Germ Cell Survival Research Articles

Single-cell transcriptome profiling reveals several LncRNAs differentially expressed in idiopathic germ cell aplasia.

Mechanisms underlying severe male infertility are still largely elusive. However, recently, a single-cell transcription study by our group identified several differentially expressed coding genes in all the somatic cell types in testes of patients with idiopathic germ cell aplasia (iGCA). Here, we leverage this work by extending the analysis also to the non-coding portion of the genome. As a result, we found that 43 LncRNAs were differentially expressed in the somatic cells of these patients. Interestingly, a significant portion of the overexpressed LncRNAs was found to be a target of TAF9B, a transcription factor known to be involved in germ cell survival. Moreover, several overexpressed LncRNAs were also found to be activated in a mouse model of Sertoli cells treated with bisphenol A, a widespread environmental contaminant, long suspected to impair male fertility. Finally, a literature search for MEG3, a maternally imprinted LncRNA overexpressed as well in our patients, found it to be involved, among other things, in obesity and inflammation, known comorbidities of iGCA, ultimately suggesting that our findings deepen the understanding of the molecular insights coupled not only to the pathogenesis, but also to the clinical course of this class of patients.

Testicular Sertoli Cell Hormones in Differences in Sex Development.

The Sertoli cells of the testes play an essential role during gonadal development, in addition to supporting subsequent germ cell survival and spermatogenesis. Anti-Müllerian hormone (AMH) is a member of the TGF-β superfamily, which is secreted by immature Sertoli cells from the 8th week of fetal gestation. lnhibin B is a glycoprotein, which is produced by the Sertoli cells from early in fetal development. In people with a Difference or Disorder of Sex Development (DSD), these hormones may be useful to determine the presence of testicular tissue and potential for spermatogenesis. However, fetal Sertoli cell development and function is often dysregulated in DSD conditions and altered production of Sertoli cell hormones may be detected throughout the life course in these individuals. As such this review will consider the role of AMH and inhibin B in individuals with DSD.

P-468 Organotypic culture of testicular tissue from infant boys with cryptorchidism

Abstract Study question Can organotypic culture support the survival and maturation of germ cells and niche-related cells within testicular tissue from infant boys with cryptorchidism? Summary answer The testicular structure and the number of germ cells were maintained during organotypic culture, whereas Sertoli cells and peritubular myoid cells (PTMCs) matured. What is known already Testicular tissue cryopreservation (TTC) is a strategy to safeguard the fertility of prepubertal boys who face a risk of infertility. Organotypic culture of immature testicular tissue from mice achieved production of spermatozoa. Similarly, the culture of human fetal gonads resulted in the generation of competent spermatids. However , in vitro spermatogenesis by organotypic culture of human prepubertal testicular tissue has not been achieved. It is also unknown whether germ cells as well as its niche-related cells, in testicular tissue from infant boys with cryptorchidism, can maintain and mature under in vitro conditions. Study design, size, duration Testicular tissue was cryopreserved from four infant boys with bilateral cryptorchidism undergoing orchidopexy (age range: 0.5-1.4 years), as part of a fertility preservation program. Culture media with and without retinoic acid were tested. Testicular fragments were harvested at 30 days and 60 days after culture and evaluated by histological assessment of tissue structure, germ cell development, and immunohistochemical staining for germ cell and somatic cell markers. Participants/materials, setting, methods Cryopreserved-thawed testicular tissue was cut into fragments (1-2 mm3) and placed on top of agarose gel stands and cultured at 34oC with 5% CO2 in Minimum Essential Medium-alpha supplemented with knockout serum replacement, human umbilical cord plasma, Activin A, hormones, growth factors, with or without retinoic acid. Immunohistochemical analyses were performed using germ cell markers (MAGE-A, GAGE, and VASA), Sertoli cell maturation markers (AMH, AR, SOX9), PTMC marker (alpha-SMA). Main results and the role of chance Following the 60-day culture, the lumen of the seminiferous tubules had developed. The number of germ cells per tubule remained stable during this period. However, no further germ cell maturation was observed. Germ cells showed different phenotypes of MAGEA, GAGE, and VASA expression with no significant difference in number. The number of SOX9-positive Sertoli cells was significantly increased from 30 days to 60 days of culture (p <0.0001). No difference in AMH expression was observed, while AR expression in Sertoli cells was induced during culture. Alpha-SMA expression was detected in the PTMCs surrounding the seminiferous tubules. The two different culture conditions, with and without retinoic acid in the culture media, did not affect cell survival or maturation. Limitations, reasons for caution The small number of testicular biopsies available is a limitation. Wider implications of the findings Our organotypic culture conditions support the long-term survival of germ cells in testicular tissue from infant boys with cryptorchidism. Thus, further studies are needed to induce the maturation of germ cells under similar experimental conditions. Trial registration number not applicable

