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Development of a syncytial germline for gamete formation requires complex regulation of cytokinesis and cytoplasmic remodeling. Recently, several uncovered cellular events have been investigated in the Caenorhabditis elegans (C. elegans) germline. In these cellular processes, the factors involved in contractility are highly conserved with those of mitosis and meiosis. However, the underlying regulatory mechanisms are far more complicated than previously thought, likely due to the single syncytial germline structure. In this review, we highlight how the proteins involved in contractility ensure faithful cell division in different cellular contexts and how they contribute to maintaining intercellular bridge stability. In addition, we discuss the current understanding of the cellular events of cytokinesis and cytoplasmic remodeling during the development of the C. elegans germline, including progenitor germ cells, germ cells, and spermatocytes. Comparisons are made with relevant systems in Drosophila melanogaster (D. melanogaster) and other animal models.

Melphalan significantly contributes to the increase in childhood cancer survival rate. It acts as a gonadotoxic agent and leads to testes damage, dysbalance in gonadal hormones, and impairment in the germ cell proliferation. Therefore, it might be a potent threat to male fertility in individuals who have undergone melphalan treatment during childhood cancer. However, the molecular mechanisms of melphalan-induced gonadal damage are not yet fully explored and they need to be investigated to determine the benefit-risk profile. In the present study, juvenile male SD rats were subjected to single and intermittent cycles of melphalan exposure in a dose-dependent (0.375, 0.75 and 1.5 mg/kg) manner. Methods of end-points evaluations were quantification of micronuclei formation in peripheral blood, sperm count, sperm motility and head morphology, sperm and testicular DNA damage, histological studies in testes, oxidative/nitrosative stress parameters. A single cycle of exposure at high dose (1.5 mg/kg) produced significant effect on micronuclei formation only after the first week of exposure, whereas failed to produce significant effect at the end of the sixth week. Intermittent cycles of exposure at the dose of 1.5 mg/kg produced significant alterations in all the parameters (micronuclei in peripheral blood, testes and epididymides weight and length, MDA, GSH and nitrite levels, sperm count and motility, sperm head morphology, testicular and sperm DNA damage, protein expression in testes and histological parameters). So, time of exposure as well as the amount of exposure (total dosage administered) is critical in determining the magnitude of the damage in germ cell risk assessment.

Germ cells reflect the evolutionary history and future potential of a species. Understanding how the genome is organised in gametocytes is fundamental to understanding fertility and its impact on genetic diversity and evolution of species. Here, we explore principles of chromatin remodelling during the formation of germ cells and how these are affected by genome reshuffling.

Genome editing technology facilitates the creation of specific and precise genetic changes to unravel gene function and rapidly transfer unique alleles between chicken breeds in contrast to lengthy traditional crossbreeding methods for the study of poultry genetics. Innovations in genome sequencing technology have made it possible to map polymorphisms associated with both monogenic and multigenic traits in livestock species. We, and many others, have demonstrated the use of genome editing to introduce specific monogenic traits in chicken through targeting of cultured primordial germ cells. In this chapter, we describe materials and protocols for performing heritable genome editing in the chicken through targeting of in vitro propagated chicken primordial germ cells.

Post-transcriptional RNA modifications critically regulate various biological processes. N4-acetylcytidine (ac4C) is an epi-transcriptome, which is highly conserved in all species. However, the in vivo physiological functions and regulatory mechanisms of ac4C remain poorly understood, particularly in mammals. In this study, we demonstrate that the only known ac4C writer, N-acetyltransferase 10 (NAT10), plays an essential role in male reproduction. We identified the occurrence of ac4C in the mRNAs of mouse tissues and showed that ac4C undergoes dynamic changes during spermatogenesis. Germ cell-specific ablation of Nat10 severely inhibits meiotic entry and leads to defects in homologous chromosome synapsis, meiotic recombination and repair of DNA double-strand breaks during meiosis. Transcriptomic profiling revealed dysregulation of functional genes in meiotic prophase I after Nat10 deletion. These findings highlight the crucial physiological functions of ac4C modifications in male spermatogenesis and expand our understanding of its role in the regulation of specific physiological processes in vivo.

Gonocytes are germline stem cells in the neonatal testis with important potential applications in fertility restoration and transgenesis. Using stepwise experiments, we examined the effects of different media combined with fetal bovine serum (FBS) and/or knockout serum replacement (KSR) on the in vitro proliferation, colony-formation, ultrastructure, and expression of pluripotency markers of porcine gonocytes. Testis cells from 1-wk-old piglets were cultured for 28 days in 6 different culture media (DMEM, DMEM/F12, GMEM, α-MEM, StemPro, and RPMI), each supplemented with 5%, 10%, or 15% FBS and/or 5%, 10%, or 15% KSR. The media and FBS/KSR combination leading to the maximum number of gonocytes, and their colonies were selected for further analyses. KSR supplementation resulted in a reduced somatic cell propagation and increased gonocyte colony formation (P 0.001). Culturing in DMEM+15%FBS led to the greatest number of gonocytes (P 0.001), while the largest diameter and greatest number of colonies were formed in DMEM+5%FBS+10%KSR cultures (P 0.001). Gonocytes and their colonies in DMEM+15%FBS expressed all the examined gonocyte and pluripotency markers. KSR alone did not support gonocyte propagation, likely due to a reduced somatic cell proliferation; however, the combination of FBS and KSR increased gonocyte colony formation and their size.

Centrosomes are microtubule-organizing centers comprised of a pair of centrioles and the surrounding pericentriolar material. Abnormalities in centriole number are associated with cell division errors and can contribute to diseases such as cancer. Centriole duplication is limited to once per cell cycle and is controlled by the dosage-sensitive Polo-like kinase 4 (PLK4). Here, we show that PLK4 abundance is translationally controlled through conserved upstream open reading frames (uORFs) in the 5′ UTR of the mRNA. Plk4 uORFs suppress Plk4 translation and prevent excess protein synthesis. Mice with homozygous knockout of Plk4 uORFs (Plk4Δu/Δu) are viable but display dramatically reduced fertility because of a significant depletion of primordial germ cells (PGCs). The remaining PGCs in Plk4Δu/Δu mice contain extra centrioles and display evidence of increased mitotic errors. PGCs undergo hypertranscription and have substantially more Plk4 mRNA than somatic cells. Reducing Plk4 mRNA levels in mice lacking Plk4 uORFs restored PGC numbers and fully rescued fertility. Together, our data uncover a specific requirement for uORF-dependent control of PLK4 translation in counterbalancing the increased Plk4 transcription in PGCs. Thus, uORF-mediated translational suppression of PLK4 has a critical role in preventing centriole amplification and preserving the genomic integrity of future gametes.

