The molecular evolution of spermatogenesis across mammals.

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Comparative analysis of porcine iPSCs derived from Sertoli cells and fibroblasts.

Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor ß signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming.

4-[1-Ethyl-1-methylhexy]-phenol induces apoptosis and interrupts Ca2+ homeostasis via ROS pathway in Sertoli TM4 cells.

Biological effect of an individual nonylphenol (NP) isomer extremely relies upon the side chain structure. This research was designed to evaluate the impact of NP isomer, 4-[1-ethyl-1-methylhexy]-phenol (NP65), on Sertoli cells in vitro. Sertoli TM4 cells were exposed to various concentration (0, 0.1, 1, 10, or 20 µM) of NP65 for 24 h, and the outcomes indicated that treatment of NP65 induced reactive oxygen species (ROS) generation, oxidative stress, and apoptosis for Sertoli TM4 cells. In addition, it was found that NP65 exposure affected homeostasis of Ca2+ in Sertoli TM4 cells by increasing cytoplasm [Ca2+]i, inhibiting Ca2+-ATPase activity and decreasing cyclic adenosine monophosphate (cAMP) concentration. Pretreatment with ROS scavenger, N-acetylcysteine (NAC), attenuated NP65-induced oxidative stress as well as apoptosis for TM4 cells. Furthermore, NAC blocked NP65-induced disorders of Ca2+ homeostasis by attenuating the growth of intracellular [Ca2+]i and the inhibition of Ca2+-ATPase and cAMP activities. Thus, we have demonstrated that NP65 induced apoptosis as well as acted as a potent inhibitor of Ca2+-ATPase activity and resulted in disorder of Ca2+ homeostasis in Sertoli TM4 cells; ROS participated in the process. Our results supported the view that oxidative stress acted an essential role within the development of apoptosis and Ca2+ overload in TM4 cells as a consequence of NP65 stimulation.

Follicle-stimulating hormone-mediated decline in miR-92a-3p expression in pubertal mice Sertoli cells is crucial for germ cell differentiation and fertility.

Sertoli cells (Sc) are the sole target of follicle-stimulating hormone (FSH) in the testis and attain functional maturation post-birth to significantly augment germ cell (Gc) division and differentiation at puberty. Despite having an operational microRNA (miRNA) machinery, limited information is available on miRNA-mediated regulation of Sc maturation and male fertility. We have shown before that miR-92a-3p levels decline in pubertal rat Sc. In response to FSH treatment, the expressions of FSH Receptor, Claudin11 and Klf4 were found to be elevated in pubertal rat Sc coinciding with our finding of FSH-induced decline in miR-92a-3p levels. To investigate the association of miR-92a-3p and spermatogenesis, we generated transgenic mice where such pubertal decline of miR-92a-3p was prevented by its overexpression in pubertal Sc under proximal Rhox5 promoter, which is known to be activated specifically at puberty, in Sc. Our in vivo observations provided substantial evidence that FSH-induced decline in miR-92a-3p expression during Sc maturation acts as an essential prerequisite for the pubertal onset of spermatogenesis. Elevated expression of miR-92a-3p in post-pubertal testes results into functionally compromised Sc, leading to impairment of the blood-testis barrier formation and apoptosis of pre-meiotic Gc, ultimately culminating into infertility. Collectively, our data suggest that regulation of miR-92a-3p expression is crucial for Sc-mediated induction of active spermatogenesis at puberty and regulation of male fertility.

Pyroglutamylated RFamide peptide (QRFP): Role in early testicular development in relation to Sertoli cell maturation in prepubertal mice.

QRFP, an orexigenic neuropeptide, binds to its cognate receptor GPR103 and regulates various biological functions. We have recently shown that QRFP and its receptor are present in mice testes and that their expression is high during early postnatal period. The present study aimed to investigate the effect of sustained high level of QRFP on Sertoli cells proliferation and differentiation and to relate these events with germ cell differentiation and lumen formation in the seminiferous tubules in mice testes during prepubertal period. QRFP was injected intraperitoneally to male mice from postnatal day 5 to 16. Morphometric analysis and various markers related to Sertoli cell maturation (WT1, p27kip1, AMH, AR and CYP19A1) and germ cell proliferation and differentiation (PCNA, GDNF and c-Kit) were evaluated. QRFP administration caused an early lumen formation in the seminiferous tubules in testis of treated mice. Further, there was a significant increase in p27kip1 expression and a marked decrease in AMH expression in QRFP-treated mice compared to controls. However, no appreciable change was noted in AR expression in treated mice. QRFP treatment also caused an increase in c-Kit expression in treated mice compared to controls, suggesting an accelerated spermatogonial differentiation in testis of QRFP-treated mice. Taken together, the present results suggest that the prolonged high level of QRFP increases Sertoli cell maturation, which, in turn, plays a contributory role in increasing the pace of germ cell differentiation and formation of lumen in the seminiferous tubules.

