Germ Cell Proliferation Research Articles

A Potential Role for the Gsdf–eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective

AbstractGonadal soma–derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag “pull-down” assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor β–dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf–dynein and Gsdf–eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor β) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.

BRAF Controls the Effects of Metformin on Neuroblast Cell Divisions in C. elegans.

Metformin has demonstrated substantial potential for use in cancer treatments. Liver kinase B (LKB)-AMP-activated protein kinase (AMPK) and mTOR are reported to be the main targets of metformin in relation to its ability to prevent cancer cell proliferation. However, the role of metformin in the control of neoplastic cancer cell growth is possibly independent of LKB-AMPK and mTOR. Using C. elegans as a model, we found that the neuronal Q-cell divisions in L1-arrested worms were suppressed following metformin treatment in AMPK-deficient mutants, suggesting that the mechanism by which metformin suppresses these cell divisions is independent of AMPK. Our results showed that the mTOR pathway indeed played a role in controlling germ cell proliferation, but it was not involved in the neuronal Q-cell divisions occurring in L1-arrested worms. We found that the neuronal Q-cells divisions were held at G1/S cell stage by metformin in vivo. Additionally, we demonstrated that metformin could reduce the phosphorylation activity of BRAF and block the BRAF-MAPK oncogenesis pathway to regulate neuronal Q-cell divisions during L1 arrest. This work discloses a new mechanism by which metformin treatment acts to promote neuronal cancer prevention, and these results will help promote the study of the anticancer mechanisms underlying metformin treatments.

High temperature-induced masculinization in yellow catfish Tachysurus fulvidraco: A potential approach for environmental-friendly mono-sex production

Many environmental stressors, including stress temperature, high density, hypoxia, low food availability, high social pressure, and bright background colour, have been found to cause masculinization in fish. The environmental-friendly factors-induced masculinization provides a possibility of environmental-friendly mono-sex production for aquaculture. In the present work, we found high temperature (33.5 °C) or cortisol administration (300 mg kg−1 feed) covering the critical period of ovarian differentiation (15–38 days post-fertilization, DPF) caused partial masculinization of XX genotype in yellow catfish, via the application of sex-linked markers and successive histological analysis containing critical time window of ovarian and testicular differentiation and differentiated gonads, including 25 DPF, 35 DPF, 45 DPF, 65 DPF, and 125 DPH of XX female, XX male, and XY male. The masculinization modes in high temperature and cortisol administration groups were similar, including repression of primordial germ cells proliferation and retention of the ovarian cavity in XX pseudo-male. XX male was similar to XY male at both transcriptional and morphological levels, via RNA-seq analysis and morphological characteristics and gonadal histology respectively, providing a possibility to produce functional XX males and all-female XX population on a large-scale through an environmental-friendly approach for yellow catfish. The establishment and application of an environmental-friendly approach for mono-sex production will reduce the usage of steroid hormones and functionally similar chemicals in the aquaculture industry.

A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis elegans Somatic Gonad.

In Caenorhabditis elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. A strong loss-of-function mutation (T239I) affects the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. We conducted a genetic screen for suppressor mutations that restore germ cell proliferation in the T239I mutant background and isolated seven intragenic mutations, located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky channels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three of the suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. The mutations described here may be useful for elucidating the biochemical pathways that produce the active biomolecules transiting through soma–germline gap junctions.

Reactive oxygen species-evoked genotoxic stress mediates arsenic-induced suppression of male germ cell proliferation and decline in sperm quality