Interspecific hybridization may trigger the transition from sexual reproduction to asexuality, but mechanistic reasons for such a change in a hybrid’s reproduction are poorly understood. Gametogenesis of many asexual hybrids involves a stage of premeiotic endoreplication (PMER), when gonial cells duplicate chromosomes and subsequent meiotic divisions involve bivalents between identical copies, leading to production of clonal gametes. Here, we investigated the triggers of PMER and whether its induction is linked to intrinsic stimuli within a hybrid’s gonial cells or whether it is regulated by the surrounding gonadal tissue. We investigated gametogenesis in the Cobitis taenia hybrid complex, which involves sexually reproducing species (Cobitis elongatoides and C. taenia) as well as their hybrids, where females reproduce clonally via PMER while males are sterile. We transplanted spermatogonial stem cells (SSCs) from C. elongatoides and triploid hybrid males into embryos of sexual species and of asexual hybrid females, respectively, and observed their development in an allospecific gonadal environment. Sexual SSCs underwent regular meiosis and produced normally reduced gametes when transplanted into clonal females. On the other hand, the hybrid’s SSCs lead to sterility when transplanted into sexual males but maintained their ability to undergo asexual development (PMER) and production of clonal eggs, when transplanted into sexual females. This suggests that asexual gametogenesis is under complex control when somatic gonadal tissue indirectly affects the execution of asexual development by determining the sexual differentiation of stem cells and once such cells develop to female phenotypes, hybrid germ cells trigger the PMER from their intrinsic signals.

Metabolism is an important cellular process necessary not only for producing energy and building blocks for cells, but also for regulating various cell functions, including intracellular signaling, epigenomic effects, and transcription. The regulatory roles of metabolism have been extensively studied in somatic cells, including stem cells and cancer cells, but data regarding germ cells are limited. Because germ cells produce individuals of subsequent generations, understanding the role of metabolism and its regulatory functions in germ cells is important. Although limited information concerning the specific role of metabolism in germ cells is available, recent advances in related research have revealed specific metabolic states of undifferentiated germ cells in embryos as well as in germ cells undergoing oogenesis and spermatogenesis. Studies have also elucidated the functions of some metabolic pathways associated with germ cell development and the non-genomic heritable machinery of germ cells. In this review, we summarized all the available knowledge on the characteristic metabolic pathways in germ cells, focusing on their regulatory functions, while discussing the issues that need to be addressed to enhance the understanding of germ cell metabolism.

STUDY QUESTION Can transgender women cryopreserve germ cells obtained from their orchiectomy specimen for fertility preservation, after having used puberty suppression and/or hormonal treatment? SUMMARY ANSWER In the vast majority of transgender women, there were still immature germ cells present in the orchiectomy specimen, and in 4.7% of transgender women—who all initiated medical treatment in Tanner stage 4 or higher—mature spermatozoa were found, which would enable cryopreservation of spermatozoa or testicular tissue after having used puberty suppression and/or hormonal treatment. WHAT IS KNOWN ALREADY Gender affirming treatment (i.e. puberty suppression, hormonal treatment, and subsequent orchiectomy) impairs reproductive function in transgender women. Although semen cryopreservation is generally offered during the transition process, this option is not feasible for all transgender women (e.g. due to incomplete spermatogenesis when initiating treatment in early puberty, in case of inability to masturbate, or when temporary cessation of hormonal treatment is too disruptive). Harvesting mature spermatozoa, or testicular tissue harboring immature germ cells, from orchiectomy specimens obtained during genital gender-affirming surgery (gGAS) might give this group a chance of having biological children later in life. Previous studies on spermatogenesis in orchiectomy specimens showed conflicting results, ranging from complete absence of germ cells to full spermatogenesis, and did not involve transgender women who initiated medical treatment in early- or late puberty. STUDY DESIGN, SIZE, DURATION Histological and immunohistochemical analyses were performed on orchiectomy specimens from 214 transgender women who underwent gGAS between 2006 and 2018. Six subgroups were identified, depending on pubertal stage at initiation of medical treatment (Tanner stage 2-3, Tanner stage 4-5, adult), and whether hormonal treatment was continued or temporarily stopped prior to gGAS in each of these groups. PARTICIPANTS/MATERIALS, SETTING, METHODS All transgender women used a combination of estrogens and testosterone suppressing therapy. Orchiectomy specimen sections were stained with Mayer’s hematoxylin and eosin and histologically analyzed to assess the Johnsen score and the ratio of most advanced germ cell types in at least 50 seminiferous tubular cross-sections. Subsequently, immunohistochemistry was used to validate these findings using spermatogonia, spermatocytes or spermatids markers (MAGE-A3/A4, γH2AX, Acrosin, respectively). Possibilities for fertility preservation were defined as: preservation of spermatozoa, preservation of spermatogonial stem cells or no possibilities (in case no germ cells were found). Outcomes were compared between subgroups and logistic regression analyses were used to assess the association between the duration of hormonal treatment and the possibilities for fertility preservation. MAIN RESULTS AND THE ROLE OF CHANCE Mature spermatozoa were encountered in 4.7% of orchiectomy specimens, all from transgender women who had initiated medical treatment in Tanner stage 4 or higher. In 88.3% of the study sample orchiectomy specimens only contained immature germ cells (round spermatids, spermatocytes or spermatogonia, as most advanced germ cell type). In 7.0%, a complete absence of germ cells was observed, all these samples were from transgender women who had initiated medical treatment in adulthood. Cessation of hormonal treatment prior to gGAS did not affect the presence of germ cells or their maturation stage, nor was there an effect of the duration of hormonal treatment prior to gGAS. LIMITATIONS, REASONS FOR CAUTION Since data on serum hormone levels on the day of gGAS were not available, we were unable to verify if the transgender women who were asked to temporarily stop hormonal treatment 4 weeks prior to surgery actually did so, and if people with full spermatogenesis were compliant to treatment. WIDER IMPLICATIONS OF THE FINDINGS There may still be options for fertility preservation in orchiectomy specimens obtained during gGAS since a small percentage of transgender women had full spermatogenesis, which could enable cryopreservation of mature spermatozoa via a testicular sperm extraction procedure. Furthermore, the vast majority still had immature germ cells, which could enable cryopreservation of testicular tissue harboring spermatogonial stem cells. If maturation techniques like in vitro spermatogenesis become available in the future, harvesting germ cells from orchiectomy specimens might be a promising option for those who are otherwise unable to have biological children. STUDY FUNDING/COMPETING INTEREST None. TRIAL REGISTRATION NUMBER N/A.

