Germ Cell Development Research Articles

Development of a novel testis-on-a-chip that demonstrates reciprocal crosstalk between Sertoli and Leydig cells in testicular tissue

The reciprocal crosstalk between testicular Sertoli and Leydig cells plays a vital role in supporting germ cell development and maintaining testicular characteristics and spermatogenesis. Conventional 2D and the recent 3D assay systems fail to accurately replicate the dynamic interactions between these essential endocrine cells. Furthermore, most in vitro testicular tissue models lack the ability to capture the complex multicellular nature of the testis. To address these limitations, we developed a 3D multicellular testis-on-a-chip platform that effectively demonstrates the reciprocal crosstalk between Sertoli cells and the adjacent Leydig cells while incorporating various human testicular tissue constituent cells and various natural polymers infused with blood coagulation factors. Additionally, we identified SERPINB2 as a biomarker of male reproductive toxicity that is activated in both Sertoli and Leydig cells upon exposure to various toxicants. Leveraging this finding, we designed a fluorescent reporter-conjugated toxic biomarker detection system that enables both an intuitive and quantitative assessment of material toxicity by measuring the converted fluorescence intensity. By integrating this fluorescent reporter system into the Sertoli and Leydig cells within our 3D multicellular chip platform, we successfully developed a testis-on-chip model that can be utilized to evaluate the male reproductive toxicity of potential drug candidates. This innovative approach holds promise for advancing toxicity screening and reproductive research.

A new type of spermiogenesis in teleost fish: Formation of the aflagellate sperm in Campylomormyrus compressirostris (Osteoglossomorpha: Mormyridae)

Osteoglossomorpha, the bony tongue fishes, show great variation in morphology, behavioural strategies, reproductive biology and gamete ultrastructure. The order Osteoglossiformes is the only vertebrate taxon, in which four types of sperm (monoflagellate, biflagellate and aflagellate aquasperm and the complex introsperm) have been described. It is also the only vertebrate lineage in which aflagellate spermatozoa exist. The aim of this study was to analyse the structure of the testis and the process of spermiogenesis in the mormyrid Campylomormyrus compressirostris during the breeding season using light and electron microscopy (transmission and scanning). Males of this species have a single testis of the anastomosing tubular type. The tubules of the anterior part of the testis contain cysts with developing germ cells, and this region is much wider than the posterior part, which consists of efferent ducts filled with sperm cells. The cysts are filled with single or mitotic spermatogonia, primary and secondary spermatocytes and early spermatids. At the stage of spermatids with fine granular chromatin, the cysts rupture and successive stages of spermatid differentiation take place in the testicular lumen; we therefore characterise this process as ‘extracystic spermiogenesis’. Sperm development in C. compressirostris is extremely simple and involves chromatin condensation in the central region of the nucleus, a slight decrease in nuclear volume, the appearance of numerous vesicles in the cytoplasm that form a tubular-vesicular system at the base of the nucleus. Both centrioles and mitochondria are translocated to the peripheral region of the midpiece, which forms the opposite pole to the nucleus. There are many differences between the types of spermiogenesis described so far in teleosts and that found in C. compressirostris, including the loss of flagellum formation. This unique type of spermiogenesis is restricted to species of the families Mormyridae and Gymnarchidae, all of which possess aflagellate spermatozoa. Our data demonstrate that the spermatid differentiation and existence of the aflagellate spermatozoon are a unique phenomena not only among teleosts but also in the whole vertebrate lineage.

DDX5 Can Act as a Transcription Factor Participating in the Formation of Chicken PGCs by Targeting BMP4.