This study aimed to investigate whether genotoxic stress mediates arsenic (As)-induced decline in sperm quality. Mice drank ultrapure water containing NaAsO2 (15 mg/L) for 70 days. The mature seminiferous tubules and epididymal sperm count were reduced in As-exposed mice. Cell proliferation, determined by immunostaining with Ki67, was suppressed in As-exposed seminiferous tubules and GC-1 cells. PCNA, a proliferation marker, was reduced in As-exposed mouse testes. Cell growth index was decreased in As-exposed GC-1 cells. Flow analysis showed that As-exposed GC-1 cells were retarded at G2/M phase. CDK1 and cyclin B1 were reduced in As-exposed GC-1 cells and mouse testes. Additional experiment revealed that p-ATR, a marker of genotoxic stress, was elevated in As-exposed mouse testes and GC-1 cells. Accordingly, p-p53 and p21, two downstream molecules of ATR, were increased in As-exposed GC-1 cells. Excess reactive oxygen species (ROS), measured by immunofluorescence, and DNA-strand break, determined by Comet assay, were observed in As-exposed GC-1 cells. γH2AX, a marker of DNA-strand break, was elevated in As-exposed seminiferous tubules and GC-1 cells. NAC alleviated As-evoked DNA damage, genotoxic stress, cell proliferation inhibition and sperm count reduction. In conclusion, ROS-evoked genotoxic stress mediates As-induced germ cell proliferation inhibition and decline in sperm quality.

Igf3 is essential for ovary differentiation in zebrafish†.

Zebrafish gonadal sexual differentiation is an important but poorly understood subject. Previously, we have identified a novel insulin-like growth factor (Igf) named insulin-like growth factor 3 (Igf3) in teleosts. The importance of Igf3 in oocyte maturation and ovulation has been recently demonstrated by us in zebrafish. In this study, we have further found the essential role of Igf3 in gonadal sexual differentiation of zebrafish. A differential expression pattern of igf3 between ovary and testis during sex differentiation (higher level in ovary than in testis) was found in zebrafish. An igf3 knockout zebrafish line was established using TALENs-mediated gene knockout technique. Intriguingly, all igf3 homozygous mutants were males due to the female-to-male sex reversal occurred during sex differentiation. Further analysis showed that Igf3 did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells. Oocyte development was arrested at primary growth stage, and the ovary was gradually sex-reversed to testis before 60 day post fertilization (dpf). Such sex reversal was likely due to decreased germ cell proliferation by suppressing PI3K/Akt pathway in early ovaries of igf3 mutants. Estrogen is considered as a master regulator in fish sex differentiation. Here, we found that igf3 expression could be upregulated by estrogen in early stages of ovarian follicles as evidenced in in vitro treatment assays and cyp19a1a mutant zebrafish, and E2 failed to rescue the defects of igf3 mutants in ovarian development, suggesting that Igf3 may serve as a downstream factor of estrogen signaling in sex differentiation. Taken together, we demonstrated that Igf3 is essential for ovary differentiation in zebrafish.

Cisplatin and carboplatin result in similar gonadotoxicity in immature human testis with implications for fertility preservation in childhood cancer

BackgroundClinical studies indicate chemotherapy agents used in childhood cancer treatment regimens may impact future fertility. However, effects of individual agents on prepubertal human testis, necessary to identify later risk, have not been determined. The study aimed to investigate the impact of cisplatin, commonly used in childhood cancer, on immature (foetal and prepubertal) human testicular tissues. Comparison was made with carboplatin, which is used as an alternative to cisplatin in order to reduce toxicity in healthy tissues.MethodsWe developed an organotypic culture system combined with xenografting to determine the effect of clinically-relevant exposure to platinum-based chemotherapeutics on human testis. Human foetal and prepubertal testicular tissues were cultured and exposed to cisplatin, carboplatin or vehicle for 24 h, followed by 24–240 h in culture or long-term xenografting. Survival, proliferation and apoptosis of prepubertal germ stem cell populations (gonocytes and spermatogonia), critical for sperm production in adulthood, were quantified.ResultsCisplatin exposure resulted in a significant reduction in the total number of germ cells (− 44%, p < 0.0001) in human foetal testis, which involved an initial loss of gonocytes followed by a significant reduction in spermatogonia. This coincided with a reduction (− 70%, p < 0.05) in germ cell proliferation. Cisplatin exposure resulted in similar effects on total germ cell number (including spermatogonial stem cells) in prepubertal human testicular tissues, demonstrating direct relevance to childhood cancer patients. Xenografting of cisplatin-exposed human foetal testicular tissue demonstrated that germ cell loss (− 42%, p < 0.01) persisted at 12 weeks. Comparison between exposures to human-relevant concentrations of cisplatin and carboplatin revealed a very similar degree of germ cell loss at 240 h post-exposure.ConclusionsThis is the first demonstration of direct effects of chemotherapy exposure on germ cell populations in human foetal and prepubertal testis, demonstrating platinum-induced loss of all germ cell populations, and similar effects of cisplatin or carboplatin. Furthermore, these experimental approaches can be used to determine the effects of established and novel cancer therapies on the developing testis that will inform fertility counselling and development of strategies to preserve fertility in children with cancer.