Boosting effect of testosterone on GDNF expression in Sertoli cell line (TM4); comparison between TM3 cells-produced and exogenous testosterone.

The Glial cell-derived neurotrophic factor (Gdnf) and testosterone induce the spermatogonial stem cells (SSCs) self-renewal and spermatogenesis, respectively. In present study the stimulating role of testosterone on Sertoli cells to produce Gdnf, and the possible effect of Gdnf on Gfrα1 and c-RET expressions were investigated. The TM4 cells (line Sertoli cells) were co-cultured with [0.1, 0.2 and 0.4 (ng/ml)] of exogenous and TM3 (line Leydig cells)-produced testosterones, and consequently the TM4-produced Gdnf concentration was evaluated. Next, the SSCs were co-cultured with the TM-4 derived media (endogenous Gdnf) and exogenous Gdnf [0.1, 0.2, and 0.4 ng/ml)]. The 0.1 and 0.2 ng/ml endogenous and 3 concentrations of exogenous testosterone up-regulated the Gdnf expression versus non-treated Sertoli cells. The TM4-produced and exogenous Gdnfs, in all concentrations, up-regulated the receptors expression. In conclusion, the testosterone, solely, stimulates the Gdnf synthesis and the Gdnf, individually, amplifies its receptor's expression at mRNA and protein levels.

TNF-α/ENO1 signaling facilitates testicular phagocytosis by directly activating Elmo1 gene expression in mouse Sertoli cells.

Phagocytic clearance of apoptotic germ cells (GCs), as well as residual bodies released from developing spermatids, is critical for Sertoli cells (SCs) to maintain inner environment homeostasis within the testis. However, the molecular mechanisms controlling the phagocytosis are ill defined. Here, we identify a new role for alpha-enolase (ENO1), a key enzyme during glycolysis, as a molecule that facilitates testicular phagocytosis via transactivation of the engulfment and cell motility 1 (Elmo1) gene. Using immunohistochemistry and double-labeling immunofluorescence, ENO1 was observed to be expressed exclusively in the nuclei of SCs and its expression correlated with the completion of SC differentiation. By incubating TM4 cells with different pharmacological inhibitors and establishing TM4Tnfr1-/- cells, we demonstrated that SC-specific expression of ENO1 was under a delicate paracrine control from apoptotic GCs. In turn, persistent blockade of ENO1 expression by a validated small interfering RNA protocol resulted in the disturbance of spermatogenesis and impairment of male fertility. Furthermore, using ChIP, electrophoretic mobility shift and luciferase reporter assays, we showed that, in the presence of apoptotic GCs, ENO1 binds to the distal region of the Elmo1 promoter and facilitates transactivation of the Elmo1 gene. In agreement, overexpression of ELMO1 ameliorated ENO1 deficiency-induced impairment of phagocytosis in TM4 cells. These data reveal a novel role for SC-specific expression of ENO1 in regulating phagocytosis in testis, identify tumor necrosis factor-α and ELMO1 as critical upstream and downstream factors in mediating ENO1 action, and have important implications for our understanding of paracrine control of SC function by adjacent GCs.

Therapeutic application of Sertoli cells for treatment of various diseases.

Sertoli cells (SCs) are immune privileged cells found in the testis that function to immunologically protect maturing germ cells from immune destruction. This immune protection is due to the blood-testis-barrier, which prevents infiltration of cytotoxic immune cells and antibodies, and SC production of immunomodulatory factors, that favor a tolerogenic environment. The ability of SCs to create an immune privileged environment has led to the exploration of their potential use in the treatment of various diseases. SCs have been utilized to create a tolerogenic ectopic microenvironment, to protect co-grafted cells, and to deliver therapeutic proteins through gene therapy. To date, numerous studies have reported the potential use of SCs for the treatment of diabetes, neurodegenerative disorders, and restoration of spermatogenesis. Additionally, SCs have been investigated as a delivery vehicle for therapeutic products to treat other diseases like Laron syndrome, muscular dystrophy, and infections. This review will provide an overview of these therapeutic applications.

Protein kinase A drives paracrine crisis and WNT4-dependent testis tumor in Carney complex.

