Search

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell typespecific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies. [ABSTRACT FROM AUTHOR]

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltrans- ferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified. [ABSTRACT FROM AUTHOR]

Correct fetal testis development underpins adult male fertility, and TGFb superfamily ligands control key aspects of this process. Transcripts encoding one such ligand, activin A, are upregulated in testes after sex determination and remain high until after birth. Testis development requires activin signalling; mice lacking activin A (Inhba KO) display altered somatic and germ cell proliferation, disrupted cord elongation and altered steroid synthesis. In human pregnancies with pre-eclampsia, the foetus is inappropriately exposed to elevated activin A. To learn how this affects testis development, we examined mice lacking the potent activin inhibitor, inhibin, (Inha KO) at E13.5, E15.5 and PND0. At E13.5, testes appeared similar in WT and KO littermates, however E15.5 Inha KO testes displayed two germline phenotypes: (1) multinucleated germ cells within cords, and (2) germ cells outside of cords, both of which are documented following in utero exposure to endocrine disrupting phthalates in rodents. Quantitation of Sertoli and germ cells in Inha KO (modelling elevated activin A) and Inhba KO (low activin A) testes using immunofluorescence demonstrated activin A bioactivity determines the Sertoli/germ cell ratio. The 50% reduction in gonocytes in Inha KO testes at birth indicates unopposed activin A has a profound impact on embryonic germ cells. Whole testis RNAseq on Inha KO mice revealed most transcripts affected at E13.5 were present in Leydig cells and associated with steroid biosynthesis/metabolism. In agreement, androstenedione (A4), testosterone (T), and the A4:T ratio were reduced in Inha KO testes at E17.5, confirming unopposed activin A disrupts testicular steroid production. E15.5 testes cultured with either activin A and/or mono-2-ethylhexyl phthalate (MEHP) generated common histological and transcriptional outcomes affecting germline and Leydig cells, recapitulating the phenotype observed in Inha KO testes. Cultures with activin A and MEHP together provided evidence of common targets. Lastly, this study extends previous work focussed on the Inhba KO model to produce a signature of activin A bioactivity in the fetal testis. These outcomes show the potential for elevated activin A signalling to replicate some aspects of fetal phthalate exposure prior to the masculinization programming window, influencing fetal testis growth and increasing the risk of testicular dysgenesis. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are essential for male fertility. Here, we report that mouse SSC generation is driven by a transcription factor (TF) cascade controlled by the homeobox protein, RHOX10, which acts by driving the differentiation of SSC precursors called pro-spermatogonia (ProSG). We identify genes regulated by RHOX10 in ProSG in vivo and define direct RHOX10-target genes using several approaches, including a rapid temporal induction assay: iSLAMseq. Together, these approaches identify temporal waves of RHOX10 direct targets, as well as RHOX10 secondary-target genes. Many of the RHOX10-regulated genes encode proteins with known roles in SSCs. Using an in vitro ProSG differentiation assay, we find that RHOX10 promotes mouse ProSG differentiation through a conserved transcriptional cascade involving the key germ-cell TFs DMRT1 and ZBTB16. Our study gives important insights into germ cell development and provides a blueprint for how to define TF cascades.