P-804 Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal mammalian testicular tissue: a comparative study

Abstract Study question What is the impact of different static and dynamic organotypic culture systems on the outcome of prepubertal porcine immature testicular tissue (ITT) culture? Summary answer Using a hybrid silicone-agarose static culture system improves germ cell survival and differentiation in ITT ex-vivo. What is known already In mice, organotypic culture of ITT led to the birth of healthy fertile pups. In other larger mammals, including humans, it remains challenging to efficiently reproduce spermatogenesis ex-vivo. With the advent of tissue engineering, culture systems are becoming more complex to better mimic the in vivo environment by modifying and/or improving physical culture conditions (i.e. tissue perfusion, oxygen diffusion, direct/indirect contact with medium). Recent studies in rodents showed that by using hybrid static and dynamic silicone-based organotypic culture systems, the efficiency of spermatogenesis can be significantly improved. Such culture systems have not been reported in large mammals yet. Study design, size, duration Testes were collected from three 5-7 days old piglets as a by-product of castration for a Belgian farm. The fresh ITT fragments were immediately used for culture experiments. Fragments were cultured for a 30 days duration in the different static i.e. polytetrafluoroethylene membrane inserts (CT), agarose gel (AG) and agarose gel with polydimethylsiloxane cover (AGPC) and dynamic i.e. microfluidic (MF) systems using a single medium. The CT system served as control. Participants/materials, setting, methods Cultured ITT fragments were monitored every 4-5 days by serial imaging. Analyses were performed at days 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity and diameters were evaluated on Periodic Acid Schiff-stained slides. Immunohistochemistry was used to identify: germ (PGP9.5, VASA, SYCP3, CREM), somatic (SOX9, INSL3) and proliferating (Ki67) cells and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured using ELISA. A two-way ANOVA was used for statistical analyses. Main results and the role of chance All systems supported the survival, growth and maturation of ITT fragments in vitro. Between days 0 and 30, ST diameters increased in all systems (p < 0.000) (day 0: 61.2±6.5μm; day 30: CT: 75.3±18.1μm, AG: 77.7±15.7μm, AGPC: 84.9±13.6μm, 66.9±12.9μm). Sertoli cell (SOX9) numbers also increased while spermatogonia (PGP9) numbers per ST decreased (day 0: 6.94±0.31, day 30: CT: 1.84±0.64, AG: 2.65±0.88, AGPC: 3.47±0.7, MF: 2.11±0.84). Meiotic germ cells (SYCP3, CREM) were absent at day 0 and were generated starting day 5 (SYCP3) and 10 (CREM) in the different systems and maintained till day 30 (SYCP3 per ST; CT: 0.31±0.21, AG: 0.5±0.31, AGPC: 0.69±0.20, MF: 0.38±0.20 and CREM per ST: CT: 0.18±0.18, AG: 0.25±0.13, AGPC: 0.34±0.32, MF: 0.14±0.14). When compared to control (CT), significantly larger STs (days 10 to 30), a higher number of spermatogonia (PGP9) (day 30; AGPC: 3.72±0.70, CT: 1.84±0.64, p = 0.04), meiotic SYCP3 + (day 20, AGPC: 0.95±0.49, CT: 0.46±0.39, p = 0.01) and CREM+ cells (day 20, AGPC: 0.49±0.21, CT: 0.23±0.64, p = 0.02) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cells (Ki67/VASA), Leydig cells (INSL3) and testosterone levels were not significantly different between the different systems over the culture period. Limitations, reasons for caution The small sample size and inter-tissue variability could have limited the results of this study. Also it is not excluded that further differences, in terms of tissue growth, germ cell survival and differentiation, could be observed between the different systems if a more complex culture medium is used. Wider implications of the findings In contrast to rodents, modifying physical culture conditions minimally impacts the outcome of porcine ITT maturation ex-vivo. Due to anatomo-physiological similarities between pigs and humans, it is likely for similar results to be observed if applied to human ITT. Studies aiming at determining optimal culture medium components are rather needed. Trial registration number not applicable