17\u03b1-ethynylestradiol prevents the natural male-to-female sex change in gilthead seabream (Sparus aurata L.)

Exposure to 17α-ethynylestradiol (EE2, 5 μg/g food) impairs some reproductive events in the protandrous gilthead seabream and a short recovery period does not allow full recovery. In this study, spermiating seabream males in the second reproductive cycle (RC) were fed a diet containing 5 or 2.5 μg EE2/g food for 28 days and then a commercial diet without EE2 for the remaining RC. Individuals were sampled at the end of the EE2 treatment and then at the end of the RC and at the beginning of the third RC, 146 and 333 days after the cessation of treatment, respectively. Increased hepatic transcript levels of the gene coding for vitellogenin (vtg) and plasma levels of Vtg indicated both concentrations of EE2 caused endocrine disruption. Modifications in the histological organization of the testis, germ cell proliferation, plasma levels of the sex steroids and pituitary expression levels of the genes coding for the gonadotropin β-subunits, fshβ and lhβ were detected. The plasma levels of Vtg and most of the reproductive parameters were restored 146 days after treatments. However, although 50% of the control fish underwent sex reversal as expected at the third RC, male-to female sex change was prevented by both EE2 concentrations.

Cortisol induces masculinization of XX medaka through gonadal soma-derived growth factor (GSDF) and anti-Müllerian hormone receptor type 2 (AMHR2)

The medaka Oryzias latipes is a teleost fish with an XX/XY sex determination system similar to that of mammals. However, under high-temperature conditions, XX medaka are masculinized by an elevation of the key teleost glucocorticoid, cortisol. Cortisol inhibits female-type proliferation of germ cells and induces masculinization of XX medaka during gonadal sex differentiation. To identify masculinization mechanisms downstream of cortisol, we analysed the functions of gonadal soma-derived growth factor (gsdf) and anti-Müllerian hormone receptor type 2 (amhr2); these genes are known to play important roles in the inhibition of germ cell proliferation and male differentiation. We investigated the impact of gsdf and amhr2 on the proliferation of germ cells using gsdf knockout (KO) and amhr2 KO medaka. At hatching stage, loss of gsdf or amhr2 function recovered female-type proliferation in germ cells under cortisol treatment. Moreover, cortisol treatment of gsdf KO or amhr2 KO medaka did not induce masculinization of XX medaka. These results suggest that cortisol inhibits female-type proliferation of germ cells and induces masculinization of XX medaka through GSDF and AMHR2. This study thereby provides the first evidence that GSDF and AMHR2 are involved in cortisol-induced masculinization.

Loss of Drosophila E3 Ubiquitin Ligase Hyd Promotes Extra Mitosis in Germline Cysts and Massive Cell Death During Oogenesis.

The Drosophila hyperplastic disc (hyd) gene is the ortholog of mammalian tumor suppressor EDD, which is implicated in a wide variety of cellular processes, and its regulation is impaired in various tumors. It is a member of the highly conserved HECT family of E3 ubiquitin ligases, which directly attach ubiquitin to targeted substrates. In early works, it was shown that Drosophila Hyd may be a tumor suppressor because it is involved in the control of imaginal-disc cell proliferation and growth. In this study, we demonstrated that Hyd is also important for the regulation of female germ cell proliferation and that its depletion leads to additional germline cell mitoses. Furthermore, we revealed a previously unknown Hyd function associated with the maintenance of germ cells’ viability. A reduction in hyd expression by either mutations or RNA interference resulted in large-scale germ cell death at different stages of oogenesis. Thus, the analysis of phenotypes arising from the hyd deficiency points to Hyd’s role in the regulation of germline metabolic processes during oogenesis.

