat treatment (43 °C, 30 min), and the percentage of ROS-positive cells, the malonaldehyde (MDA) concentration, and the activity of the antioxidases, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were measured. Next, the Sertoli cells were treated with N-acetyl-l-cysteine (NAC) (1 mmol/L, 2 h), an antioxidant agent, before they were exposed to heat stress. The effects of NAC on ROS accumulation, MDA levels, antioxidase activity, the CaMKKß-AMPK signaling pathway and expression of tight junction proteins were assessed. The results showed that heat stress reversibly increased the percentage of ROS-positive cells and MDA levels, and decreased the activity of SOD, GSH-Px, and CAT. Pretreatment with NAC abrogated these effects of heat stress. Additionally, NAC reversed the heat stress-induced decrease in the expression of CaMKKß and dephosphorylation of AMPK. NAC also obviously rescued the heat stress-induced downregulation of tight junction proteins (claudin-11, JAM-A, occludin, and ZO-1) both at the mRNA and protein level. In conclusion, the findings indicate that oxidative damage participates in heat stress-induced downregulation of tight junction proteins in Sertoli cells by inhibiting the CaMKKß-AMPK axis. Further, NAC reversed the effects of heat stress on tight junction proteins; this means that it has potential as a protective agent that can prevent reproductive dysfunction in boars under conditions of heat stress.

Transcriptome profile of bovine iPSCs derived from Sertoli Cells.

Although induced pluripotent stem cells (iPSCs) had been generated from several somatic cell types in cattle, their pluripotency and differentiation capacities after freezing/thawing, and the dysregulated transcripts involved in pathways critical for reprogramming were not investigated. Additionally, selection of proper source cells is critical for iPSC derivation because the residual influence of the somatic origin may variegate their differentiation propensity. Sertoli cells (SCs) have special properties suitable for iPSCs derivation. Herein bovine SCs were enriched from the cryopreserved testicular tissues and reprogrammed into iPSCs using lentivirus carrying yamanaka factors (OSKM). These iPSCs have typical morphology resembling human iPSCs and remain normal karyotypes. They can express alkaline phosphatase activity and common pluripotency markers with a low methylation in the promoter region of Nanog. They can also form embryoid bodies and teratomas that give rise to cells/tissues from three embryonic germ layers. Transcriptome profiling showed that the exogenous OSKM were silenced and 8009 dysregulated mRNAs were identified. The pluripotency, methyldioxygenase and anti-apoptosis genes were all upregulated but the apoptotic gene downregulated in these iPSCs. Bunch of pathways related to the reprogramming, inflammation and viral infection pathways were upregulated, while pathways associated with the differentiation, senescence, metabolism and apoptosis were downregulated in these cells. After cryopreservation/thawing, the recovered iPSCs remain strong pluripotency and differentiation capabilities. Together, iPSCs were derived from the bovine SCs isolated from the cryopreserved neonatal bull testis, pluripotency and differentiation capacities verified, iPSCs cryopreserved, cultured and again reverified for pluripotency and differentiation capacities.

Assessment of fresh and cryopreserved testicular tissues from (pre)pubertal boys during organ culture as a strategy for in vitro spermatogenesis.