O-095 The persistent Y hypothesis

Abstract The Y chromosome has been touted as a wimpy relic of the X, with its survival dependent on a few critical functions in spermatogenesis and sex determination, the loss of which would signal its demise. Why then has it survived since its origin (∼165 MYA) in all but a handful of therian mammal species? This is in stark contrast to the high turnover of sex chromosomes observed in other vertebrate linages, so the mammal Y turns out to be an exception of persistence, rather than the rule. Here we propose a novel explanation for such perseverance. The Y chromosome bears so-called ‘executioner genes’ that are critical for successful meiotic progression. Their expression is required to initiate meiotic sex chromosome inactivation, but then must be subject to this very silencing they induce to ensure germ cell survival. Whenexecutioners are translocated to an autosome they escape meiotic silencing and, being pachytene-lethal, cease meiosis. Therefore, these meiotic executioners act as their own judge, jury and executioner, posing strong evolutionary constraint for the Y chromosome to persist in eutherian mammals.

P-088 Standardized fabrication of murine testicular organoids with improved germ cell survival

Abstract Study question Can our newly developed testicular organoid (TO) growth platform advance the robustness of murine TOs? Summary answer The platform resulted in more consistent TO histology. Moreover, improved germ cell survival was observed after a two-week culture with numbers comparable to fresh samples. What is known already Organ cultures have traditionally been used for in-vitro spermatogenesis (IVS) in rodents because they best preserve the testicular architecture which is pivotal in achieving IVS. However, organ cultures do not offer the ability to access and manipulate single cells, making it an inefficient model for mechanistic studies. Organoids made from testicular cell suspensions offer these features. Although TO cultures can result in organoids with compartmentalized testicular architecture, histological heterogeneity between individual TOs limits reproducibility of the results, offering unreliable readouts. Moreover, germ cell loss is characteristic during the reorganization phase. Study design, size, duration Here, we tested a new TO growth platform. Firstly, the focus was put on improving germ cell survival in TOs during tubulogenesis in the first two weeks of culture. For this, four different growth media (A-D), supplemented with other combinations or concentrations of growth factors, were compared. Next, five cell seeding densities (I-V) were tested for their ability to recreate the testicular architecture in TOs in the selected culture media. Participants/materials, setting, methods Testicular cells from 5 days old C57BL/6J mice were grown in our TO platform with alpha-MEM-based medium, previously found to support TO generation in mice (medium A). Three additional conditions were tested in their ability to improve germ cell survival during tubulogenesis (B-D). Finally, the ideal cell density (I-V) was determined based on histological resemblance to native tissue: one tubule-like structure and surrounding interstitium. Cellular reorganization and germ cell maintenance were characterized by (immuno)histochemistry. Main results and the role of chance During short-term cultures of 2 weeks, testicular cells self-assembled and compacted into organoids in our platform. Interestingly, media B and D resulted in the highest amount of germ cells (p < 0.05), comparable to the fresh control. Particularly TOs cultured in medium D also exhibited the largest surface area, indicative for better in-vitro growth. Finally, TOs that were cultured in condition D had the best histology when grown at cell density IV and V (p < 0.05). Limitations, reasons for caution Candidate factors have to be tested in their ability to elevate the meiotic blockage of germ cells typically observed in organ culture, but also in TOs. Finally, results obtained with rodents remain to be confirmed in further human studies. Wider implications of the findings The opportunities TOs offer to manipulate cells (genetic modification, inclusion and exclusion) are essential for the study of male infertility and the search for potential therapies. Moreover, they permit high-throughput screening of chemicals, thereby substantially reducing the number of animals for the high demanding reproductive toxicity and drug discovery studies. Trial registration number not applicable

Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.

Background: In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.

Syntaxin binding protein 2 in sertoli cells regulates spermatogonial stem cell maintenance through directly interacting with connexin 43 in the testes of neonatal mice.

Interactions between Sertoli and germ cells are essential for spermatogenesis and male fertility. However, the mechanism of action underlying these interactions in the testes remains largely unknown. In this study, we investigated the distribution and function of syntaxin binding protein 2 (STXBP2) in the mammalian testis. METHODS ANDRESULTS: First, we found that STXBP2 was mainly expressed in Sertoli cells. Then, to explore the function of STXBP2 in the testes, we assessed the effects of Stxbp2 knockdown on neonatal testicular and spermatogonial stem cell (SSC) development. Our results revealed that STXBP2 was required for the migration of Sertoli cells and germ cell survival. Mechanistically, we found that STXBP2 interacted with connexin 43 (Cx43) and regulated its expression. Taken together, our results demonstrated a novel regulatory mechanism in which the STXBP2/Cx43 complex is essential for the maintenance of Sertoli-germline interactions.