Optimization of porcine embryonic germ cell culture system.

Homologous feeder culture system can efficiently promote the proliferation of embryonic germ (EG) cells or embryonic stem (ES) cells while avoiding contamination by exogenous proteins and pathogens. In this study, we compared the potency of using homologous porcine embryonic fibroblasts (PEFs), gonadal stromal cells (GSCs), porcine adipose-derived stem cells (PASCs), or porcine amniotic fluid stem (PAFS) cells as feeder cells for porcine EG growth, with the commonly used mouse embryonic fibroblasts (MEFs). We compared the feeder cell growth rates; secretion of growth factors including stem cell factor (SCF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF); the effects of growth factors on porcine PGC growth; and EG growth rates when individual cells were used as feeders. Our results showed that feeder cells secreted limited amounts of growth factors, and supplementation of growth factors can significantly improve the formation of EG colonies and number of passages (P < 0.05). GSC and PEF were more suitable for EG growth because of their faster growth rate and their support on EG growth. In conclusion, this study identified novel homologous cells that can be used for EG production.

PRMT7 is involved in regulation of germ cell proliferation during embryonic stage

Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7−/− mice at E11.5. We also find that the size of testes is reduced in Prmt7−/− mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-β singling pathway is significantly changed in germ cells of Prmt7−/− mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.

Transcriptional Regulation of Müllerian Inhibiting Substance (MIS) and Establishment of a Gonadal Somatic Cell Line Using mis-GFP Transgenic Medaka (Oryzias latipes).

In vertebrate germ cell differentiation, gonadal somatic cells and germ cells are closely related. By analyzing this relationship, it has recently been reported in mammals that primordial germ cells (PGCs), induced from pluripotent stem cells and germline stem cells, can differentiate into functional gametes when co-cultured in vitro with fetal gonadal somatic cells. In some fish species, differentiation into functional sperm by reaggregation or co-culture of gonadal somatic cells and germ cells has also been reported; however, the relationship between gonadal somatic cells and germ cells in these species is not well-understood. Here, we report the transcriptional regulation of Müllerian inhibiting substance (MIS) and the establishment of a gonadal somatic cell line using mis-GFP transgenic fish, in medaka (Oryzias latipes)—a fish model which offers many advantages for molecular genetics. MIS is a glycoprotein belonging to the transforming growth factor β superfamily. In medaka, mis mRNA is expressed in gonadal somatic cells of both sexes before sex differentiation, and MIS regulates the proliferation of germ cells during this period. Using luciferase assays, we found that steroidogenic factor 1 (SF1) and liver receptor homolog 1 (LRH1) activate medaka mis gene transcription, probably by binding to the mis promoter. We also report that mis-GFP transgenic medaka emit GFP fluorescence specific to gonadal somatic cells in the gonads. By fusing Sertoli cells from transgenic medaka with a cell line derived from medaka hepatoma cancer, we produced a hybridoma cell line that expresses gonadal somatic cell-specific markers, including Sertoli and Leydig cell markers. Moreover, embryonic PGCs co-cultured with the established hybridoma, as feeder cells, proliferated and formed significant colonies after 1 week. PGCs cultured for 3 weeks expressed a germ cell marker dnd, as well as the meiotic markers sycp1 and sycp3. Thus, we here provide the first evidence in teleosts that we have successfully established a gonadal somatic cell-derived hybridoma that can induce both the proliferation and meiosis of germ cells.