Correct fetal testis development underpins adult male fertility, and TGFβ superfamily ligands control key aspects of this process. Transcripts encoding one such ligand, activin A, are upregulated in testes after sex determination and remain high until after birth. Testis development requires activin signalling; mice lacking activin A (Inhba KO) display altered somatic and germ cell proliferation, disrupted cord elongation and altered steroid synthesis. In human pregnancies with pre-eclampsia, the foetus is inappropriately exposed to elevated activin A. To learn how this affects testis development, we examined mice lacking the potent activin inhibitor, inhibin, (Inha KO) at E13.5, E15.5 and PND0. At E13.5, testes appeared similar in WT and KO littermates, however E15.5 Inha KO testes displayed two germline phenotypes: (1) multinucleated germ cells within cords, and (2) germ cells outside of cords, both of which are documented following in utero exposure to endocrine disrupting phthalates in rodents. Quantitation of Sertoli and germ cells in Inha KO (modelling elevated activin A) and Inhba KO (low activin A) testes using immunofluorescence demonstrated activin A bioactivity determines the Sertoli/germ cell ratio. The 50% reduction in gonocytes in Inha KO testes at birth indicates unopposed activin A has a profound impact on embryonic germ cells. Whole testis RNAseq on Inha KO mice revealed most transcripts affected at E13.5 were present in Leydig cells and associated with steroid biosynthesis/metabolism. In agreement, androstenedione (A4), testosterone (T), and the A4:T ratio were reduced in Inha KO testes at E17.5, confirming unopposed activin A disrupts testicular steroid production. E15.5 testes cultured with either activin A and/or mono-2-ethylhexyl phthalate (MEHP) generated common histological and transcriptional outcomes affecting germline and Leydig cells, recapitulating the phenotype observed in Inha KO testes. Cultures with activin A and MEHP together provided evidence of common targets. Lastly, this study extends previous work focussed on the Inhba KO model to produce a signature of activin A bioactivity in the fetal testis. These outcomes show the potential for elevated activin A signalling to replicate some aspects of fetal phthalate exposure prior to the masculinization programming window, influencing fetal testis growth and increasing the risk of testicular dysgenesis.

Résumé La spermatogenèse chez les mammifères adultes est. sensible à la chaleur et en conséquence incompatible avec la température corporelle typique des mammifères de 37 °C. On pense que ça soit la raison pour laquelle les testicules de la plupart des mammifères mâles se trouvent dans le scrotum à l’extérieur du corps où ils fonctionnent à environ 33 °C. Il a été suggéré que l’exposition des testicules cryptorchidés à une température anormalement élevée pourrais empêcher le développement testiculaire normal et ainsi être à l’origine de l’infertilité chez l’adulte. Je résume des données cliniques, génétiques et histologiques en faveur de l’hypogonadisme hypogonadotrope et contre le stress thermique comme cause sous-jacente de l’infertilité adulte dans la cryptorchidie. Résumé patient: L’infertilité et un risque accru de développer un cancer des testicules chez les patients diagnostiqués avec un testicule non descendu, sont la conséquence d’un déficit hormonal plutôt que de dommages cellulaires induits par la température. La cryptorchidie nécessite donc un traitement à la fois chirurgical et hormonal.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7 days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10 ng/mL bFGF and 10 ng/mL bFGF + 100 ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on the growth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications. Methods: Testis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1). Results: Following xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes. Conclusion: Xenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time. [ABSTRACT FROM AUTHOR]

Primordial germ cells nesting in the fetal testis give rise to gonocytes. The gonocytes then transform into spermatogenic stem cells (SSCs) during the neonatal period and thereafter serve as a lifetime source of spermatogenesis. Therefore, gonocyte to SSC transformation is quite an important process that supports fertility in males. During the gonocyte to SSC transformation, morphological and transcriptomic changes sequentially occur and gonocytes migrate from the center to the peripheral region of the seminiferous tubules. However, extrinsic signals which trigger the transcriptomic changes as well as the migration are not yet fully clarified. Recent studies have drawn attention to the temporal activation of the hypothalamic-pituitary-gonadal axis during the neonatal stage which occurs concurrently with SSC formation. This phenomenon is called mini-puberty, and recent studies on human cryptorchid patients as well as animal models partially support the hypothesis that mini-puberty plays pivotal roles in gonocyte-to-SSC transformation. Focusing on this point, here, we aimed to discuss the latest knowledge on the importance of mini-puberty in spermatogenesis in this review.

BackgroundCryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs in vitro that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications.MethodsTestis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1).ResultsFollowing xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes.ConclusionXenotransplantation, without in vitro propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.