STUDY QUESTION: Can the organ culture method be applied to both fresh and cryopreserved human (pre)pubertal testicular tissue as a strategy for in vitro spermatogenesis? SUMMARY ANSWER: Although induction of spermatogenesis was not achieved in vitro, testicular architecture, endocrine function and spermatogonial proliferation were maintained in both fresh and cryopreserved testicular tissues. WHAT IS KNOWN ALREADY: Cryopreservation of a testicular biopsy is increasingly offered as a fertility preservation strategy for prepubertal cancer patients. One of the proposed experimental approaches to restore fertility is the organ culture method, which, in the mouse model, successfully allows for in vitro development of spermatozoa from testicular biopsies. However, complete spermatogenesis from human prepubertal testicular tissue in such an organ culture system has not been demonstrated. STUDY DESIGN, SIZE, DURATION: Testicular tissue was collected from nine (pre)pubertal boys diagnosed with cancer (ranging from 6 to 14 years of age) admitted for fertility preservation before treatment. Testicular biopsies were either immediately processed for culture or first cryopreserved, using a controlled slow freezing protocol, and thawed before culture. Organ culture of testicular fragments was performed in two different media for a maximum period of 5 weeks, targeting early cellular events (viability, meiosis and somatic differentiation) in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Fresh and cryopreserved-thawed testis fragments (1-2 mm3) were cultured at a gas-liquid interphase (34°C, 5% CO2) in Minimum Essential Medium alpha + 10% knock-out serum replacement medium containing 10-7 M melatonin and 10-6 M retinoic acid, with or without 3 IU/L FSH/LH supplementation. The effect of culture conditions on testicular fragments was weekly assessed by histological evaluation of germ cell development and immunohistochemical identification of spermatogonia (using MAGEA4), proliferative status of spermatogonia and Sertoli cells (using proliferating cell nuclear antigen [PCNA]), intratubular cell apoptosis (by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and Sertoli cells maturation (using Anti-Müllerian Hormone [AMH] versus Androgen Receptor [AR]). Additionally, Leydig cells' functionality was determined by measuring testosterone concentration in the culture media supernatants. MAIN RESULTS AND THE ROLE OF CHANCE: Neither fresh nor cryopreserved human (pre)pubertal testicular fragments were able to initiate spermatogenesis in our organ culture system. Nonetheless, our data suggest that fresh and cryopreserved testicular fragments have comparable functionality in the described organ culture conditions, as reflected by the absence of significant differences in any of the weekly evaluated functional parameters. Additionally, no significant differences were found between the two tested media when culturing fresh and cryopreserved human testicular fragments. Although spermatogonia survived and remained proliferative in all culture conditions, a significant reduction of the spermatogonial population (P ≤ 0.001) was observed over the culture period, justified by a combined reduction of proliferation activity (P ≤ 0.001) and increased intratubular cell apoptosis (P ≤ 0.001). We observed a transient increase in Sertoli cell proliferative activity, loss of AMH expression (P ≤ 0.001) but no induction of AR expression. Leydig cell endocrine function was successfully stimulated in vitro as indicated by increased testosterone production in all conditions throughout the entire culture period (P ≤ 0.02). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although not noticeable in this study, we cannot exclude that if an optimized culture method ensuring complete spermatogenesis in human testicular fragments is established, differences in functional or spermatogenic efficiency between fresh and cryopreserved tissue might be found. WIDER IMPLICATIONS OF THE FINDINGS: The current inability to initiate spermatogenesis in vitro from cryopreserved human testicular fragments should be included in the counselling of patients who are offered testicular tissue cryopreservation to preserve fertility. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by EU-FP7-PEOPLE-2013-ITN 603568 `Growsperm'. None of the authors have competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganization.