Selective autophagic degradation of ACLY (ATP citrate lyase) maintains citrate homeostasis and promotes oocyte maturation

ABSTRACT Macroautophagy/autophagy is a cellular and energy homeostatic mechanism that contributes to maintain the number of primordial follicles, germ cell survival, and anti-ovarian aging. However, it remains unknown whether autophagy in granulosa cells affects oocyte maturation. Here, we show a clear tendency of reduced autophagy level in human granulosa cells from women of advanced maternal age, implying a potential negative correlation between autophagy levels and oocyte quality. We therefore established a co-culture system and show that either pharmacological inhibition or genetic ablation of autophagy in granulosa cells negatively affect oocyte quality and fertilization ability. Moreover, our metabolomics analysis indicates that the adverse impact of autophagy impairment on oocyte quality is mediated by downregulated citrate levels, while exogenous supplementation of citrate can significantly restore the oocyte maturation. Mechanistically, we found that ACLY (ATP citrate lyase), which is a crucial enzyme catalyzing the cleavage of citrate, was preferentially associated with K63-linked ubiquitin chains and recognized by the autophagy receptor protein SQSTM1/p62 for selective autophagic degradation. In human follicles, the autophagy level in granulosa cells was downregulated with maternal aging, accompanied by decreased citrate in the follicular fluid, implying a potential correlation between citrate metabolism and oocyte quality. We also show that elevated citrate levels in porcine follicular fluid promote oocyte maturation. Collectively, our data reveal that autophagy in granulosa cells is a beneficial mechanism to maintain a certain degree of citrate by selectively targeting ACLY during oocyte maturation. Abbreviations: 3-MA: 3-methyladenine; ACLY: ATP citrate lyase; AMA: advanced maternal age; CG: cortical granule; CHX: cycloheximide; CQ: chloroquine; CS: citrate synthase; COCs: cumulus-oocyte-complexes; GCM: granulosa cell monolayer; GV: germinal vesicle; MII: metaphase II stage of meiosis; PB1: first polar body; ROS: reactive oxygen species; shRNA: small hairpin RNA; SQSTM1/p62: sequestosome 1; TCA: tricarboxylic acid; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild-type

Desmoplakin (Dsp) conditional knockout in NR5A1+ somatic cells affects germ cell survival in developing mouse gonads.

Cell to cell interactions are crucial for morphogenesis and tissue formation. Desmoplakin (encoded by the Dsp gene) is a component of desmosomes and anchors the transmembrane adhesion proteins to the cytoskeleton. Its role in gonad development remains vague. To study the role of desmoplakin in gonad development, we used a tissue-specific knockout of the Dsp gene in the NR5A1+ somatic cells of the gonads. We show here that desmoplakin is necessary for the survival of germ cells in fetal testes and ovaries. The Dspknockout in NR5A1+ somatic cells in testes decreased the number of germ cells, and thus the size of the testes, but did not affect the Sertoli cells or the structure of testis cords and interstitium. The Dspknockout in NR5A1+ somatic cells in ovaries decreased the number of female germ cells and drastically reduced the formation of ovarian follicles. Dsp knockout in NR5A1+ somatic cells did not affect the sex determination and sexual differentiation of the gonads, as judged from an unchanged expression of genes essential for these processes. We conclude that mediation by desmoplakin cell adhesion between the gonadal cells is necessary for germ cell survival.

Small-Molecule-Driven Direct Reprogramming of Fibroblasts into Functional Sertoli-Like Cells as a Model for Male Reproductive Toxicology.

Sertoli cells (SCs) are vital to providing morphological and nutritional support for spermatogenesis. Defects in SCs often lead to infertility. SCs transplantation is a promising potential strategy to compensate for SC dysfunction. However, isolation of SCs from testes is impractical due to obvious and ethical limitations. Here, a molecular cocktail is identified comprising of pan-BET family inhibitor (I-BET151), retinoic acid, and riluzole that enables the efficient conversion of fibroblasts into functional Sertoli-like cells (CiSCs). The gene expression profiles of CiSCs resemble those of mature SCs and exhibit functional properties such as the formation of testicular seminiferous tubules, engulfment of apoptotic sperms, supporting the survival of germ cells, and suppressing proliferation of primary lymphocytes in vitro. Moreover, CiSCs are sensitive to toxic substances, making them an alternative model to study the deleterious effects of toxicants on SCs. The study provides an efficient approach to reprogram fibroblasts into functional SCs by using pure chemical compounds.