Stromal cell-derived factor-1α predominantly mediates the ameliorative effect of linagliptin against cisplatin-induced testicular injury in adult male rats

Stromal cell-derived factor-1α (SDF-1α) plays a key role in trafficking of stem cells and regeneration of injured tissue through interaction with its receptor, CXCR4. This study investigated the probable therapeutic effect of linagliptin (LG) against cisplatin (CP)-induced testicular injury and the underlying mechanisms. 12 week old male Sprague-Dawley rats were randomly assigned into 6 groups (n = 10 each) as follow: (i) Control, (ii) LG-treated control, (iii) CP-exposed rats, (iv) CP-exposed rats received LG, (v) CP-exposed rats received AMD3100, as CXCR4 antagonist, and (vi) CP-exposed rats received AMD3100 prior to LG. After 15 days, blood, testes and epididymides were collected for analyses. There were significant increases in both circulatory and testicular levels of SDF-1α in LG-treated rats. Conversely, higher levels of incretin hormones were found in serum but not in testicular tissue of rats, following LG therapy. CP injection significantly reduced body, testicular and epididymal weights of rats, and were restored by LG therapy. Treatment of CP-exposed rats with LG improved the deteriorated testicular architecture, reconstructed spermatogenesis, increased sperm count and quality, and normalized testosterone levels. LG therapy increased gene expression of Lin28a and Mvh, but did not alter the expressions of somatic-related genes. Additionally, LG therapy promoted germ cells survival and proliferation likely via activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. These positive effects of LG therapy were almost blunted by administration of AMD3100. These results provided mechanistic insights into the ameliorative effect of LG on CP-induced testicular injury, through activation of SDF-1α/CXCR4 signaling pathway. Our findings suggest that LG can be a promising therapeutic candidate for CP-induced testicular injury.

Assessment of Yangtze sturgeon as recipient for the production of American paddlefish gametes through spermatogonia transplantation

The Chinese paddlefish (Psephurus gladius), one of the world’s largest freshwater fish, was last seen alive in 2003; they are presumed now to be extinct. In fish, germ cell transplantation is currently known as one of the most powerful assisted reproductive technologies for the conservation of endangered species. In the event that a Chinese paddlefish is unexpectedly caught in the near future, we aimed to develop an experimental strategy to produce paddlefish gametes in the gonads of surrogate sturgeon. Spermatogonia were collected from the testes of 2.5-year-old immature male American paddlefish (Polyodon spathula), the species most closely related to the Chinese paddlefish, by Percoll gradient centrifugation, and transplanted into the peritoneal cavity of Yangtze sturgeon (Acipenser dabryanus) larvae at 7–8 days post-hatch. At two months post-transplantation, donor-derived spermatogonia had efficiently colonized in the recipient gonads and proliferated. A PCR analysis developed to detect xenogenic donor-derived mtDNA sequences in recipient gonads revealed that American paddlefish germ cells survived for at least seven months after transplantation in the gonads of Yangtze sturgeon recipients. These results show that the somatic microenvironment of Yangtze sturgeon gonads was able to support the colonization, proliferation, and survival of xenogeneic germ cells from a different taxonomic family. This study provides key information that could lead to future restoration of Chinese paddlefish using germ cell transplantation.

Chronic exposure of BPA impairs male germ cell proliferation and induces lower sperm quality in male mice