Primordial germ cells (PGCs) play an important role in sexual fate determination and gonadal development in gonochoristic fish, such as zebrafish and medaka. However, little is known about the function of PGCs in hermaphroditic fish. Rice field eel (Monopterus albus), a protogynous hermaphroditic fish, is an economically valuable aquaculture species. We eliminated PGCs in rice field eels during embryogenesis via morpholino-mediated knockdown dead end (dnd). The PGCs-depleted gonads developed into testis-like structures with Sertoli cells and Leydig cells. The gene expression pattern of 15-month-old PGCs-depleted gonads showed that male-biased genes, dmrt1, sox9a, gsdf, and amh, were significantly higher than that of the WT, whereas female-biased genes, foxl2 and cyp19a1a, were significantly decreased. These results indicate that PGCs are essential for ovarian differentiation in rice field eel, and PGCs-depleted gonads develop into sterile males without undergoing the female and intersex stages. Our study is the first to identify the role of PGCs in sex differentiation in rice field eel, a protogynous hermaphrodite teleost. And it is of great significance in rice field eel for discovering the underlying mechanism of sex differentiation and establishing sex control technology.

Oogenesis and folliculogenesis are considered as complex and species-specific cellular differentiation processes, which depend on the in vivo ovarian follicular environment and endocrine cues. Considerable efforts have been devoted to driving the differentiation of female primordial germ cells toward mature oocytes outside of the body. The recent experimental attempts have laid stress on offering a suitable microenvironment to assist the in vitro folliculogenesis and oogenesis. Despite developing a variety of bioengineering techniques and generating functional mature gametes through in vitro oogenesis in earlier studies, we still lack knowledge of appropriate microenvironment conditions for building biomimetic culture systems for female fertility preservation. Therefore, this review paper can provide a source for a large body of scientists developing cutting-edge in vitro culture systems for female germ cells or setting up the next generation of reproductive medicine as feasible options for female infertility treatment. The focal point of this review outlines advanced bioengineering technologies such as 3D biofabricated hydrogels/scaffolds and microfluidic systems utilized with female germlines for fertility preservation through in vitro folliculogenesis and oogenesis.

Primordial germ cells (PGCs) have been used in avian genetic resource conservation and transgenic animal production. Despite their potential applications to numerous avian taxa facing extinction due to habitat loss and degradation, research has largely focused on poultry, such as chickens, in part owing to the difficulty in obtaining intact PGCs from other species. Recently, phenotypic differences between PGCs of chicken and zebra finch, a wild bird with vocal learning, in early embryonic development have been reported. In this study, we used advanced single-cell RNA sequencing (scRNA-seq) technology to evaluate zebra finch and chicken PGCs and surrounding cells, and to identify species-specific characteristics. We constructed single-cell transcriptome landscapes of chicken gonadal PGCs for a comparison with previously reported scRNA-seq data for zebra finch. We identified interspecific differences in several signaling pathways in gonadal PGCs and somatic cells. In particular, NODAL and insulin signaling pathway activity levels were higher in zebra finch than in chickens, whereas activity levels of the downstream FGF signaling pathway, involved in the proliferation of chicken PGCs, were higher in chickens. This study is the first cross-species single-cell transcriptomic analysis targeting birds, revealing differences in germ cell development between phylogenetically distant Galliformes and Passeriformes. Our results provide a basis for understanding the reproductive physiology of avian germ cells and for utilizing PGCs in the restoration of endangered birds and the production of transgenic birds.

Coilia nasus is an endangered fish species in the Yangtze River, and there is urgent need to know the genes related to its reproduction and fertility. The DAZ family including boule, daz and dazl, plays an important role in germ cells development. In this study, the cDNA sequence of boule (Cnbol) and dazl (Cndazl) genes were cloned and their expression patterns were characterized in Coilia nasus. RT-PCR showed that the expression of Cnbol and Cndazl mRNAs was restricted in adult gonads. The section in situ hybridization indicated that the temporospatial expression patterns of Cnbol and Cndazl were significantly different. In the testis, Cnbol was mainly detected in spermatocytes and spermatids, while Cndazl was prominently expressed in s permatogonia. In the ovary, Cnbol and Cndazl were highly expressed in the early stages of oocytes. Interestingly, Cndazl was also concentrated in perinuclear speckle and then localized to the Balbiani body in late stages of oocytes. In addition, Cnbol and Cndazl 3′ UTR can specifically label medaka Primordial Germ Cells (PGCs). According to our results, Cnbol and Cndazl are marker genes of germ cells and may play a vital role in the development and differentiation of germ cells in Coilia nasus.

Emerging evidence suggests that selenium plays an essential role in sperm maturation. However, the specific signaling pathway by which selenium exerts effect has not been elucidated. To evaluate the effect of selenium on GPX4-mediated lipid peroxidation and apoptosis in germ cells, selenium deficiency was modeled by culturing GC2-spd cells in serum-free medium. Treatment with 0.5-μM sodium selenite (NaSe) or 5.0-μM selenomethionine (SeMet) significantly improved the proliferation rate and GPX4 protein expression after selenium deficiency. Moreover, NaSe and SeMet decreased the MDA content and lipid peroxidation. When adenovirus was used to knockdown the expression of the GPX4 gene (shRNA-GPX4), the early apoptosis rate of the shRNA-GPX4 cells was significantly higher than that of the EGFP cells. Increased expression of Caspase3 and Bax, as well as MDA content were observed in the shRNA-GPX4 cells compared with EGFP cells. In further, overexpression of the GPX4 gene (ORF-GPX4) cells exhibited increased cell proliferation and decreased MDA content. However, there was no significant difference in 12/15-lox expression both in ORF-GPX4 cells and shRNA-GPX4 cells. Conclusively, GPX4 was involved in the regulation of lipid peroxidation and apoptosis in GC2-spd cells. Selenium played a role in promoting cell proliferation by mediating GPX4. The regulation of GPX4 may occur independently of 12/15-Lox. These findings confirmed the effect of selenium on spermatogenesis and offered a potential target for treating abnormal semen quality in men.