As an RNA binding protein (RBP), DDX5 is widely involved in the regulation of various biological activities. While recent studies have confirmed that DDX5 can act as a transcriptional cofactor that is involved in the formation of gametes, few studies have investigated whether DDX5 can be used as a transcription factor to regulate the formation of primordial germ cells (PGCs). In this study, we found that DDX5 was significantly up-regulated during chicken PGC formation. Under different PGC induction models, the overexpression of DDX5 not only up-regulates PGC markers but also significantly improves the formation efficiency of primordial germ cell-like cells (PGCLC). Conversely, the inhibition of DDX5 expression can significantly inhibit both the expression of PGC markers and PGCLC formation efficiency. The effect of DDX5 on PGC formation in vivo was consistent with that seen in vitro. Interestingly, DDX5 not only participates in the formation of PGCs but also positively regulates their migration and proliferation. In the process of studying the mechanism by which DDX5 regulates PGC formation, we found that DDX5 acts as a transcription factor to bind to the promoter region of BMP4-a key gene for PGC formation-and activates the expression of BMP4. In summary, we confirm that DDX5 can act as a positive transcription factor to regulate the formation of PGCs in chickens. The obtained results not only enhance our understanding of the way in which DDX5 regulates the development of germ cells but also provide a new target for systematically optimizing the culture and induction system of PGCs in chickens in vitro.

Sterilization of fish through adaptable gRNAs targeting dnd1 using CRISPR-Cas13d system

Sterilizing fish is a valuable technique not only for preventing genetic contamination from escaped aquaculture fish but also for enhancing fish meat quality. The clustered regularly interspaced short palindromic repeats-CRISPR-associated 13d (CRISPR-Cas13d) system is capable of RNA-targeted gene knockdown with reduced off-target effects. Unlike morpholino knockdown, which is limited to specific target sites, the CRISPR-Cas13d system utilizes the entire mRNA as a target site for gRNA. This enables the design of adaptable gRNAs by targeting the conserved sequences across fish species, leading to promising sterilization for a wide variety of aquaculture fish. In this study, we demonstrated that the effectiveness of this system in fish sterilization by the knockdown of dead end1 (dnd1) and nanos3, which are crucial for germ cell development. Initially, we demonstrated that gRNAs designed using the cas13design tool, which was developed based on studies of cultured mammalian cells, effectively induced germ cell-deficient medaka with low-cytotoxicity. Additionally, we identified the nucleotide positions of the gRNA (seed sequence) that are crucial for knockdown in teleost fish. These data will be valuable for designing adaptable gRNA in teleost fish and targeting multiple paralogous genes. To develop an adaptable sterilization technique using the CRISPR-Cas13d system, we aligned 81 dnd1 sequences from 70 fish species and identified a highly conserved region of the gRNA targets. Despite not finding a perfectly matched target region in the dnd1 alignment, we designed 15 adaptable gRNAs that potentially targeting dnd1 in 55 out of 70 fish species, leveraging sequences from representative aquaculture and aquarium fish (salmon, tilapia, puffer fish, amberjack, tuna, guppy and carp) and experimental model fish (medaka, zebrafish, and stickleback. To confirm whether adaptable gRNAs had a knockdown effect, we initially injected the gRNAs into medaka embryos. Adaptable gRNAs with a single-nucleotide mismatch against medaka dnd1 resulted in the complete loss of germ cells with little cytotoxicity, suggesting that mismatches are unlikely to induce off-target effects crucial for embryonic development. Additionally, adaptable gRNAs designed based on the salmon dnd1 sequence, which was 100% identical in the Oncorhynchus, Salvelinus, and Salmo groups, effectively generated germ cell-deficient rainbow trout. These results indicate that the knockdown of dnd1 using the CRISPR-Cas13d system is effective for sterilizing both aquaculture and experimental model fish species. Moreover, the adaptable gRNAs designed in this study can potentially sterilize a diverse range of fish species.