Large-cell calcifying Sertoli cell tumors (LCCSCTs) are among the most frequent lesions occurring in male Carney complex (CNC) patients. Although they constitute a key diagnostic criterion for this rare multiple neoplasia syndrome resulting from inactivating mutations of the tumor suppressor PRKAR1A, leading to unrepressed PKA activity, LCCSCT pathogenesis and origin remain elusive. Mouse models targeting Prkar1a inactivation in all somatic populations or separately in each cell type were generated to decipher the molecular and paracrine networks involved in the induction of CNC testis lesions. We demonstrate that the Prkar1a mutation was required in both stromal and Sertoli cells for the occurrence of LCCSCTs. Integrative analyses comparing transcriptomic, immunohistological data and phenotype of mutant mouse combinations led to the understanding of human LCCSCT pathogenesis and demonstrated PKA-induced paracrine molecular circuits in which the aberrant WNT4 signal production is a limiting step in shaping intratubular lesions and tumor expansion both in a mouse model and in human CNC testes.

KATNB1 is a master regulator of multiple katanin enzymes in male meiosis and haploid germ cell development.

Katanin microtubule-severing enzymes are crucial executers of microtubule regulation. Here, we have created an allelic loss-of-function series of the katanin regulatory B-subunit KATNB1 in mice. We reveal that KATNB1 is the master regulator of all katanin enzymatic A-subunits during mammalian spermatogenesis, wherein it is required to maintain katanin A-subunit abundance. Our data shows that complete loss of KATNB1 from germ cells is incompatible with sperm production, and we reveal multiple new spermatogenesis functions for KATNB1, including essential roles in male meiosis, acrosome formation, sperm tail assembly, regulation of both the Sertoli and germ cell cytoskeletons during sperm nuclear remodelling, and maintenance of seminiferous epithelium integrity. Collectively, our findings reveal that katanins are able to differentially regulate almost all key microtubule-based structures during mammalian male germ cell development, through the complexing of one master controller, KATNB1, with a 'toolbox' of neofunctionalised katanin A-subunits.

Sumoylation and its regulation in testicular Sertoli cells.

The molecular regulation of Sertoli cells and their crosstalk with germ cells has not been fully characterized. SUMO proteins are essential for normal development and are expressed in mouse and human Sertoli cells; However, the cell-specific role of sumoylation in those cells has only started to be elucidated. In other cell types, including granulosa cells, sumoylation is regulated by a SUMO ligase KAP1/Trim28. Deletion of KAP1 in Sertoli cells causes testicular degeneration; However, the role of KAP1 in those cells has not been identified. Here we show that both mouse and human Sertoli undergo apoptosis upon inhibition of sumoylation with a chemical inhibitor or via a siRNA technology. We have additionally detected changes in the Sertoli cell proteome upon the inhibition of sumoylation, and our data suggest that among others, the expression of ER/stress-related proteins is highly affected by this inhibition. Sumoylation may also regulate the NOTCH signaling which is important for the maintenance of the developing germ cells. Furthermore, we show that a siRNA-down-regulation of KAP1 in a Sertoli-derived cell line causes an almost complete inactivation of sumoylation. In conclusion, sumoylation regulates important survival and signaling pathways in Sertoli cells, and KAP1 can be a major regulator of sumoylation in these cells.

Direct reprogramming of human Sertoli cells into male germline stem cells with the self-renewal and differentiation potentials via overexpressing DAZL/DAZ2/BOULE genes.

We propose a new concept that human somatic cells can be converted to become male germline stem cells by the defined factors. Here, we demonstrated that the overexpression of DAZL, DAZ2, and BOULE could directly reprogram human Sertoli cells into cells with the characteristics of human spermatogonial stem cells (SSCs), as shown by their similar transcriptomes and proteomics with human SSCs. Significantly, human SSCs derived from human Sertoli cells colonized and proliferated in vivo, and they could differentiate into spermatocytes and haploid spermatids in vitro. Human Sertoli cell-derived SSCs excluded Y chromosome microdeletions and assumed normal chromosomes. Collectively, human somatic cells could be converted directly to human SSCs with the self-renewal and differentiation potentials and high safety. This study is of unusual significance, because it provides an effective approach for reprogramming human somatic cells into male germ cells and offers invaluable male gametes for treating male infertility.

Two distinct Sertoli cell states are regulated via germ cell crosstalk†.

