Germ Cell Differentiation Research Articles

This study investigated the role of vasotocin (AVT) in the multifaceted regulation of spermatogenesis in zebrafish, with a focus on its interaction with gonadotropin hormones. Using an ex vivo cultured testis, we explored the interaction of AVT with gonadotropins, LH and FSH, to regulate different stages of germ cell development. In this study, we used recombinant zebrafish FSH and hCG to stimulate the LH-induced response due to the limited availability of zebrafish LH, as it is known to exert similar effects on testicular function. Treatment with AVT enhanced FSH-induced proliferation of early undifferentiated spermatogonia (Aund) germ cells, as well as promoting the proliferation of later-stage type B germ cells when combined with LH/hCG. Additionally, AVT significantly decreased FSH-induced 11-ketotestosterone (11-KT) production and Leydig-derived factors, including cyp17a1 and insl3 , without significantly affecting LH/hCG-induced androgen production. Based on these findings, we hypothesize that in the presence of FSH, AVT drives early germ cell proliferation and differentiation while simultaneously inhibiting premature progression through spermiogenesis. This stage-specific modulation of gonadotropin signaling by AVT underscores its dual role in fine-tuning testicular function and germ cell maturation in male zebrafish. Overall, our findings suggest that AVT contributes to the complex multifactorial network regulating spermatogenesis in zebrafish.

Testicular organoids (TOs) offer an opportunity to preserve fertility, but current TO protocols are limited by suboptimal maintenance of the organoid structure, meiotic defects, and incomplete in vitro spermatogenesis (IVS). Here, a strategy is developed for self-reconstitution of single-cell suspensions of neonatal mouse testes into TOs containing the major testicular cell types and generation of tubular-like structures and haploid spermatids. Morphological and lineage-specific marker analyses revealed that these TOs met the criteria for organoids, including germ cell differentiation and recapitulation of key events in meiosis (e.g., chromosome recombination and synapsis). Notably, the spatiotemporal characteristics of spermatogenesis in the TOs are comparable to those in testes, and their derived haploids resembled step-4/5-like round spermatids in vivo. Further scRNA-seq analysis confirmed that spermatid-like cells accounted for ≈2.43% of the total germ cells. Next, undifferentiated germ cells are able to develop into haploid spermatids even when chimeric TOs are reconstructed via testicular somatic cells (Sertoli cells or Leydig cells) from mice with different genetic backgrounds. Collectively, the TO-based findings provide a promising platform for studies on testicular microenvironment construction, IVS, and fertility preservation.

This study investigated the modulation of cellular stress in the male reproductive system of Daurian ground squirrels (Spermophilus dauricus) across distinct phases of hibernation under extreme environmental conditions. Morphological and volumetric changes in the testes and epididymides were assessed through paraffin-embedded sections and hematoxylin-eosin (H&E) staining, while oxidative stress (OS) and endoplasmic reticulum stress (ERS) markers were quantified using western blotting, colorimetric assays, and immunofluorescence histochemistry. Relative protein expression within key signaling pathways was also evaluated. Compared to the summer active stage, (1) reproductive hormone concentrations and testicular and epididymal visceral mass increased during hibernation and post-hibernation, indicating sustained reproductive activity. (2) Malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels remained stable in the testes and epididymides during hibernation, suggesting effective suppression of oxidative stress during this stage. (3) Superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) increased in the testes during pre-hibernation, potentially supporting germ cell differentiation. (4) Protein expression of unfolded protein response (UPR) markers, including CHOP, p-PKR, GRP78, p-IRE1, and p-PERK, was elevated during hibernation in both the testes and epididymides, implicating UPR activation in the preservation of reproductive tissue integrity and subsequent recovery. Despite stage-specific variations, the testes and epididymides of Daurian ground squirrels maintained a well-regulated cellular stress response throughout hibernation, preventing structural degeneration of the reproductive system. Furthermore, hibernation markedly reduced the abundance of common internal reference proteins such as β-actin and GAPDH, underscoring the need to use total protein normalization for accurate quantification in hibernation-related western blot analyses.