PIWI proteins play important roles in germline development in the mammals. However, the functions of PIWIs in crustaceans remain unknown. In the present study, we identified three Piwis from the testis of Eriocheir sinensis (E. sinensis). Three Piwi genes encoded proteins with typical features of PIWI subfamilies and were highly expressed in the testis. Three PIWIs could be detected in the cytoplasm of spermatocytes and spermatids, while in spermatozoa, we could only detect PIWI1 and PIWI3 in the nucleus. The knockdown of PIWIs by dsRNA significantly affected the formation of the nuclei in spermatozoa, which resulted in deviant and irregular nuclei. PIWI defects significantly inhibited the apoptosis of abnormal germ cells through the caspase-dependent apoptosis pathway and p53 pathway. Knockdown of PIWIs inhibited the expression of caspase (Casp) 3, 7, 8, and p53 without affecting Bcl2 (B-cell lymphoma gene 2), Bax (B-cell lymphoma-2-associated X), and BaxI (B-cell lymphoma-2-associated X inhibitor), which further significantly increased abnormal spermatozoa in the knockdown-group crabs. These results show a new role of PIWI proteins in crustaceans that is different from that in mammals. In summary, PIWIs play roles in the formation of the germline nucleus and can maintain apoptosis in abnormal germ cells to remove abnormal germ cells in E. sinensis.

It is now recognized that maternal environmental factors, including chemical exposure and nutritional conditions, alter DNA methylation patterns in fetal germ cells, subsequently affecting germ cell development as well as offspring phenotypes. Here, we describe steps for detecting DNA methylation changes in mouse germ cells isolated from both embryonic and spermatogenic stages after maternal exposure to a chemical compound. For complete details on the use and execution of this protocol, please refer to Tando et al. (2021).

In birds, sex is determined through cell-autonomous mechanisms and various factors, such as the dosage of DMRT1. While the sex-determination mechanism in gonads is well known, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism underlying sex determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also exhibited sex-biased expression, and the number of female-biased genes detected was higher than that of male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Retinoic acid stimulation of cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination in avian PGCs involves aspects of both cell-autonomous and somatic-cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

Preserving a high degree of genome integrity and stability in germ cells is of utmost importance for reproduction and species propagation. However, the regulatory mechanisms of maintaining genome stability in the developing primordial germ cells (PGCs), in which rapid proliferation is coupled with global hypertranscription, remain largely unknown. Here, we find that mouse PGCs encounter a constitutively high frequency of transcription–replication conflicts (TRCs), which lead to R-loop accumulation and impose endogenous replication stress on PGCs. We further demonstrate that the Fanconi anemia (FA) pathway is activated by TRCs and has a central role in the coordination between replication and transcription in the rapidly proliferating PGCs, as disabling the FA pathway leads to TRC and R-loop accumulation, replication fork destabilization, increased DNA damage, dramatic loss of mitotically dividing mouse PGCs, and consequent sterility of both sexes. Overall, our findings uncover the unique source and resolving mechanism of endogenous replication stress during PGC proliferation, provide a biological explanation for reproductive defects in individuals with FA, and improve our understanding of the monitoring strategies for genome stability during germ cell development.

Perchlorate is a chemical compound commonly used in military artillery and equipment. It has been detected in drinking water, air, soil, and breast milk. Exposure of humans to perchlorate can occur in the theater of war and areas adjacent to military training grounds. A high concentration of perchlorate has been found to affect reproduction in vertebrates, including fish. However, whether environmental concentrations of perchlorate can affect primordial germ cells (PGCs), the founders of sperm and eggs, is not clearly understood. In the present study, we examined the effects of 0, 10, 100, and 1000 μg/L potassium perchlorate exposure on the embryonic development of medaka and their PGCs. Perchlorate exposure delayed hatching time, reduced heartbeat, inhibited migration of PGCs, and increased developmental deformities in the larvae. The 10 and 20 mg/L concentrations of perchlorate were lethal to embryos, whereas vitamin C co-treatment (1 mg/L) completely blocked perchlorate-induced mortality. RNA-seq analysis of isolated PGCs showed a non-linear pattern in expression profiles of differentially altered genes. Significantly upregulated genes were found in PGCs from the 10 and 1000 μg/L groups, whereas the 100 μg/L groups showed the highest number of significantly downregulated genes. Gene ontology analysis predicted differentially expressed genes to be involved in proteolysis, metabolic processes, peptides activity, hydrolase activity, and hormone activity. Among the cellular components, extracellular, intracellular, sarcoplasmic, and 6-phosphofructokinase and membrane-bounded processes were affected. Ingenuity Pathway Analysis of PGC transcriptomes revealed thyroid hormone signaling to be affected by all concentrations of perchlorate. The present results suggested that perchlorate affected the development of medaka larvae and vitamin C was able to ameliorate perchlorate-induced embryo mortality. Additionally, perchlorate altered the global transcriptional network in PGCs in a non-linear fashion suggesting its potential effects on developing germ cells and fertility.