BackgroundBisphenol A (BPA) is a well-known endocrine disruptor that affects male fertility. However, the main biological events through which BPA affects spermatogenesis remain to be identified. MethodsAdult male mice were treated by feeding with drinking water containing BPA (0.2 μg/ml, 20 μg/ml, 200 μg/ml, respectively) for two months. Testes were collected for protein extraction or for immunohistochemical analysis. Epididymal spermatozoa were collected for sperm quality evaluation and male fertility assay by in vitro fertility (IVF). Serums were collected for detection of testosterone levels. Proteins associated with germ cell proliferation, meiosis, blood-testis barrier, and steroidogenesis production were examined in BPA-treated and control mice testes. CCK8 assay was used to detect the effect of BPA on the proliferation of GC-1 and GC-2 cells. ResultsThe BPA-treated mice were characterized by decreased sperm quality, serum testosterone levels and, sub-fertile phenotype characterizing with low pregnancy rates and reduced fertilization efficiency. In lower BPA (0.2 μg/ml) treatment, PCNA and PLZF were down-expressed that indicated impaired germ cell proliferation. SYCP3 was down-expressed in BPA-treated mice, but expressions of other proteins associated with meiosis and blood-testis barrier were not significantly altered. CYP11A1 and HSD3B1 were down-expressed in BPA-treated mice that demonstrated reduced steroidogenesis activity. BPA has a concentration-dependent inhibition effect on the proliferation of GC-1 and GC-2 cells. Conclusively, low doses BPA exposure reduced mice sperm quality mainly by impairing germ cell proliferation, leading to reduced male fertility. The study would provide relevant information for investigation on molecular mechanisms and protective strategy on male production.

Anti-Müllerian hormone (Amh/amh) plays dual roles in maintaining gonadal homeostasis and gametogenesis in zebrafish

Anti-Müllerian hormone (AMH/Amh) plays a role in gonadal differentiation and function across vertebrates. In zebrafish we demonstrated that Amh deficiency caused severe gonadal dysgenesis and dysfunction. The mutant gonads showed extreme hypertrophy with accumulation of early germ cells in both sexes, namely spermatogonia in the testis and primary growth oocytes in the ovary. In amh mutant females, the folliculogenesis was normal in young fish but receded progressively in adults, which was accompanied by progressive decrease in follicle-stimulating hormone (fshb) expression. Interestingly the expression of fshb increased in the pituitary of juvenile amh mutant males but decreased in adults. The upregulation of fshb in mutant male juveniles was likely one of the mechanisms for triggering gonadal hypergrowth, whereas the downregulation of fshb in adults might involve a negative feedback by gonadal inhibin. Further analysis using mutants of fshb and growth differentiation factor 9 (gdf9) provided evidence for a role of FSH in triggering ovarian hypertrophy in young female amh mutant as well. In summary, the present study provided comprehensive genetic evidence for dual roles of Amh in controlling zebrafish gonadal homeostasis and gametogenesis in both sexes. Amh suppresses proliferation or accumulation of early germ cells (spermatogonia in testis and primary growth oocytes in ovary) while promoting their exit to advanced stages, and its action may involve both endocrine and paracrine pathways.

Stem cell niche exit in C. elegans via orientation and segregation of daughter cells by a cryptic cell outside the niche.

Stem cells reside in and rely upon their niche to maintain stemness but must balance self-renewal with the production of daughters that leave the niche to differentiate. We discovered a mechanism of stem cell niche exit in the canonical C. elegans distal tip cell (DTC) germ stem cell niche mediated by previously unobserved, thin, membranous protrusions of the adjacent somatic gonad cell pair (Sh1). A disproportionate number of germ cell divisions were observed at the DTC-Sh1 interface. Stem-like and differentiating cell fates segregated across this boundary. Spindles polarized, pairs of daughter cells oriented between the DTC and Sh1, and Sh1 grew over the Sh1-facing daughter. Impeding Sh1 growth by RNAi to cofilin and Arp2/3 perturbed the DTC-Sh1 interface, reduced germ cell proliferation, and shifted a differentiation marker. Because Sh1 membrane protrusions eluded detection for decades, it is possible that similar structures actively regulate niche exit in other systems.

Next-generation sequencing: toward an increase in the diagnostic yield in patients with apparently idiopathic spermatogenic failure.