Noncollagenous domain 1 (NC1)-peptide is a biologically active peptide derived from the C-terminal region of collagen α3(IV) chain, a structural constituent protein at the basement membrane in the rat testis, likely via proteolytic cleavage of matrix metalloproteinase 9. Studies have shown that this NC1 peptide regulates testis function by inducing Sertoli cell blood-testis barrier (BTB) remodeling and is also capable of inducing elongate spermatid exfoliation through its disruptive effects on the organization of actin- and microtubule (MT)-based cytoskeletons at these cell adhesion sites. However, the underlying molecular mechanism remains unknown. NC1 peptide was found to exert its biologic effects through an activation of small GTPase cell division control protein 42 homolog (Cdc42) because cooverexpression of the dominant negative mutant of Cdc42 [namely, Cdc42-T17N (via a single mutation of amino acid residue 17 from the N terminus from Thr to Asn by site-directed mutagenesis, making it constitutively inactive)] and NC1 peptide was able to block the NC1 peptide-induced Sertoli cell tight junction-permeability barrier disruption. Their cooverexpression also blocked the NC1 peptide-induced misdistribution of BTB-associated proteins at the cell-cell interface and also disruptive cytoskeletal organization of F-actin and MTs through changes in spatial expression of the corresponding actin and MT regulatory proteins. Interestingly, NC1 peptide was also found to induce an up-regulation of phosphorylated (p)-ribosomal protein S6 (rpS6) (namely, p-rpS6-S235/S236) and a concomitant down-regulation of p-Akt1/2 (namely, p-Akt1-S473 and p-Akt2-S474), but these changes could not be blocked by overexpression of Cdc42-T17N. More importantly, NC1 peptide-induced Cdc42 activation was effectively blocked by treatment of Sertoli cell epithelium with a p-Akt1/2 activator SC79, which is also capable of blocking NC1 peptide-induced down-regulation of p-Akt1-S473 and p-Akt2/S474, but not p-rpS6-S235/S236 up-regulation. In summary, these findings illustrate that Cdc42 is working downstream of the mammalian target of rapamycin complex 1/rpS6/Akt1/2 signaling pathway to support NC1 peptide-mediated effects on Sertoli cell function in the testis using the rat as an animal model.-Su, W., Cheng, C. Y. Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganization.

Assessment of Sertoli Cell Proliferation by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide and Sulforhodamine B Assays.

The correct functioning of Sertoli cells (SCs) is pivotal for successful spermatogenesis. They are major targets for hormones, endocrine disruptors, and other substances that men are subjected to every day. One of the main SC functions that quickly responds to a deleterious stimulus is proliferation. This is directly related to the in vivo capacity of these cells to sustain a good number of developing germ cells. The protocols in this article can be tested on SCs of different origin. For the case of human SCs from small human testicular biopsies, a short and simple protocol to isolate and culture these cells is provided. The other protocols discussed herein represent two different procedures, somewhat complementary, to assess SC proliferation. In brief, the sulforhodamine B assay allows the investigator to dye healthy fixed SCs maintained in culture. In the MTT assay, on the other hand, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by live SCs. These methods are mostly used to evaluate how SC proliferative activity responds to exposure to compounds such as toxicants or hormones. © 2019 by John Wiley & Sons, Inc.

Influence of Wilms' tumor suppressor gene WT1 on bovine Sertoli cells polarity and tight junctions via non-canonical WNT signaling pathway.

Sertoli cells (SCs) are polarized epithelial cells and provide a microenvironment for the development of germ cells (GCs). The Wilms' tumor suppressor gene WT1 which support spermatogenesis is expressed explicitly in SCs. This study investigated the effect of WT1 on the polarity and blood-testis barrier (BTB) formation of bovine SCs in order to provide theoretical and practical bases for the spermatogenic process in mammals. In this study, newborn calf SCs were used as research material, and the RNAi technique was used to knockdown the endogenous WT1. The results show that WT1 knockdown did not affect the proliferation ability of SCs, but down-regulated the expression of polarity-associated proteins (Par3, Par6b, and E-cadherin), junction-associated protein (occludin) and inhibits transcription of downstream genes (WNT4, JNK, αPKC, and CDC42) in non-canonical WNT signaling pathway. WT1 also altered ZO-1 and occludin protein distribution. Overexpression of WNT1 did not affect the expression of Par6b, E-cadherin, and occludin, whereas the non-canonical WNT signaling pathway inhibitors wnt-c59, CCG-1423, and GO-6983 down-regulated the expression of Par6b, E-cadherin, and occludin respectively. This study indicates that WT1 mediates the regulation of several proteins involved in bovine SCs polarity maintenance and intercellular tight junctions (TJ) by non-canonical WNT signaling pathway.