Sertoli cells are a critical component of the testis environment for their role in maintaining seminiferous tubule structure, establishing the blood-testis barrier, and nourishing maturing germ cells in a specialized niche. This study sought to uncover how Sertoli cells are regulated in the testis environment via germ cell crosstalk in the mouse. We found two major clusters of Sertoli cells as defined by their transcriptomes in Stages VII-VIII of the seminiferous epithelium and a cluster for all other stages. Additionally, we examined transcriptomes of germ cell-deficient testes and found that these existed in a state independent of either of the germ cell-sufficient clusters. Altogether, we highlight two main transcriptional states of Sertoli cells in an unperturbed testis environment, and a germ cell-deficient environment does not allow normal Sertoli cell transcriptome cycling and results in a state unique from either of those seen in Sertoli cells from a germ cell-sufficient environment.

Long-Term Maintenance of Viable Adult Rat Sertoli Cells Able to Establish Testis Barrier Components and Function in Response to Androgens.

A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-ß3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions.

Genetic Regulation of Avian Testis Development.

As in other vertebrates, avian testes are the site of spermatogenesis and androgen production. The paired testes of birds differentiate during embryogenesis, first marked by the development of pre-Sertoli cells in the gonadal primordium and their condensation into seminiferous cords. Germ cells become enclosed in these cords and enter mitotic arrest, while steroidogenic Leydig cells subsequently differentiate around the cords. This review describes our current understanding of avian testis development at the cell biology and genetic levels. Most of this knowledge has come from studies on the chicken embryo, though other species are increasingly being examined. In chicken, testis development is governed by the Z-chromosome-linked DMRT1 gene, which directly or indirectly activates the male factors, HEMGN, SOX9 and AMH. Recent single cell RNA-seq has defined cell lineage specification during chicken testis development, while comparative studies point to deep conservation of avian testis formation. Lastly, we identify areas of future research on the genetics of avian testis development.

Temperature response of enriched pre-pubertal caprine male germline stem cells in vitro.

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-ß1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (ß1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.

AKAP9 supports spermatogenesis through its effects on microtubule and actin cytoskeletons in the rat testis.

In mammalian testes, extensive remodeling of the microtubule (MT) and actin cytoskeletons takes place in Sertoli cells across the seminiferous epithelium to support spermatogenesis. However, the mechanism(s) involving regulatory and signaling proteins remains poorly understood. Herein, A-kinase anchoring protein 9 (AKAP9, a member of the AKAP multivalent scaffold protein family) was shown to be one of these crucial regulatory proteins in the rat testis. Earlier studies have shown that AKAP9 serves as a signaling platform by recruiting multiple signaling and regulatory proteins to create a large protein complex that binds to the Golgi and centrosome to facilitate the assembly of the MT-nucleating γ-tubulin ring complex to initiate MT polymerization. We further expanded our earlier studies based on a Sertoli cell-specific AKAP9 knockout mouse model to probe the function of AKAP9 by using the techniques of immunofluorescence analysis, RNA interference (RNAi), and biochemical assays on an in vitro primary Sertoli cell culture model, and an adjudin-based animal model. AKAP9 robustly expressed across the seminiferous epithelium in adult rat testes, colocalizing with MT-based tracks, and laid perpendicular across the seminiferous epithelium, and prominently expressed at the Sertoli-spermatid cell-cell anchoring junction (called apical ectoplasmic specialization [ES]) and at the Sertoli cell-cell interface (called basal ES, which together with tight junction [TJ] created the blood-testis barrier [BTB]) stage specifically. AKAP9 knockdown in Sertoli cells by RNAi was found to perturb the TJ-permeability barrier through disruptive changes in the distribution of BTB-associated proteins at the Sertoli cell cortical zone, mediated by a considerable loss of ability to induce both MT polymerization and actin filament bundling. A considerable decline in AKAP9 expression and a disruptive distribution of AKAP9 across the seminiferous tubules was also noted during adjudin-induced germ cell (GC) exfoliation in this animal model, illustrating AKAP9 is essential to maintain the homeostasis of cytoskeletons to maintain Sertoli and GC adhesion in the testis.

Estradiol ameliorates metformin-inhibited Sertoli cell proliferation via AMPK/TSC2/mTOR signaling pathway.