A large proportion of patients with idiopathic spermatogenic failure (SPGF; oligozoospermia or nonobstructive azoospermia [NOA]) do not receive a diagnosis despite an extensive diagnostic workup. Recent evidence has shown that the etiology remains undefined in up to 75% of these patients. A number of genes involved in germ-cell proliferation, spermatocyte meiotic divisions, and spermatid development have been called into play in the pathogenesis of idiopathic oligozoospermia or NOA. However, this evidence mainly comes from case reports. Therefore, this study was undertaken to identify the molecular causes of SPGF. To accomplish this, 15 genes (USP9Y, NR5A1, KLHL10, ZMYND15, PLK4, TEX15, TEX11, MEIOB, SOHLH1, HSF2, SYCP3, TAF4B, NANOS1, SYCE1, and RHOXF2) involved in idiopathic SPGF were simultaneously analyzed in a cohort of 25 patients with idiopathic oligozoospermia or NOA, accurately selected after a thorough diagnostic workup. After next-generation sequencing (NGS) analysis, we identified the presence of rare variants in the NR5A1 and TEX11 genes with a pathogenic role in 3/25 (12.0%) patients. Seventeen other different variants were identified, and among them, 13 have never been reported before. Eleven out of 17 variants were likely pathogenic and deserve functional or segregation studies. The genes most frequently mutated were MEIOB, followed by USP9Y, KLHL10, NR5A1, and SOHLH1. No alterations were found in the SYCP3, TAF4B, NANOS1, SYCE1, or RHOXF2 genes. In conclusion, NGS technology, by screening a specific custom-made panel of genes, could help increase the diagnostic rate in patients with idiopathic oligozoospermia or NOA.

MOTILIPERM Ameliorates Immobilization Stress-Induced Testicular Dysfunction via Inhibition of Oxidative Stress and Modulation of the Nrf2/HO-1 Pathway in SD Rats.

It is well established that physiological stress has an adverse effect on the male reproductive system. Experimental studies have demonstrated the promising effects of MOTILIPERM in male infertility. MOTILIPERM extract is composed of three crude medicinal herbs: Morinda officinalis How (Rubiaceae) roots, Allium cepa L. (Liliaceae) outer scales, and Cuscuta chinensis Lamark (convolvulaceae) seeds. The present study aimed to investigate the possible mechanisms responsible for the effects of MOTILIPERM on testicular dysfunction induced by immobilization stress. Fifty male Sprague Dawley rats were divided into five groups (10 rats each): a normal control group (CTR), a control group administered MOTILIPERM 200 mg/kg (M 200), an immobilization-induced stress control group (S), an immobilization-induced stress group administered MOTILIPERM 100 mg/kg (S + M 100), and MOTILIPERM 200 mg/kg (S + M 200). Stressed rats (n = 30) were subjected to stress by immobilization for 6 h by placing them in a Perspex restraint cage, while controls (n = 20) were maintained without disturbance. Rats were administrated 100 or 200 mg/kg MOTILIPERM once daily for 30 days 1 h prior to immobilization. At the end of the treatment period, we measured body and reproductive organ weight; sperm parameters; histopathological damage; reproductive hormone levels; steroidogenic acute regulatory protein (StAR); biomarkers of oxidative stress; and apoptosis markers. MOTILIPERM treatment improved testicular dysfunction by up-regulating (p < 0.05) sperm count, sperm motility, serum testosterone level, StAR protein level, Johnsen score, and spermatogenic cell density in stressed rats. MOTILIPERM decreased oxidative stress by increasing (p < 0.05) testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione peroxidase-4 (GPx 4), catalase, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) levels and decreasing (p < 0.05) malondialdehyde (MDA) and reactive oxygen species/reactive nitrogen species (ROS/RNS) levels. Furthermore, MOTILIPERM down-regulated (p < 0.05) cleaved caspase 3 and BCL2 associated X protein (Bax) levels; increased pro caspase-3 and B-cell lymphoma 2 (Bcl-2) levels; and upregulated testicular germ cell proliferation in stressed rats. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels also significantly (p < 0.05) decreased after pretreatment with MOTILIPERM in stressed rats. Collectively, our results suggest that, in immobilization-mediated stress-induced testicular dysfunction, MOTILIPERM sustains normal spermatogenesis via antioxidant and anti-apoptotic activities by activating the NRF/HO-1 signaling pathway.