In vivo effect of recombinant Fsh and Lh administered to meagre (Argyrosomus regius) at the initial stages of sex differentiation

Recombinant gonadotropins, follicle stimulating (rFsh) and luteinizing hormone (rLh), offer the potential to induce gametogenesis in prepubertal fish. This study aimed to determine the in vivo effect of the administration of Argyrosomus regius rFsh and rLh on the reproductive development of prepubertal meagre juveniles at the initial stages of sexual differentiation. Juvenile meagre, 9-months old with mean weight of 219 ± 3.9 g (mean ± SEM) were randomly distributed into nine groups (n = 8 per group). Experimental groups were treated weekly with an acute injection of either rFsh or rLh. Control groups were injected with saline solution. In a 3-week experiment, different groups were administered with different doses 6, 12 or 18 µg kg−1 of rFsh or rLh or saline solution. In a 6-week experiment a group was administered with 12 µg kg−1 of rFsh and a second group with saline solution. The fish were held in a single 10 m3 tank with natural photoperiod (Feb. – March) and temperature 16.1 ± 0.4 °C. At the start of the experiment (n = 8) and at the end of the 3-week experiment, fish were blood sampled and sacrificed. Blood was analysed for 17β-estradiol (E2) and 11-ketotestosterone (11-KT). Gonads and liver were dissected and weighed. Gonads were fixed in Bouińs solution and processed for histological analysis. Juvenile meagre at the start of the experiment were in the initial stages of sexual differentiation, indicated by the presence of the ovarian cavity or testes duct that was surrounded by undifferentiated embryonic germ stem cells and somatic cells. At the end of the 3-week experiment, there was no significant difference in gonadosomatic index (GSI) amongst control (initial and saline treated) and the experimental groups. After three weeks of application of rFsh, rLh or saline all fish presented a similar gonadal structure as at the start of the experiment. However, the incidence of sporadic developing germ cells (principally spermatogonia, spermatocytes, spermatids, but also perinucleolar stage oocytes) generally increased in rGth treated meagre. A mean of 44 % of meagre treated with rFsh or rLh presented sporadic isolated developing germ cells, mainly male cells. Plasma steroid levels of E2 decreased significantly from the start of the experiments to the end. At the end of the experiments there were no differences in plasma E2 amongst Control fish and rGth treated fish. Plasma 11-KT showed no change from the start of the experiment to week 3. However, a significant increase was observed in a proportion of the rFsh group after six weeks of treatment compared to the start of the experiment and the saline control group on week 6. The application of rFsh or rLh to meagre at the initial stages of sex differentiation did not stimulate steroid production until week six (11-KT) and had a limited, but evident effect on the development of sporadic isolated germ cells. However, we conclude that rGth, rFsh or rLh did not stimulate large developmental changes in sexually undifferentiated meagre gonads.

Dzip1 is dynamically expressed in the vertebrate germline and regulates the development of Xenopus primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperms and oocytes. Proper development of PGCs is crucial for the survival of the species. In many organisms, factors responsible for PGC development are synthesized during early oogenesis and assembled into the germ plasm. During early embryonic development, germ plasm is inherited by a few cells, leading to the formation of PGCs. While germline development has been extensively studied, how components of the germ plasm regulate PGC development is not fully understood. Here, we report that Dzip1 is dynamically expressed in vertebrate germline and is a novel component of the germ plasm in Xenopus and zebrafish. Knockdown of Dzip1 impairs PGC development in Xenopus embryos. At the molecular level, Dzip1 physically interacts with Dazl, an evolutionarily conserved RNA-binding protein that plays a multifaced role during germline development. We further showed that the sequence between amino acid residues 282 and 550 of Dzip1 is responsible for binding to Dazl. Disruption of the binding between Dzip1 and Dazl leads to defective PGC development. Taken together, our results presented here demonstrate that Dzip1 is dynamically expressed in the vertebrate germline and plays a novel function during Xenopus PGC development.