BackgroundPrimordial germ cells (PGCs) are the precursor cells of gametes and pivotal in understanding reproductive and developmental biology. Importantly, having a thorough understanding of PGC specification is leading to critical advances in sterility induction in aquaculture species. In shellfish, however, the ability to develop these approaches is hampered by the lack of information available regarding germ cell specification. The goal of this study was to identify genes uniquely expressed in these earliest germ cells of the economically and ecologically important bivalve mollusc, the Pacific oyster (Crassostrea (Magallana) gigas).ResultsTo capture specification of the PGCs - which represent a rare cell type - during embryonic development, we analyzed single-cell transcriptomes during cleavage, blastula, and gastrulation stages of C. gigas development. We identified cells in gastrulae that likely represent developing, distinct larval tissue types and organs, including muscles and shell gland, as well as undifferentiated cells. Using expression of the germ cell marker gene vasa, we identified cells in blastulae that likely represent the developing germ cell lineage that had yet to fully differentiate and segregate from somatic cell types. However, by the gastrula stage, vasa expression was limited primarily to a single cluster of cells. Other genes uniquely expressed in these vasa-positive cells include those with functions in transcriptional repression, chromatin architecture, and DNA repair, suggesting these cells represent oyster PGCs. Interestingly, some genes with no known homologies are also uniquely expressed in this cluster, perhaps representing novel PGC-associated genes in bivalves.ConclusionsWe identified a suite of candidate genes that can be explored for their role in oyster PGC specification and advance efforts to develop methods to achieve reproductive sterility via germ cell disruption in cultured shellfish. In addition, this effort produced a transcriptional atlas of early developmental cell states in bivalve embryos, providing a wealth of information on genes contributing to other important developmental processes, such as tissue differentiation and shell production. These data represent the earliest developmental stages examined via single-cell RNA sequencing in a lophotrochozoan.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12122-7.

Extracellular-vesicle-mediated transfer of let-7b/7c promotes the proliferation of transition-state spermatogonia in neonatal mouse testis.

A central question in developmental biology is how germ lines are established. We studied the specification of male germ cells (GCs) within the anther. Here, we focused on the potential role of mechanics, an aspect of anther development that has been very poorly characterized. Using a combination of live imaging and mechanical measurements, we showed that GCs emerge within a special micromechanical niche, where the inner tissues exert pressure on the outer cell layers, placing themselves under compression. Mechanical perturbations impair tissue expansion patterns and severely compromise GC specification. Further investigations revealed that the master genetic regulator SPOROCYTELESS/NOZZLE (SPL/NZZ) was central in establishing this micromechanical environment via cell wall softening. In turn, the mechanical cues stabilized the transcription of SPL/NZZ. Here, we propose an intrinsic growth-derived mechanochemical feedback loop that safeguards the fate of GCs.

DNA methylation was the earliest epigenetic mark discovered-it is essential for mammalian development and forms a molecular memory that can transcend generations, as in the phenomenon of genomic imprinting. Set against this long-term potential, methylation is dynamic across the life cycle, with genome-wide changes at germ-cell specification, gametogenesis, and preimplantation development accompanying major shifts in cell potency. With a tool kit of precision genetic reagents, the mouse has been a mainstay in developing mechanistic understanding of how methylation is targeted to the genome and in exploring its susceptibility to environmental factors, such as parental diet. The availability of genome sequence from many more species combined with the ability to profile methylation and other epigenetic marks in very small numbers of cells now provides rich epigenomic information from other mammals. This information has begun to reveal both similarities as well as surprising differences in the way in which methylation is patterned across the genome among mammals. Such knowledge will be critical in assessing the outcomes of interventions during assisted reproduction in human clinical practice and livestock production.