Copyright of Basic & Clinical Andrology is the property of BioMed Central and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Pre- and/or post-natal administrations of di(2-ethylhexyl) phthalate (DEHP) in experimental animals cause alterations in the spermatogenesis. However, the mechanism by which DEHP affects fertility is unknown and could be through alterations in the survival and differentiation of the gonocytes. The aim of the present study was to evaluate the effect of a single administration of DEHP in newborn mice on gonocytic proliferation, differentiation and survival and its long-term effects on seminiferous epithelium and sperm quality. BALB/c mice distributed into Control and DEHP groups were used. Each animal in the DEHP group was given a single dose of 500 mg/Kg at birth. The animals were analyzed at 1, 2, 4, 6, 8, 10 and 70 days postpartum (dpp). Testicular tissues were processed for morphological analysis to determine the different types of gonocytes, differentiation index, seminiferous epithelial alterations, and immunoreactivity to Stra8, Pcna and Vimentin proteins. Long-term evaluation of the seminiferous epithelium and sperm quality were carried out at 70 dpp. The DEHP animal group presented gonocytic degeneration with delayed differentiation, causing a reduction in the population of spermatogonia (Stra8 +) in the cellular proliferation (Pcna+) and disorganization of Vimentin filaments. These events had long-term repercussions on the quality of the seminiferous epithelium and semen. Our study demonstrates that at birth, there is a period that the testes are extremely sensitive to DEHP exposure, which leads to gonocytic degeneration and delay in their differentiation. This situation can have long-term repercussions or permanent effects on the quality of the seminiferous epithelium and sperm parameters., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Objectives: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD‐box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. Materials and Methods: Germ cell‐specific Ddx5 knockout (Ddx5‐/‐) mice were generated. The morphology of testes and epididymides and fertility in both wild‐type and Ddx5‐/‐ mice were analysed. Single‐cell RNA sequencing (scRNA‐seq) was used to profile the transcriptome in testes from wild‐type and Ddx5‐/‐ mice at postnatal day (P) 2. Dysregulated genes were validated by single‐cell qRT‐PCR and immunofluorescent staining. Results: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell‐specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5‐/‐ mice from E18.5 to P6. Single‐cell transcriptome analysis showed that genes involved in cell cycle and glial cell line‐derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5‐deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. Conclusions: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility. [ABSTRACT FROM AUTHOR]

Abstract Background Spermatogenesis is a cellular differentiation process that includes three major events: mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis. Steady-state spermatogenesis relies on functions of spermatogonial stem cells (SSCs). Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals. Currently, our knowledge about SSC and spermatogenesis is severely limited in domestic animals. Results In the present study, we examined transcriptomes of testes from domestic yaks at four different stages (3, 5, 8 and 24 months of age) and attempted to identify genes that are associated with key developmental events of spermatogenesis. Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3, 5, 8 and 24 months old yaks were gonocytes, spermatogonia, spermatocytes and elongated spermatids, respectively. RNA-sequencing (RNA-seq) analyses revealed that 11904, 4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition, the mitosis to meiosis transition and the meiosis to post-meiosis transition. Further analyses identified a list of candidate genes than may regulate these important cellular processes. CXCR4, a previously identified SSC niche factor in mouse, was one of the up-regulated genes in the 5 months old yak testis. Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis. Conclusions Together, these findings demonstrated histological changes of postnatal testis development in the domestic yak. During development of spermatogonial lineage, meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression. Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.