Ago4-piRNA complex is a key component of genomic immune system against transposon expression in Penaeus monodon

Argonaute proteins are key constituents of small RNA-guided regulatory pathways. In crustaceans, members of the AGO subfamily of Argonaute proteins that play vital roles in immune defense are well studied, while proteins of the PIWI subfamily are less established. PmAgo4 of the black tiger shrimp, Penaeus monodon, though phylogenetically clustered with the AGO subfamily, has distinctive roles of the PIWI subfamily in safeguarding the genome from transposon invasion and controlling germ cell development. This study explored a molecular mechanism by which PmAgo4 regulates transposon expression in the shrimp germline. PmAgo4-associated small RNAs were co-immunoprecipitated from shrimp testis lysate using a PmAgo4-specific polyclonal antibody. RNA-seq revealed a majority of 26–27 nt long small RNAs in the PmAgo4-IP fraction suggesting that PmAgo4 is predominantly associated with piRNAs. Mapping of these piRNAs on nucleotide sequences of two gypsy and a mariner-like transposons of P. monodon suggested that most piRNAs were originated from the antisense strand of transposons. Suppression of PmAgo4 expression by a specific dsRNA elevated the expression levels of the three transposons while decreasing the levels of transposon-related piRNAs. Taken together, these results imply that PmAgo4 exerts its suppressive function on transposons by controlling the biogenesis of transposon-related piRNAs and thus, provides a defense mechanism against transposon invasion in shrimp germline cells.

Characteristics of the Vasa Gene in Silurus asotus and Its Expression Response to Letrozole Treatment.

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.

P38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells.

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1–7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

Identification of RNA‐dependent liquid‐liquid phase separation proteins using an artificial intelligence strategy

RNA-dependent liquid-liquid phase separation (LLPS) proteins play critical roles in cellular processes such as stress granule formation, DNA repair, RNA metabolism, germ cell development, and protein translation regulation. The abnormal behavior of these proteins is associated with various diseases, particularly neurodegenerative disorders like amyotrophic lateral sclerosis and frontotemporal dementia, making their identification crucial. However, conventional biochemistry-based methods for identifying these proteins are time-consuming and costly. Addressing this challenge, our study developed a robust computational model for their identification. We constructed a comprehensive dataset containing 137 RNA-dependent and 606 non-RNA-dependent LLPS protein sequences, which were then encoded using amino acid composition, composition of K-spaced amino acid pairs, Geary autocorrelation, and conjoined triad methods. Through a combination of correlation analysis, mutual information scoring, and incremental feature selection, we identified an optimal feature subset. This subset was used to train a random forest model, which achieved an accuracy of 90% when tested against an independent dataset. This study demonstrates the potential of computational methods as efficient alternatives for the identification of RNA-dependent LLPS proteins. To enhance the accessibility of the model, a user-centric web server has been established and can be accessed via the link: http://rpp.lin-group.cn.

Morphofunctional characteristics of the gonads in females of the pontic shad Alosa immaculata in modern ecological conditions of the lower Nistru in the spring-summer period

Pontic shad (Alosa immaculata) is a migratory gregarious fish that spawns in the Don, Dnieper, Dniester, Danube and other rivers. The species is of valuable commercial importance. This work analyzes changes in the reproductive functions of the commercially valuable species of pontic shad (Alosa immaculata) under conditions of fragmentation of the river Dniester. Currently, the age composition, as well as the linear weight indicators of the spawning pontic shad population, turned out to be similar to the data of previous years. Oogenesis of Dniester’s pontic shad is characterized by asynchronous development of germ cells and portioned release of eggs. Spawning of spawners begins in the third ten days of May, at a water temperature of 18°C and ends in July. In the lower Dniester in April and July were revealed in some females’ destructive changes in the development of vitellogenin oocytes, which will negatively affect the population size of migratory herring.

Spermatocytes are the terminals of germ cell differentiation in plateau zokor (Eospalax baileyi) during the non-breeding season.