Primordial germ cells (PGCs) are highly specialized cells that play a relevant role in the maintenance and evolution of the species, since they create new combinations of genetic information between the organisms. Amphibians are a class of amniote vertebrates that are divided into three subclasses, the anurans (frogs and toads), the urodeles (salamanders and newts), and the gymnophiones (caecilians). The study of PGCs in amphibians has been addressed in more detail in anurans while little is known about the biology of this cell lineage in urodeles. Studies in some urodeles species have suggested that PGCs are of mesodermal origin, specifying in the lateral plate mesoderm at the late gastrula stage. With classical experiments it shown that, there is an induction of mesoderm, therefore most likely urodeles PGCs develop from unspecialized mesodermal tissue that responds to extracellular signals. However, some fundamental biological processes of PGCs such as the analysis of their specification, arrival, and colonization to the gonads, and their maintenance and differentiation into mature and fertile gametes remain to be elucidated. Therefore, knowledge about the biology of PGCs is of great importance to ensure the perpetuation of urodeles amphibians, as some species are in danger of becoming extinct.

1. A series of experiments were conducted to examine the developmental potential of cryopreserved gonadal germ cells (GGCs) recovered from both males and females on embryo day 7 (7 d-GGCs) using the PBS(-) method. Germline chimeras were produced by transferring 200 frozen/unfrozen 7 d-GGCs recovered from female/male Rhode Island Red (RIR) embryos into the dorsal aorta of 2-day-old female and male white leghorn (WL) embryos.2. Germ-cell recipient embryos were hatched and raised to sexual maturity and progeny testing was conducted by mating with RIR of the opposite sex. Brown-feathered progeny chicks were hatched in all eight possible progeny testing combinations, except for male GGC recipients produced by transferring female GGCs. Furthermore, brown-feathered progeny chicks were hatched when frozen-thawed sperm from male germline chimeras, produced by transferring unfrozen 7d-GGCs, were inseminated in normal female RIR and female WL germline chimeras.3. The results indicated that cryopreserved female/male GGCs from 7-day-old chick embryos, recovered using the PBS(-) method, were fully capable of developing into normal spermatozoa and ova in the gonad of recipient embryos under appropriate GGC donor/recipient combinations.

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell–like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

In many non-mammalian organisms, a population of germ-line stem cells supports continuing production of gametes during post-natal life, and germ-line stem cells are also present and functional in male mammals. Traditionally, however, they have been thought not to exist in female mammals, who instead generate all their germ cells during fetal life. Over the last several years, this dogma has been challenged by several reports, while being supported by others. We describe and compare these conflicting studies with the aim of understanding how they came to opposing conclusions. We first consider studies that, by examining marker-gene expression, the fate of genetically marked cells, and consequences of depleting the oocyte population, addressed whether ovaries of post-natal females contain oogonial stem cells that give rise to new oocytes. We next discuss whether ovaries contain cells that, even if inactive under physiological conditions, nonetheless possess oogonial stem cell properties that can be revealed through cell culture. We then examine studies of whether cells harvested after long-term culture of cells obtained from ovaries can, following transplantation into ovaries of recipient females, give rise to oocytes and offspring. Finally, we note studies where somatic cells have been re-programmed to acquire a female germ-cell fate. We conclude that the weight of evidence strongly supports the traditional interpretation that germ-line stem cells do not exist post-natally in female mammals. However, the ability to generate germ cells from somatic cells in vitro establishes a method to generate new gametes from cells of post-natal mammalian females.

Genome diversity is essential for evolution and is of fundamental importance to human health. Generating genome diversity requires phases of DNA damage and repair that can cause genome instability. Humans have a high incidence of de novo congenital disorders compared to other organisms. Recent access to eggs, sperm and preimplantation embryos is revealing unprecedented rates of genome instability that may result in infertility and de novo mutations that cause genomic imbalance in at least 70% of conceptions. The error type and incidence of de novo mutations differ during developmental stages and are influenced by differences in male and female meiosis. In females, DNA repair is a critical factor that determines fertility and reproductive lifespan. In males, aberrant meiotic recombination causes infertility, embryonic failure and pregnancy loss. Evidence suggest germ cells are remarkably diverse in the type of genome instability that they display and the DNA damage responses they deploy. Additionally, the initial embryonic cell cycles are characterized by a high degree of genome instability that cause congenital disorders and may limit the use of CRISPR-Cas9 for heritable genome editing.

The goal of this study is to examine bisphenol AF (BPAF)-induced multinucleation (MNC) in comparison with dibutyl phthalate (DBP), known to induce MNC in mouse gonocytes in vivo. We performed image-based single-cell high content analysis (HCA) in the mouse spermatogonia C18-4 cells treated with various concentrations of BPAF and DBP. BPAF as low as 5 µM was cytotoxic and resulted in 40% cell death of the C18-4 cells after 72 h. HCA revealed that 5 µM of BPAF significantly increased the number of MNC by an average of 3.6-fold. DBP did not induce MNC in the doses we tested. Cytokinesis is tightly regulated by various small GTPase-signaling pathways. We, therefore, tested 5 selective GTPase inhibitors and found that Y27632, a ROCK inhibitor, reduced the BPAF-induced MNC by nearly 30%. Inhibition of Cdc42 by ML141 conversely increased the number of BPAF-induced MNC. We performed a hierarchical cluster analysis of the HCA data and demonstrated that the cytoskeletal disruption by BPAF was reversely modified by Y27632. We found that mRNA expression of genes regulating Rho and Rac GTPase activities, p190RhoGap and MgcRacGap, was altered in BPAF-treated C18-4 cells in a time-dependent manner. Multinucleated gonocytes are often indicators of disease pathologies. Our results provided the first evidence of mechanisms of the dual toxicity by BPAF to male germ cells, which induces chromosome endoreplication without the coordinated cytokinetic cellular components. The unique genotoxic mechanism of forming multinucleated germ cells suggests a novel mode of action in the male repro-toxicity concern over the increasingly ubiquitous presence of BPA analogs.

Understanding the molecular and cellular mechanisms of human primordial germ cells (hPGCs) is essential in studying infertility and germ cell tumorigenesis. Many RNA-binding proteins (RBPs) and non-coding RNAs are specifically expressed and functional during hPGC developments. However, the roles and regulatory mechanisms of these RBPs and non-coding RNAs, such as microRNAs (miRNAs), in hPGCs remain elusive. In this study, we reported a new regulatory function of DAZL, a germ cell-specific RBP, in miRNA biogenesis and cell proliferation. First, DAZL co-localized with miRNA let-7a in human PGCs and up-regulated the levels of >100 mature miRNAs, including eight out of nine let-7 family, miR21, miR22, miR125, miR10 and miR199. Purified DAZL directly bound to the loops of precursor miRNAs with sequence specificity of GUU. The binding of DAZL to the precursor miRNA increased the maturation of miRNA by enhancing the cleavage activity of DICER. Furthermore, cell proliferation assay and cell cycle analysis confirmed that DAZL inhibited the proliferation of in vitro PGCs by promoting the maturation of these miRNAs. Evidently, the mature miRNAs up-regulated by DAZL silenced cell proliferation regulators including TRIM71. Moreover, DAZL inhibited germline tumor cell proliferation and teratoma formation. These results demonstrate that DAZL regulates hPGC proliferation by enhancing miRNA processing.