Abstract Due to their significant economic impact, reproductive traits are crucial in beef cattle breeding programs. However, identifying the genomic regions associated with these traits requires large datasets to capture the complex genetic architecture underlying their variability. Traits such as age at first calving (AFC), stayability (STAY), and scrotal circumference measured at 365 and 450 days (SC365 and SC450, respectively) are highly polygenic, making it essential to leverage robust genomic methodologies to uncover meaningful associations. In this study we aimed to identify candidate genes and genomic regions associated with reproductive traits in Nellore cattle. A dataset containing genomic estimated breeding values (GEBVs) for 304,782 Nellore animals genotyped with 437,650 SNPs (after quality control) was made available by the Brazilian Association of Zebu Breeders (ABCZ). The Algorithm for Proven and Young (APY), implemented in the PREGSF90 software, was used to compute the matrix using 36,000 core animals. Subsequently, the SNP solutions were estimated by back-solving the GEBVs predicted by ABCZ using the single-step GBLUP method. We identified genomic regions associated with these traits using sliding windows of 175 consecutive SNPs, and the top 1% of genomic windows were used to annotate positional candidate genes. The top 1% genomic windows for these traits explained between 2.8% (STAY) to 3.0% (AFC) of the additive genetic variance, highlighting their polygenic nature. Functional analysis of the candidate genes within these genomic regions provided valuable insights into the genetic architecture underlying reproductive traits in Nellore cattle. For instance, our results revealed genes with important functions for each trait, such as SERPINA14 (plays a key role for the endometrial epithelium) and CCNB1 (essential for spindle checkpoint regulation, meiosis, and mitosis) identified for AFC. ARHGAP18 (involved in maintaining endothelial cell alignments) and KCNC1 (regulates potassium ion flow crucial for animal longevity) were identified for STAY. Additionally, genes such as EVI5 (important for centrosome stability), BRDT (essential for male germ cell differentiation), KIT (associated with the male germ cells), and (involved in spermatogenesis and intraflagellar transport) were identified for both SC365 and SC450. We identified genomic regions and candidate genes, some of which have been previously reported in the literature, while others are novel discoveries that warrant further investigation. These findings contribute to gene prioritization efforts, facilitating the identification of functional candidate genes that can enhance genomic selection strategies for economically important traits in Nellore cattle.

Abstract Boar fertility is critical for swine reproductive efficiency. Sperm production capacity is driven by Sertoli cell numbers within the testis. Sertoli cell proliferation is malleable in early life but ceases post-puberty. Extensive Sertoli cell proliferation occurs in neonates, a period termed mini-puberty that overlaps temporally with nursing. Previous data demonstrates that colostrum consumption promotes neonatal Sertoli cell proliferation and subsequent sperm production in the boar, a phenomenon termed lactocrine programming. For example, boars who consumed more colostrum as neonates produced 17 billion more sperm per ejaculate (15% increase) compared to boars with lower colostrum intake. However, the biological mechanisms underlying this effect are unclear. The objective of this study was to determine how consumption of sow milk affects testicular development and reproductive endocrinology in neonatal pigs. After birth, white crossbred neonatal boars nursed colostrum ad libitum for 2 days prior to treatment. Boars were randomly assigned to consume either sow milk (n = 4) or milk replacer (n = 4; NutraStart Liqui-Wean) three times daily (90 ml/feeding) for eight days. At 10 days of age, boars were weighed and euthanized prior to testis and blood collection. Testes were weighed and snap frozen for subsequent proteomics. Serum concentrations of thyroxine (T4), gonadal steroids (Leydig cell products), and anti-müllerian hormone (AMH; Sertoli cell product) were quantified. Milk-fed boars had 35% heavier testes (P = 0.031) compared with replacer-fed pigs, despite no difference in body weight (P > 0.05). Serum concentrations of T4 were 30% greater in milk-fed boars compared with replacer-fed animals (P = 0.0339). Circulating concentrations of AMH and gonadal steroids (e.g., testosterone, 17β-estradiol) did not differ between treatments (P > 0.05). Regarding testicular proteomics, a total of 1,517 proteins were detected. Of these, 49 proteins were upregulated and 721 proteins were downregulated within the testes of milk-fed boars compared to replacer-fed (P < 0.05). Downregulated proteins within testes of milk-fed boars included those important for germ cell differentiation (e.g., DDX4), steroidogenesis (e.g., HSD3B), and apoptosis (e.g., PDCD4). In addition, AMH was also downregulated within testes of milk-fed boars, indicating an effect on Sertoli cells. Upregulated testicular proteins included mediators of cellular metabolism (e.g., IDH1), steroidogenesis (e.g., StAR), cell proliferation (e.g., CHD5), and antioxidant defenses (e.g., GSTO1). Notably, INSL3 – a biomarker of Leydig cell functionality – was also upregulated within milk-fed versus replacer-fed testes. Interestingly, transthyretin (thyroid hormone transporter) was also elevated, consistent with increased circulating T4 concentrations in milk-fed boars. To conclude, these data elucidate the biological mechanisms underlying lactocrine programming of the porcine testis. Neonatal intake of sow milk increased testicular weight, indicative of a proliferative effect on Sertoli cells. Therefore, manipulation of neonatal diet may be a novel intervention to develop boars with greater reproductive potential.