Plateau zokor (Eospalax baileyi) is a subterranean rodent and seasonal breeder. During the non-breeding season, the testicles regress, leading to the arrest of spermatogenesis and loss of fertility. The identification of the specific germ cell type at which spermatogenesis is arrested, as well as potential regulatory factors during the non-breeding season, is important for understanding seasonal spermatogenesis in subterranean species. This study analyzed genes in spermatocytes of plateau zokor by referring to single-cell RNA results in mice. We discovered that spermatogenesis is arrested at the spermatocyte during the non-breeding season, which was corroborated via immunofluorescence staining results. The analysis of gene expression during different stages of meiotic prophase I has revealed that germ cell development may be arrested, starting from zygonema, during the non-breeding season. Meanwhile, we discovered that the apoptosis genes were up-regulated, leading to apoptosis in spermatocytes. To confirm that the germ cell differentiation was blocked during the non-breeding season due to a decrease in the androgen level, we used androgen receptor antagonist (flutamide) to intervene in the breeding season and found that the inner diameter of the seminiferous tubules was significantly reduced, spermatogenesis was arrested, and spermatocytes underwent apoptosis. This study revealed that spermatocytes are the terminal of germ cell differentiation in plateau zokor during the non-breeding season and that the arrest of differentiation is attributed to a decline in androgen levels. Our results complement the theoretical basis of seasonal reproduction in plateau zokor.

A Case Report of 46 XY Partial Gonadal Dysgenesis with Dual Gonadal Tumours

46XY gonadal dysgenesis is known as a disorder of sex development (DSD) that occurs due to abnormality in gonadal development. There is a conflict between one’s genotype, phenotype, and gonadal development, which influences the wide range of presentation and clinical appearance of 46XY females. This was a case of a 46XY female who presented with primary amenorrhea with delayed puberty at the age of 18 years old. She had ambiguous genitalia. Her breast and pubic hair were at Tanner Stage 2. Transabdominal ultrasound found a small uterus with a vagina; however, the gonads were not seen. A magnetic resonance imaging (MRI) showed a 20mm right gonad located extra-pelvis near the right iliac vessels while the 9mm streak left gonad was at the usual location next to the sigmoid colon. All her tumour markers were normal except lactate dehydrogenase (LDH) was elevated. She was given estrogen therapy for pubertal induction and underwent laparoscopy prophylactic bilateral gonadectomy. The intraoperative findings were similar to the MRI findings, and the histopathology examination (HPE) results showed left gonadoblastoma and right dysgerminoma FIGO stage 1A. She continued taking estrogen therapy until she had withdrawal bleeding, and oral progesterone was added. 46XY-DSD has high risk of developing germ cell tumour and requires prophylactic gonadectomy as soon as the diagnosis is established. However, delay in presentation and surgery may affect the outcomes and prognosis.

Effect of parental ploidy on reproductive system development in sturgeon hybrids in the second year of life

The paper presents the data on germ cell development in various sturgeon hybrids with various ploidy levels. The study was performed at experimental industrial sites of the Branch for Freshwater Fisheries of the Russian Federal Research Institute of Fisheries and Oceanography (VNIIPRKH). Histology samples were taken in the winter, summer and autumn. Histology slides were made following the standard procedure. The figures show all stages of germ cell development in males and females of various hybrids, depending on rearing conditions (thermal constant). Two-year-old Russian sturgeon × Kaluga fish hybrids with similar ploidy levels reached gonad maturity stage I at a thermal constant of 8,571 degree-days (547 days). Female Russian sturgeon × beluga hybrids aged 631 days (10,191 degree-days) showed accelerated germ cell development characteristic of stage I to early stage II. Male Russian sturgeon × beluga and Siberian sturgeon × beluga hybrids had sexually indifferent germ cells. These species are expected to be sterile. Male Russian sturgeon × Kaluga fish hybrids aged 374 days had sexually indifferent germ cells with occasional primary spermatogonia; cell division was observed on Day 547. The study found that distant hybrids show better growth than hybrids with similar ploidy levels, where more intensive germ cell development is observed. Further research will elucidate the effect of gonad development during reproductive system formation on the growth of sturgeon hybrids with various ploidy levels.