Metformin is a commonly used for treating type 2 diabetes and it acts on a variety of organs including the male reproductive system. 17ß-estradiol plays an important role in Sertoli cell (SC) proliferation which determines the germ cell development and spermatogenesis. The aim of this study is to investigate the effect of metformin on immature chicken SC proliferation and the potential mechanisms by which 17ß-estradiol regulate this process. Results showed that metformin significantly inhibited SC proliferation, whereas 17ß-estradiol weakened the inhibitory effects of metformin on SC viability, cell growth, and cell cycle progression. SC proliferation-inhibiting effect of metformin exposure was regulated by decreasing adenosine triphosphate level and respiratory enzyme activity in the mitochondria; this process was possibly mediated by the adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) signaling pathway, which was regulated by the down-expressed miR-1764 and by the decreased antioxidant enzyme activity and excessive reactive oxygen species generation. In addition, SCs transfected with the miR-1764 agomir led to an improvement of proliferation capacity through down-regulating AMPKα2 level, which further decreased TSC2 expression and induced mTOR activation. However, the anti-proliferative effect of miR-1764 antagomir can be alleviated by 17ß-estradiol treatment via the up-expression of miR-1764 in transfected SCs. Our findings suggest appropriate dose of exogenous 17ß-estradiol treatment can ameliorate the inhibitory effect of metformin on SC proliferation via the regulation of AMPK/TSC2/mTOR signaling pathway, this might reduce the risk of poor male fertility caused by the abuse of anti-diabetic agents.

circBTBD7 Promotes Immature Porcine Sertoli Cell Growth through Modulating miR-24-3p/MAPK7 Axis to Inactivate p38 MAPK Signaling Pathway.

Sertoli cells are the crucial coordinators to guarantee normal spermatogenesis and male fertility. Although circular RNAs (circRNAs) exhibit developmental-stage-specific expression in porcine testicular tissues and have been thought of as potential regulatory molecules in spermatogenesis, their functions and mechanisms of action remain largely unknown, especially in domestic animals. A novel circBTBD7 was identified from immature porcine Sertoli cells using reverse transcription PCR, Sanger sequencing, and fluorescence in situ hybridization assays. Functional assays illustrated that circBTBD7 overexpression promoted cell cycle progression and cell proliferation, as well as inhibited cell apoptosis in immature porcine Sertoli cells. Mechanistically, circBTBD7 acted as a sponge for the miR-24-3p and further facilitated its target mitogen-activated protein kinase 7 (MAPK7) gene. Overexpression of miR-24-3p impeded cell proliferation and induced cell apoptosis, which further attenuated the effects of circBTBD7 overexpression. siRNA-induced MAPK7 deficiency resulted in a similar effect to miR-24-3p overexpression, and further offset the effects of miR-24-3p inhibition. Both miR-24-3p overexpression and MAPK7 knockdown upregulated the p38 phosphorylation activity. The SB202190 induced the inhibition of p38 MAPK pathway and caused an opposite effect to that of miR-24-3p overexpression and MAPK7 knockdown. Collectively, circBTBD7 promotes immature porcine Sertoli cell growth through modulating the miR-24-3p/MAPK7 axis to inactivate the p38 MAPK signaling pathway. This study expanded our knowledge of noncoding RNAs in porcine normal spermatogenesis through deciding the fate of Sertoli cells.

Age-related histomorphometric and ultrastructural changes in the Sertoli cells of Japanese quail (Coturnix Coturnix japonica).

Prepared sections from pre-pubertal, pubertal, adult, and aged Japanese quail testes were examined by light microscopy and transmission electron microscopy (TEM) and measurements of seminiferous tubular diameter (STD), luminal diameter (SLD), epithelial height (SEH) and cross-sectional area of the seminiferous tubules (AST) were taken using an image analyzer. Apoptotic Sertoli cells with features such as cell shrinkage and chromatin condensation were observed in pre-pubertal and aged quail. There was a significant difference between the mean Sertoli cell number (SCN), SLD, SEH, STD and AST among the four age groups (P < 0.001). The highest SCN (mean ± standard error) was recorded in the adult (30.53 ± 0.42), with the aged group displaying the lowest mean (11.80 ± 0.27) SCN. Spearman's rho correlation coefficients demonstrated a strong relationship between the SCN and SEH in the pubertal (ρ=0.915; P < 0.001), adult (ρ=0.878; P < 0.001), and aged (ρ=0.858; P < 0.001) groups, while a significant moderate correlation was observed in the pre-pubertal (ρ=0.606; P < 0.001) group. There were significant moderate correlations between the SCN and STD in the pre-pubertal (ρ=0.445; P < 0.001), pubertal (ρ=0.653; P < 0.001), adult (ρ=0.440; P < 0.001), and aged (ρ=0.514; P < 0.001) groups. Furthermore, significant moderate correlations were estimated between the SCN and AST in the pre-pubertal (ρ=0.453; P < 0.001), pubertal (ρ=0.661; P < 0.001), adult (ρ=0.393; P = 0.001), and aged (ρ=0.498; P < 0.001) groups. This study provides baseline data on the morphology and development of the Sertoli cell, as well as testicular morphometry in avian species during the pre-pubertal, pubertal, adult, and aged stages using the Japanese quail as a model.