Myclobutanil-induced testis damage and apoptotic germ cell death through ER stress and autophagy in mouse testes.

Primordial germ cells (PGCs) are common precursors of all male and female germline cells. PGCs arise in peri-implantation mammalian embryos and become gender-specific germ cell precursors upon sexual differentiation of the gonadal anlage. The in vitro model commonly known as PGC-Like Cells (PGCLCs), generated from human pluripotent stem cells, is a useful surrogate of the human embryonic PGCs, providing a unique opportunity to explore human gametogenesis in vitro. The protocol presented here supports long-term in vitro expansion of human PGCLCs. Freshly isolated PGCLCs are maintained initially on a STO feeder layer and then expanded in a feeder-free condition on basement membrane extract. The long-term culture PGCLCs (LTC-PGCLCs) can be Fluorescence-Activated Cell Sorting (FACS)-enriched as CD38-positive cells and are readily amplified to tens of millions of cells as pure, feeder-free, and serum-free monotonous culture without apparent limitations such as senescence. It is practically feasible to obtain a pure population of more than 1 million human PGCLCs from a few thousand freshly isolated PGCLCs. The human LTC-PGCLC cell culture is a useful and convenient in vitro model to study human germ cell biology and differentiation into the downstream germline cells.

There is a paucity of in vitro models to study the male reproductive system. Proper function of the reproductive system is critical for endocrine function, growth and development, and fertility. Without practical in vitro screening models, reproductive toxicities can be missed in early drug development or standard toxicological batteries. Successful in vitro models of the male reproductive system need to recapitulate the dynamic nature of the testis, considering the formation of the testicular niches from gonadal differentiation through puberty and the post-pubertal activity of the paracrine and endocrine signals that support spermatogenesis. In vitro approaches are reviewed that model primordial germ cell differentiation, gonadal morphogenesis, fetal steroidogenesis, neonatal reproductive development, and adult testicular niche dynamics to present opportunities for inclusion of male reproductive toxicity screening within a toxicological battery. The utility of cells derived from model organisms, differentiated from iPSCs, and obtained from donated human tissue are discussed. The field of reproductive and developmental toxicology is primed for expansion in in vitro model availability as complex in vitro model development continues to accelerate and fit-for-purpose model approaches are adopted in toxicological and drug development pipelines. This review highlights the current limitations and emerging opportunities in male reproductive in vitro models, providing a roadmap for integrating these systems into toxicology testing and drug development workflows. It highlights the need for developmentally benchmarked, physiologically relevant, and multicellular models to fill existing gaps and improve translatability.

Long non-coding RNAs (lncRNAs) are abundant in gonads, yet most knockout models show no fertility defects, leaving their role unclear. Here, we identify overlapping functions of three lncRNAs in Caenorhabditis elegans fertility. These lncRNAs bind and sequester FBF-2, a regulator that inhibits germ cell differentiation. Combined deletion of the lncRNAs reduces progenitor cell number and overall progeny, reflecting additive effects. In lncRNA mutants, pro-meiotic transcripts-normally destabilized by FBF-2-are significantly reduced. FBF-2 localizes to both the cytoplasm and peri-nuclear condensates in germ cells, but its peri-nuclear condensation and association with P granules-phase-separated, evolutionarily conserved structures-are diminished in lncRNA mutants. Our findings suggest that these lncRNAs cooperatively promote fertility by spatially restricting FBF-2 within granules, thus reducing its activity and allowing the expression of differentiation-promoting transcripts. The requirement for multiple knockouts to reveal this phenotype highlights the redundancy and combinatorial function of lncRNAs in regulating germline development.