Sryon the Y chromosome is the master switch of sex determination in mammals. It has been well established thatSryencodes a transcription factor that is transiently expressed in somatic cells of the male gonad, leading to the formation of testes. In the testis of adult mice,Sryis expressed as a circular RNA (circRNA) transcript. However, the physiological function ofSrycircRNA (circSRY) remains unknown since its discovery in 1993. Here we show that circSRY is mainly expressed in the spermatocytes, but not in mature sperm or somatic cells of the testis. Loss of circSRY led to germ cell apoptosis and the reduction of sperm count in the epididymis. The level of γH2AX was decreased, and failure of XY body formation was noted in circSRY KO germ cells. Further study demonstrated that circSRY directly bound to miR-138-5p in spermatocytes, and in vitro assay suggested that circSRY regulates H2AX mRNA through sponging miR-138-5p. Our study demonstrates that, besides determining sex,Sryalso plays an important role in spermatogenesis as a circRNA.

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon.

Interspecific germline chimerism mediated by transplantation of primordial germ cells (PGCs) of wild species to domestic hosts promises the conservation of wild birds. Cryopreservation of avian eggs and embryos is impracticable, and currently only frozen PGCs enable conservation of both the male and female descendants. Purebred offspring have been obtained from germline chimeras of wild avian species, proving the feasibility of such technology. In vitro propagation has been optimized for avian PGCs of domestic species; however, evidence is rather limited for successful isolation as well as long-term culture from a single embryo of wild species. With accelerating biodiversity loss, we have committed to preserving current genetic resources by freezing PGCs isolated from individual embryos in addition to their genetic material. We have devised a reliable protocol for the isolation and proliferation of PGCs from wild fowls in the family Phasianidae that are conserved in captive breeding (red junglefowl, bar-tailed pheasant, kalij pheasant, Siamese fireback pheasant, and silver pheasant). We obtained individual isolates of cultured circulating PGCs (49.7%, 79/155) as well as tissue PGCs (92.9%, 144/155). The specific co-culture conditions of autologous embryonic cells, without additional growth factors, facilitated the proliferation of so-called tissue PGCs (the remaining PGCs in embryonic tissue following blood aspiration). Only circulating PGCs left in blood vessels and of PGCs migrating to developing gonads have been previously reported. However, the present study is the first to report on the harvest of ectopic PGCs. The defined conditions sustained continuous proliferation of tissue PGCs for at least six months and maintained PGC identity following cryopreservation. Cultured tissue PGCs of these wild species were extensively characterized for their expression of the germ cell-specific proteins, chicken vasa homolog (CVH) and deleted in azoospermia-like (DAZL), as well as the ability to colonize chicken embryonic gonads. The novel protocol is practical for generating enough PGCs for cryopreservation, transplantation, and additionally, it enables isolation of PGCs from both blood circulation and embryonic tissue simultaneously. For conservation purposes, this approach is potentially applicable more widely to other non-domestic birds than those in the family Phasianidae that were investigated in the present study.

Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the underlying molecular mechanism that controls alternative mRNA expression during germ cell development remains elusive. Herein, we show that hnRNPH1 is highly expressed in the reproductive system and recruits the PTBP2 and SRSF3 to modulate the alternative splicing in germ cells. Conditional knockout Hnrnph1 in spermatogenic cells causes many abnormal splicing events, thus affecting the genes related to meiosis and communication between germ cells and Sertoli cells. This is characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, which ultimately leads to male sterility. Markedly, Hnrnph1 germline-specific mutant female mice are also infertile, and Hnrnph1-deficient oocytes exhibit a similar defective synapsis and cell-cell junction as seen in Hnrnph1-deficient male germ cells. Collectively, our data support a molecular model wherein hnRNPH1 governs a network of alternative splicing events in germ cells via recruitment of PTBP2 and SRSF3.

The effects of chemical and physical environmental factors are concerned as the main reason of diminished male fertility. The aim of the study was the investigation of the effects of low doses of Aroclor 1254 or combined exposure to low doses of Aroclor 1254 and low doses of ionizing radiation on the sperm quantity and quality of male germ cells including damage to genetic material of adult male mice. Mice were exposed for 2 weeks, 3 times per week by intraperitoneal injection with Aroclor 1254 diluted in corn oil at doses of 1, 2 and 4 mg/kg bw or to whole body X-rays irradiation at doses 0.05 Gy, 0.10 Gy and 0.15 Gy or to combination of X-rays and Aroclor 1254 at following doses 0.05 Gy + 1 mg/kg bw Aroclor 1254, 0.10 Gy + 2 mg/kg bw Aroclor 1254. The samples for sperm count, motility, morphology and DNA integrity of male germ cells estimation were taken from animals just after the end of exposure and 5 weeks later. Irradiation alone deteriorated sperm count and quality. Aroclor 1254 significantly reduced the sperm motility and increased sperm abnormality and at the highest dose also induced DNA damage of gametes. The combined exposure to 0.10 Gy + 2 mg/kg bw of Aroclor 1254 showed the increase in the sperm concentration and the decrease of percentage of abnormal spermatozoa compared to results after irradiation to 0.10 Gy alone. In conclusion, the low doses of Aroclor 1254 used in this study did not significantly reduce the sperm count, but affected the sperm motility, morphology and sometimes also DNA integrity of gametes. In combination with low doses of irradiation, low doses of Aroclor 1254 may ameliorate the harmful effect of irradiation on the male gametes.