Abstract Background Germ cells represent one of the typical cell types that moves over a long period of time and large distance within the animal body. To continue its life cycle, germ cells must migrate to spatially distinct locations for proper development. Defects in such migration processes can result in infertility. Thus, for more than a century, the principles of germ cell migration have been a focus of interest in the field of reproductive biology. Methods Based on published reports (mainly from rodents), investigations of germ cell migration before releasing from the body, including primordial germ cells (PGCs), gonocytes, spermatogonia, and immature spermatozoon, were summarized. Main findings Germ cells migrate with various patterns, with each migration step regulated by distinct mechanisms. During development, PGCs actively and passively migrate from the extraembryonic region toward genital ridges through the hindgut epithelium. After sex determination, male germline cells migrate heterogeneously in a developmental stage‐dependent manner within the testis. Conclusion During migration, there are multiple gates that disallow germ cells from re‐entering the proper developmental pathway after wandering off the original migration path. The presence of gates may ensure the robustness of germ cell development during development, growth, and homeostasis.

Mammalian spermatogenesis, probably the most complex of all cellular developmental processes, is an ideal model both for studying the specific mechanism of gametogenesis and for understanding the basic rules governing all developmental processes, as it entails both cell type-specific and housekeeping molecular processes. Spermatogenesis can be viewed as a mission with many tasks to accomplish, and its success is genetically programmed and ensured by the collaboration of a large number of genes. Here, I present an overview of mammalian spermatogenesis and the mechanisms underlying each step in the process, covering the cellular and molecular activities that occur at each developmental stage and emphasizing their gene regulation in light of recent studies., Competing Interests: Competing interests The author declares no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: The germline serves as a conduit for transmission of genetic and epigenetic information from one generation to the next. In males, spermatozoa are the final carriers of inheritance and their continual production is supported by a foundational population of spermatogonial stem cells (SSCs) that forms from prospermatogonial precursors during the early stages of neonatal development. In mammals, the timing for which SSCs are specified and the underlying mechanisms guiding this process remain to be completely understood. Objectives: To propose an evolving concept for how the foundational SSC population is established. Materials and methods: This review summarizes recent and historical findings from peer‐reviewed publications made primarily with mouse models while incorporating limited studies from humans and livestock. Results and conclusion: Establishment of the SSC population appears to follow a biphasic pattern involving a period of fate programming followed by an establishment phase that culminates in formation of the SSC population. This model for establishment of the foundational SSC population from precursors is anticipated to extend across mammalian species and include humans and livestock, albeit on different timescales. [ABSTRACT FROM AUTHOR]

Purpose: Undescended testes (UDT) are subjected to heat stress, which can disturb gonocyte transformation as well as apoptosis. This study aims to describe the apoptosis pathway occurring during minipuberty of children with unilateral (UDT), and to investigate the role of inhibin-B.Methods: Testicular biopsies at unilateral orchidopexy of 10 boys (6-9 months old) with normal inhibin-B (n = 5) or low inhibin-B (n = 5) were selected for immunohistochemistry and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling) assay. Testicular tubules were labelled with antibodies against Anti-Müllerian hormone (AMH, Sertoli cell marker), mouse Vasa Homolog (MVH) and placental alkaline phosphatase (PLAP) (both germ cell markers), cleaved caspase3 (apoptotic marker), and followed by confocal imaging and cell counting with Fiji/ImageJ. Data were analyzed with GraphPad Prism.Results: In males with low and normal inhibin-B, there was no statistical difference (p > 0.05) in the percentage of testicular tubules containing TUNEL + cells, number of cleaved caspase3 ± germ cells/tubule, total number of germ cells/tubule, and the percentage of fibrotic tubules or number of Sertoli cells/tubule.Conclusions: These results suggest that inhibin-B does not regulate cell death of gonocytes and further studies are required to uncover any role of inhibin-B in gonocyte transformation. [ABSTRACT FROM AUTHOR]