Male infertility is a global health concern, and many cases are idiopathic in nature. The development and differentiation of germ cells (Gcs) are supported by Sertoli cells (Scs). Differentiated Scs support the development of Gcs into sperm, and hence, male fertility. We previously reported on a developmental switch in Scs around 12 days of age onwards in rats. During the process of the differentiation of Scs, the differential expression of mitophagy-related genes and its role in male fertility are poorly understood. To address this gap, we evaluated the microarray dataset GSE48795 to identify 12 mitophagy-related hub genes, including B-Cell Leukemia/Lymphoma 2 (Bcl2) and FBJ murine osteosarcoma viral oncogene homolog (Fos). We identify Neuron-derived orphan receptor 1 (Nor1) as a potential mitophagy-related gene of interest due to its strong regulatory association with two hub genes, Bcl2 and Fos, which were differentially expressed during Sc maturation. To validate this finding, we generated a transgenic rat model with the Sc-specific knockdown of Nor1 during puberty. A functional analysis showed impaired spermatogenesis with reduced fertility in these transgenic rats. Our findings suggest that Nor1 may be an important mitophagy-related gene regulating the function of Scs and thereby regulating male fertility.

Human germ cells are the foundation of human reproduction and development, ensuring heredity and contributing to genetic diversity. Accordingly, their anomalies lead to critical diseases, including infertility. Recent advances in genomics and stem cell-based in vitro gametogenesis research have expanded our knowledge of how human germ cells are specified and differentiate during embryonic and fetal development, elucidating evolutionarily distinctive as well as conserved properties of human germ cell development. Here, based on the evidence from both in vivo and in vitro studies, we provide an integrated review of the progress in our understanding of human embryonic and fetal germ cell development, encompassing germ cell specification, epigenetic reprogramming and sex-specific germ cell development. Knowledge of the mechanisms of human germ cell development will enable its in vitro reconstitution, which in turn will serve as a foundation for innovative medical strategies to prevent germ cell-related diseases, including infertility.

Divergence of germ cell-less roles in germ line development across insect species.

Roles of Msx2 in exogen control: modulating the stem cell niche during the transition from hair shedding to regeneration.

Sequence variant of POU5F1 as risk factor for premature ovarian insufficiency by mis-regulating transcription.

The gonad differentiation and gametogenesis of fish is regulated by various factors. Protein phosphatase (PP) have the function of catalyzing the dephosphorylation of proteins in organisms. As a member of the protein phosphatase family, protein phosphatase type 6 (PP6) plays an important role in gonadal development and gametogenesis. The role of ppp6r3, which encodes the regulatory subunit 3 of protein phosphatase 6, in gonadal differentiation and gametogenesis is still unclear. In this study, two zebrafish ppp6r3 mutant lines were constructed using CRISPR/Cas9 technology. It was found that the absence of ppp6r3 leads to the development of only male zebrafish, and these mutants are incapable of fertilizing wild-type eggs. Further investigations revealed that in the testes of ppp6r3 mutants, the transition of spermatocytes to mature sperm was blocked, disrupting spermatogenesis. In summary, this study established a ppp6r3 mutant model, exhibiting defects in gonadal differentiation and gametogenesis, which provides a new model for further research on the mechanisms by which Ppp6r3 regulates germ cell proliferation, differentiation, and sex determination.

The absence of stem cells capable of efficiently generating both trophoblast and epiblast lineages has hindered precise recapitulation of embryonic development. Through high-content chemical screening, we established an (AS and LY)AL medium to generate mouse bidirectional pluripotent stem cells (BPSCs) characterized by concurrent expression of OCT4 and CDX2. Mouse BPSCs demonstrated highly plastic differentiation into trophoblast, epiblast and primitive endoderm (PrE) lineages in vitro within 48 h without exogenous inducing factors and efficiently contributed to embryonic and extraembryonic tissues in vivo. Mechanistically, hyperactivation of the Wnt signaling pathway breaks the early lineage differentiation barrier by initiating a Lef1-dependent bypass. Remarkably, integration of BPSCs with PrE induction system enables high-efficiency generation of E8.5-stage embryo models. These advanced models complete gastrulation and recapitulate definitive developmental milestones including brain morphogenesis, neural tube closure, cardiac contraction, somite patterning, and primordial germ cell specification. Moreover, human cells cultured under AL conditions acquire an OCT4 and CDX2 double-positive state and corresponding gene expression profiles, revealing conserved functionality of this culturing platform across species. These findings highlight BPSCs as a powerful tool for investigating early lineage specification and post-gastrulation embryonic development.