Large-Sample Genome-Wide Association Study of Resistance to Retained Placenta in U.S. Holstein Cows.

A genome-wide association study of resistance to retained placenta (RETP) using 632,212 Holstein cows and 74,747 SNPs identified 200 additive effects with p-values < 10-8 on thirteen chromosomes but no dominance effect was statistically significant. The regions of 87.61-88.74 Mb of Chr09 about 1.13 Mb in size had the most significant effect in LOC112448080 and other highly significant effects in CCDC170 and ESR1, and in or near RMND1 and AKAP12. Four non-ESR1 genes in this region were reported to be involved in ESR1 fusions in humans. Chr23 had the largest number of significant effects that peaked in SLC17A1, which was involved in urate metabolism and transport that could contribute to kidney disease. The PKHD1 gene contained seven significant effects and was downstream of another six significant effects. The ACOT13 gene also had a highly significant effect. Both PKHD1 and ACOT13 were associated with kidney disease. Another highly significant effect was upstream of BOLA-DQA2. The KITLG gene of Chr05 that acts in utero in germ cell and neural cell development, and hematopoiesis was upstream of a highly significant effect, contained a significant effect, and was between another two significant effects. The results of this study provided a new understanding of genetic factors underlying RETP in U.S. Holstein cows.

In vitro reconstitution of epigenetic reprogramming in the human germ line

Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency1. In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >1010-fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells2,3, differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine.

The matrisome landscape controlling in vivo germ cell fates

The developmental fate of cells is regulated by intrinsic factors and the extracellular environment. The extracellular matrix (matrisome) delivers chemical and mechanical cues that can modify cellular development. However, comprehensive understanding of how matrisome factors control cells in vivo is lacking. Here we show that specific matrisome factors act individually and collectively to control germ cell development. Surveying development of undifferentiated germline stem cells through to mature oocytes in the Caenorhabditis elegans germ line enabled holistic functional analysis of 443 conserved matrisome-coding genes. Using high-content imaging, 3D reconstruction, and cell behavior analysis, we identify 321 matrisome genes that impact germ cell development, the majority of which (>80%) are undescribed. Our analysis identifies key matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior. Further, our results demonstrate that germ cell development requires continual remodeling of the matrisome landscape. Together, this study provides a comprehensive platform for deciphering how extracellular signaling controls cellular development and anticipate this will establish new opportunities for manipulating cell fates.

Functional regulation of testosterone underlying state transition in seasonal spermatogenesis of Plateau Pika (Ochotona curzoniae)

Abstract Seasonal reproduction is an adaptive strategy that benefits the survival and growth of offspring. However, the regulatory mechanisms affecting spermatogenesis in male seasonal breeders are not well understood. We examined the actions of testosterone on seasonal spermatogenesis of Plateau Pika (Ochotona curzoniae), a typical long-day breeder living on the Qinghai–Tibetan Plateau. The annual dynamics of germ cell development and steroid hormone synthesis were confirmed and were consistent with testicular morphology. Furthermore, a testosterone regulation experiment showed that elevated testosterone stimulated proliferation of spermatogonia and regeneration of advanced spermatogenic cells in reproductively dormant pika, while testosterone suppression induced cell apoptosis both in reproductively active and dormant pikas. Transcriptomic analyses indicated that testosterone-regulated genes related to spermatogonial fate determination by binding to androgen receptors and inducing the production of retinoic acid, which was responsible for the initiation of spermatogonial differentiation. We show that testosterone orchestrates downstream signaling pathways to balance spermatogonial self-renewal and development. These findings provide a new perspective on seasonal regulation of mammalian spermatogenesis.

TEX13B is essential for metabolic reprogramming during germ cell differentiation.

What is the functional significance of Tex13b in male germ cell development and differentiation? Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. N/A. We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.