Spermatogonia From Cryopreserved Testes of Critically Endangered Chinese Sturgeon Efficiently Colonized and Preferentially Proliferated in the Recipient Gonads of Yangtze Sturgeon.

The critically endangered Chinese sturgeon, Acipenser sinensis, presents late sexual maturity and has a large body size. Germ cell transplantation is a powerful technique for the production of gametes from large-bodied species in closely related recipients with a smaller body size and shorter generation time. To accelerate reproduction of Chinese sturgeon, donor spermatogonia collected from the cryopreserved testes of 3-year-old Chinese sturgeon were intraperitoneally transplanted into 7-8 days post-hatch larvae of Yangtze sturgeon (Acipenser dabryanus) with shorter generation interval. At 2 months post-transplantation (mpt), donor spermatogonia had colonized in the 81.25% of recipient gonads, with average numbers about two times those of endogenous primordial germ cells. Within the next 2 months, the rate of endogenous germ cell division in females (2-3 times) was faster than that in males (once), whereas colonized donor-derived spermatogonia divided about 2-3 times and twice in recipient females and males, respectively. Furthermore, the expression of germ cell-related genes, dazl, dead end, and vasa, in transplanted fish was higher than that in non-transplanted fish, suggesting the incorporation and proliferation donor spermatogonia in recipient. At 18 mpt, donor-derived spermatogonia survived in the 75.00% of recipient gonads. These results showed that the somatic microenvironment of Yangtze sturgeon gonad can support the long-term colonization, proliferation, and survival of xenogeneic germ cells. Thus, this study suggested that small-bodied Yangtze sturgeon is promising recipient as surrogate for Chinese sturgeon gamete production.

Long-Term Maintenance and Meiotic Entry of Early Germ Cells in Murine Testicular Organoids Functionalized by 3D Printed Scaffolds and Air-Medium Interface Cultivation.

Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.

REPRODUCTIVE TOXICOLOGY: Environmental exposures, fetal testis development and function: phthalates and beyond.

Fetal development of the mammalian testis relies on a series of interrelated cellular processes: commitment of somatic progenitor cells to Sertoli and Leydig cell fate, migration of endothelial cells and Sertoli cells, differentiation of germ cells, deposition of the basement membrane, and establishment of cell-cell contacts, including Sertoli-Sertoli and Sertoli-germ cell contacts. These processes are orchestrated by intracellular, endocrine, and paracrine signaling processes. Because of this complexity, testis development can be disrupted by a variety of environmental toxicants. The toxicity of phthalic acid esters (phthalates) on the fetal testis has been the subject of extensive research for two decades, and phthalates have become an archetypal fetal testis toxicant. Phthalates disrupt the seminiferous cord formation and maturation, Sertoli cell function, biosynthesis of testosterone in Leydig cells, and impair germ cell survival and development, producing characteristic multinucleated germ cells. However, the mechanisms responsible for these effects are not fully understood. This review describes current knowledge of the adverse effects of phthalates on the fetal testis and their associated windows of sensitivity, and compares and contrasts the mechanisms by which toxicants of current interest, bisphenol A and its replacements, analgesics, and perfluorinated alkyl substances, alter testicular developmental processes. Working toward a better understanding of the molecular mechanisms responsible for phthalate toxicity will be critical for understanding the long-term impacts of environmental chemicals and pharmaceuticals on human reproductive health.

Role of autophagy in follicular development and maintenance of primordial follicular pool in the ovary.

The reproductive life span of the organism mainly depends on follicular development that maintains the primordial follicle pool in the cohort of follicles within the ovary. The total count of primordial follicles decreases with age due to ovulation and follicular atresia. Follicular atresia, a process of ovarian follicles degradation, mainly occurs via apoptosis, but recent studies also favor autophagy existence. Autophagy is a cellular and energy homeostatic response that helps to maintain the number of healthy primordial follicles, germ cell survival, and removal of corpus luteum remnants. But the excessive autophagic cell death changes both the quality and quantity of oocytes that ultimately affect female reproductive health. Autophagy regulation occurs by various autophagy-regulated genes like BECN1 and LC3-II (autophagy marker genes). Their abnormal regulation or mutation highly influences follicular development by alteration of primordial follicles formation, the decline in oocytes count, and germ cell loss. Various classical signaling pathways such as PI3K/AKT/mTOR, MAPK/ERK1/2, AMPK, and IRE1 are involved in granulosa and oocytes autophagy, while mTOR signaling is the primary mechanism. Along with basal level autophagy, chemical/hormone/stress-mediated autophagy also affects follicular development and female reproduction. In this review, we have primarily focused on granulosa cell and oocytes' autophagy, mechanism, and the role of autophagy determining marker genes in follicular development.

Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs.

The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.

The Effect of Bisphenol A on Notch Signaling Pathway in Development of Rat Testis

Bisphenol A (BPA) is the object of great concerns because of its widespread use, due to several millions of tons its production throughout the world each year. BPA is an endocrine disrupting chemical and is classified as a probable human carcinogen. Fetal and perinatal exposure to environmentally relevant doses of BPA induces multigenerational impairments of male fertility. Notch signaling functions to regulate cell-fate by modulating differentiation, proliferation, and survival of cells. Notch signaling system plays a significant role in male germ cell differentiation and survival. In this study, it was aimed to explore the alterations/changes in protein expression of Notch I receptor following BPA treatments during embriyonal (E) days 18 to 21, postnatal (P) days 0 to 3, and P4 to P7 time intervals in the rat testes in vivo. The results of study revealed that the used doses of BPA had’nt effect on Notch 1 protein expressions in rat testes during selected time preiods.

Comparative transcriptomic analysis reveals gene regulation mediated by caspase activity in a chordate organism

BackgroundApoptosis is a caspase regulated cell death present in all metazoans defined by a conserved set of morphological features. A well-described function of apoptosis is the removal of excessive cells during development and homeostasis. Recent studies have shown an unexpected signalling property of apoptotic cells, affecting cell fate and/or behaviour of neighbouring cells. In contrast to the apoptotic function of cell elimination, this new role of apoptosis is not well understood but seems caspase-dependent. To deepen our understanding of apoptotic functions, it is necessary to work on a biological model with a predictable apoptosis pattern affecting cell fate and/or behaviour. The tunicate Ciona intestinalis has a bi-phasic life cycle with swimming larvae which undergo metamorphosis after settlement. Previously, we have shown that the tail regression step during metamorphosis, characterized by a predictable polarized apoptotic wave, ensures elimination of most tail cells and controls primordial germ cells survival and migration.ResultsWe performed differential transcriptomic analysis between control metamorphosing larvae and larvae treated with the pan-caspase inhibitor Z-VAD-fmk in order to explore the transcriptional control of apoptotic cells on neighbouring cells that survive and migrate. When caspase activity was impaired, genes known to be involved in metamorphosis were downregulated along with other implicated in cell migration and survival molecular pathways.ConclusionWe propose these results as a confirmation that apoptotic cells can control surrounding cells fate and as a reference database to explore novel apoptotic functions in animals, including those related to migration and differentiation.

Crucial role of dead end gene for primordial germ cell survival in rice field eel (Monopterus albus)

The dead end gene has been identified as a essential factor for Primordial germ cells (PGCs) migration and survival in many species, but its role in Monopterus albus is unclear. In order to clarify the function of dead end gene in M.albus PGCs migration and survival, we first characterized the expression profile of M.albus dead end (Madnd) in developing embryos and various tissues. qRT-PCR revealed that Madnd transcripts were exclusively detected in gonad, including ovary, testis and ovotestis.Embryos injected with a Madnd morpholino (Madnd-MO) exhibited down-regulation of the vasa gene. Furthermore, the GFP signal show the PGCs migration in control group were injected with GFP-nanos3 3′-UTR mRNA for visualization, as described in a previous study, yet it was disappeared after embryos injected with Madnd-MO.Finally, we characterized the genomics sequence of the Madnd gene and designed five gRNAs for genome editing. Three gRNAs were selected for microinjection according to the results of in vitro tests. gRNAd1 was used for microinjection with the Cas9 protein and was confirmed to be effective. Our analysis in this study suggested that Madnd play a key role in PGCs migration and survival in M. albus. These data provide the basis for the production of fast-growing and reproductively M.albus sterile.

Specific deletion of protein phosphatase 6 catalytic subunit in Sertoli cells leads to disruption of spermatogenesis

Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in the seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.