Spermatogenesis is a complex developmental process. During this process, male germ cells from spermatogonia to sperm cells encounter a number of DNA damages. The most severe form of these damages is double-strand breaks (DSBs) deriving from exogenous and endogenous genotoxic insults. DSBs must be correctly repaired in a short time to maintain genomic integrity in the male germ cells. For this purpose, there are four pathways working in repair of DSBs: homologous recombination (HR), classical non-homologous end joining (cNHEJ), alternative end joining (aEJ), and single strand annealing (SSA). While the HR pathway repairs DSBs with a homology-based and error-free manner, the cNHEJ, aEJ, and SSA pathways join free ends in a sequence-independent mechanism. Possible impairments in these DSB repair mechanisms can lead to cell cycle arrest, abnormal meiotic recombination, and ultimately male infertility. In this review, we comprehensively introduce DSB repair pathways being used by male germ cells during spermatogenesis. Also, potential relationship between dysfunction in these pathways and male infertility development are discussed in the light of existing studies.

Human primordial germ cells (hPGCs) form around the time of implantation and are the precursors of eggs and sperm. Many aspects of hPGC specification remain poorly understood because of the inaccessibility of the early postimplantation human embryo for study. Here, we show that micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 give rise to hPGC-like cells (hPGCLC) and use these as a quantitatively reproducible and simple in vitro model to interrogate this important developmental event. We characterize micropatterned hPSCs up to 96 hr and show that hPGCLC populations are stable and continue to mature. By perturbing signaling during hPGCLC differentiation, we identify a previously unappreciated role for Nodal signaling and find that the relative timing and duration of BMP and Nodal signaling are critical parameters controlling the number of hPGCLCs. We formulate a mathematical model for a network of cross-repressive fates driven by Nodal and BMP signaling, which predicts the measured fate patterns after signaling perturbations. Finally, we show that hPSC colony size dictates the efficiency of hPGCLC specification, which led us to dramatically improve the efficiency of hPGCLC differentiation.In humans and other animals, eggs and sperm are unique cells that pass on genetic material to the next generation. They originate from a small group of cells called primordial germ cells that form early in life in the developing embryo. Several different signal molecules including ones known as BMP4, Wnt, and Nodal, instruct certain cells in the embryo to become primordial germ cells. The process by which primordial germ cells are made in humans is very different to how primordial germ cells are made in mice and other so-called model animals that are commonly used in research. This has made it more challenging to uncover the details of the process in humans. Fortunately, new methods have recently been created that mimic aspects of how human embryos develop using human stem cells in a laboratory dish, providing an opportunity to gain a deeper understanding of how human germ cells form. Jo et al. used a technique called micropatterning to control the shape and size of groups of human stem cells growing in a laboratory dish. Treating these cells with a signal known as BMP4 gave rise to cells that resembled primordial germ cells. The team then used this system as a model to study how primordial germ cells form in humans. The experiments found that reducing Wnt signals in stem cells stopped primordial germ cells from forming in response to BMP4, confirming that Wnt signals made by the cells in response to BMP4 are essential. However, this block was overcome by providing the stem cells with another signal called Nodal. This suggests that the role of Wnt signaling in primordial germ cell formation is in part indirect by switching on Nodal in stem cells. Defects in eggs and sperm may lead to infertility, therefore, the findings of Jo et al. have the potential to help researchers develop new fertility treatments that use eggs or sperm grown in a laboratory from the patients’ own stem cells. Such research would benefit from first developing a better understanding of how to make primordial germ cells.

As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.

The harmful effects of pesticides can be extended beyond the exposure time scale. Appraisals combining exposure and long-term post-exposure periods appear as an unavoidable approach in pesticide risk assessment, thus allowing a better understanding of the real impact of agrochemicals in non-target organisms. This study aimed to evaluate the progression of genetic damage in somatic and germ tissues of the crayfish Procambarus clarkii, also seeking for gender-specificities, following exposure (7 days) to penoxsulam (23 μg L

Efficient isolation of genetically modified cells that are phenotypically indistinguishable from the unmodified cells remains a major technical barrier for the broader utilization of CRISPR/Cas9. Here, we report a novel enrichment approach to select the genome engineered cells by co-targeting a genomically integrated GFP gene along with the endogenous gene of interest (GOI). Using this co-targeting approach, multiple genomic loci were successfully targeted in chicken (DF1) and quail (CEC-32) fibroblast cell lines by transient transfection of Cas9 and guide RNAs (gRNAs). Clonal isolation of co-targeted DF1 cells showed 75% of cell clones had deletion of GFP and biallelic deletion of the GOI. To assess the utility of this approach to generate genome modified animals, we tested it on chicken primordial germ cells (PGCs) expressing GFP by co-targeting with gRNAs against GFP and endogenous ovomucoid (OVM) gene. PGCs enriched for loss of GFP and confirmed for OVM deletion, derived by co-targeting, were injected into Hamburger and Hamilton stage 14-15 chicken embryos, and their ability to migrate to the genital ridge was confirmed. This simple, efficient enrichment approach could easily be applied to the creation of knock-out or edited cell lines or animals.

Blebs are cell protrusions generated by local membrane–cortex detachments followed by expansion of the plasma membrane. Blebs are formed by some migrating cells, e.g. primordial germ cells of the zebrafish. While blebs occur randomly at each part of the membrane in unpolarized cells, a polarization process guarantees the occurrence of blebs at a preferential site and thereby facilitates migration toward a specified direction. Little is known about the factors involved in the controlled and directed bleb generation, yet recent studies revealed the influence of an intracellular flow and the stabilizing role of the membrane–cortex linker molecule Ezrin. Based on this information, we develop and analyse a coupled bulk-surface model describing a potential cellular mechanism by which a bleb could be induced at a controlled site. The model rests upon intracellular Darcy flow and a diffusion–advection–reaction system, describing the temporal evolution from a homogeneous to a strongly anisotropic Ezrin distribution. We prove the well-posedness of the mathematical model and show that simulations qualitatively correspond to experimental observations, suggesting that indeed the interaction of an intracellular flow with membrane proteins can be the cause of the Ezrin redistribution accompanying bleb formation.

In brief Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other. Abstract In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

Microtubule acetylation is found in populations of stable, long-lived microtubules, occurring on the conserved lysine 40 (K40) residue of α-tubulin by α-tubulin acetyltransferases (αTATs). α-tubulin K40 acetylation has been shown to stabilize microtubules via enhancing microtubule resilience against mechanical stress. Here we show that a previously uncharacterized αTAT, Drosophila CG17003/leaky (lky), is required for α-tubulin K40 acetylation in early germ cells in Drosophila ovary. We found that loss of lky resulted in a progressive egg chamber fusion phenotype accompanied with mislocalization of germline-specific Vasa protein in somatic follicle cells. The same phenotype was observed upon replacement of endogenous α-tubulin84B with non-acetylatable α-tubulin84BK40A, suggesting α-tubulin K40 acetylation is responsible for the phenotype. Chemical disturbance of microtubules by Colcemid treatment resulted in a mislocalization of Vasa in follicle cells within a short period of time (~30 min), suggesting that the observed mislocalization is likely caused by direct leakage of cellular contents between germline and follicle cells. Taken together, this study provides a new function of α-tubulin acetylation in maintaining the cellular identity possibly by preventing the leakage of tissue-specific gene products between juxtaposing distinct cell types.

We studied the effect of antioxidants dibornol (2,6-diisobornyl-4-methylphenol) and its derivative (4-hydroxymethyl-2,6-diisobornylphenol), members of the alkylated phenols group, on the redox potential of male germ cells and their morphological and functional state in the rat model of pathospermia. Pharmacological effect was observed in animals treated with dibornol. The studied compounds led to the normalization of the antioxidant-prooxidant balance. However, the value of this indicator against the background of treatment with dibornol derivative attested to a shift in the redox balance of cells towards reduction reactions.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Sertoli cells (SCs) support and nourish germ cells (GCs) through their crosstalk during spermatogenesis. However, the underlying epigenetic mechanism that ensures SCs’ functions in this process remains unclear. Here, we report that UHRF1, a critical epigenetic regulator, is mainly expressed in human and mouse pre-mature SCs, and is essential for establishing Sertoli-Germ cell crosstalk. SC-specific UHRF1 knockout mice exhibit complete sterility with Sertoli cell (SC) proliferation and differentiation aberrance, blood-testis barrier (BTB) disruption, and immature germ cell (GC) sloughing. RNA sequencing and Whole Genome Bisulfite Sequencing (WGBS) revealed that many extracellular matrix (ECM)-related genes (e.g., Timp1, Trf, and Spp1) appeared upregulated with the DNA hypomethylation status in UHRF1-deficient SCs. Strikingly, overexpression of Timp1, Trf, and Spp1 in SCs in vitro and in vivo could phenocopy the SC-specific UHRF1-deficient mice. Our data demonstrated that UHRF1 regulates the transcriptional program of ECM-related genes in SCs and establishes SC-GC crosstalk.

Epigenetic reprogramming during perinatal germ cell development is essential for genomic imprinting and cell differentiation; however, the actors of this key event and their dynamics are poorly understood in rats. Our study aimed to characterize the expression patterns of epigenetic modifiers and the changes in histone modifications in rat gonocytes at the time of de novo DNA methylation. Using transgenic rats expressing Green Fluorescent Protein (GFP) specifically in germ cells, we purified male gonocytes by fluorescent activated cell sorting at various stages of perinatal development and established the transcriptomic profile of 165 epigenetic regulators. Using immunofluorescence on gonad sections, we tracked six histone modifications in rat male and female perinatal germ cells over time, including methylation of histone H3 on lysines 27, 9, and 4; ubiquitination of histone H2A on lysine119; and acetylation of histone H2B on lysine 20. The results revealed the dynamics in the expression of ten-eleven translocation enzymes and DNA methyltransferases in male gonocytes at the time of de novo DNA methylation. Moreover, our transcriptomic data indicate a decrease in histone ubiquitination and methylation coinciding with the beginning of de novo DNA methylation. Decreases in H2AK119Ub and H3K27me3 were further confirmed by immunofluorescence in the male germ cells but were not consistent for all H3 methylation sites examined. Together, our data highlighted transient chromatin remodeling involving histone modifications during de novo DNA methylation. Further studies addressing how these dynamic changes in histone posttranslational modifications could guide de novo DNA methylation will help explain the complex establishment of the male germ cell epigenome.

The animal germline is an immortal cell lineage that gives rise to eggs and/or sperm each generation. Fusion of an egg and sperm, or fertilization, sets off a cascade of developmental events capable of producing an array of different cell types and body plans. How germ cells develop, function, and eventually give rise to entirely new organisms is an important question in biology. A growing body of evidence suggests that phase separation events likely play a significant and multifaceted role in germ cells and development. Here, we discuss the organization, dynamics, and potential functions of phase-separated compartments in germ cells and examine the various ways in which phase separation might contribute to the development of multicellular organisms.

Spermatogenesis occurs in the seminiferous epithelium that shows the presence of estrogen receptors alpha (ERα) and beta (ERβ), both of which regulate gene transcription by binding to the DNA. Estrogen responsive phases of spermatogenesis are well documented; however, the genes regulated remain inexplicit. To study the regulation of genes by estrogen in male germ cells, we performed chromatin immunoprecipitation (ChIP) sequencing for ERα and ERβ under normal physiological conditions. A total of 27 221 DNA binding regions were enriched with ERα and 20 926 binding sites with ERβ. Majority of the peaks were present in the intronic regions and located 20 kb upstream or downstream from the transcription start site (TSS). Pathway analysis of the genes enriched by ChIP-Seq showed involvement in several biological pathways. Genes involved in pathways whose role in spermatogenesis is unexplored were validated; these included prolactin, GnRH, and oxytocin signaling. All the selected genes showed the presence of estrogen response elements (EREs) in their binding region and were also found to be significantly enriched by ChIP-qPCR. Functional validation using seminiferous tubule culture after treatment with estrogen receptor subtype-specific agonist and antagonist confirmed the regulation of these genes by estrogen through its receptors. The genes involved in these pathways were also found to be regulated by the respective receptor subtypes at the testicular level in our in vivo estrogen receptor agonist rat models. Our study provides a genome-wide map of ERα and ERβ binding sites and identifies the genes regulated by them in the male germ cells under normal physiological conditions.