As the major somatic cell type, Sertoli cells undergo active proliferation and play essential roles to establish testis cord at fetal stage. They also function to maintain germ cell development throughout the life of testicular development. However, the significance of Sertoli cell number for testis cord development and gonocyte fate is still unclear. Nuclear protein ataxia-telangiectasia (NPAT, also known as p220), a substrate of cyclin E/cyclin-dependent kinase 2, is well known as a regulator of cell proliferation through regulating histone expression. To study the role of NPAT during Sertoli cell development, we generated a mouse strain carrying conditional floxed Npat alleles, when crossing with anti-Müllerian hormone-cre, leading to the specific deletion of Npat in Sertoli cells. Npat disruption in Sertoli cells inhibited the programmed proliferation of fetal Sertoli cells resulting in disruption of developing testis cords, and subsequent postnatal mutant testes were severely hypoplastic. Germ cells, which are presumed to be in quiescent status during perinatal stage, exited G0 phase arrest and re-enter mitotic cell cycle prematurely. Of particular note, some germ cells possessed the meiotic signal in Npat-deficient testes. Our data thus indicates that the function of Npat-dependent Sertoli cells is essential at multiple steps in testis development, and this study also identifies Sertoli cells as a major regulator of germ cell development, which are required to maintain a local growth niche to repress premature mitosis and meiosis of gonocytes. [ABSTRACT FROM AUTHOR]

Neonatal testicular germ cells/gonocytes, transform into stem cells for spermatogenesis during 'minipuberty', driving change in timing of surgery. This study examined gonocyte transformation in cryptorchid testes in children ≤ 18 months of age with unilateral, bilateral undescended testes (UDT), complete or partial androgen insensitivity syndrome (CAIS, PAIS) [3,4]. Testicular biopsies were taken from patients with unilateral or bilateral UDT, PAIS or CAIS, aged 10 days–18 months. These testicular sections underwent immunohistochemistry with antibodies (Oct4, Ki67, C-Kit, Sox9) followed by confocal imaging, cell counting and statistical analysis. Both Sertoli cells/tubule and germ cells (GC)/tubule decreased with age, and % empty tubules (no GC) increased with age but with no significant differences between patient groups. Oct4+ germ cells/tubule decreased with age. There are some GCs and Sertoli cells proliferating during the first year and most proliferating Oct4+ germ cells (Oct4+/Ki67+) were located off tubular basement membrane. Our study showed that Oct4 expression gradually decreased after minipuberty and transformation into spermatogonia. Germ cells and Sertoli cells undergo mitosis during the first 12 months although not abundantly. We propose that Oct4+ gonocyte transformation into spermatogonia via proliferation and migration to the basement membrane may be delayed in UDT. [ABSTRACT FROM AUTHOR]

Background: Germ cells represent one of the typical cell types that moves over a long period of time and large distance within the animal body. To continue its life cycle, germ cells must migrate to spatially distinct locations for proper development. Defects in such migration processes can result in infertility. Thus, for more than a century, the principles of germ cell migration have been a focus of interest in the field of reproductive biology. Methods: Based on published reports (mainly from rodents), investigations of germ cell migration before releasing from the body, including primordial germ cells (PGCs), gonocytes, spermatogonia, and immature spermatozoon, were summarized. Main findings: Germ cells migrate with various patterns, with each migration step regulated by distinct mechanisms. During development, PGCs actively and passively migrate from the extraembryonic region toward genital ridges through the hindgut epithelium. After sex determination, male germline cells migrate heterogeneously in a developmental stage‐dependent manner within the testis. Conclusion: During migration, there are multiple gates that disallow germ cells from re‐entering the proper developmental pathway after wandering off the original migration path. The presence of gates may ensure the robustness of germ cell development during development, growth, and homeostasis. [ABSTRACT FROM AUTHOR]

DNA methylation regulates the organization and function of the genome. Yamanaka et al. now report that de novo methylation of male germ cells of mice involves the transient opening of heterochromatin at megabase-size differentially accessible domains (DADs). This chromatin remodeling likely facilitates de novo methylation of the germ cell genome. [ABSTRACT FROM AUTHOR]

Summary Retrotransposon genes are silenced by DNA methylation because of potential harm due to insertional mutagenesis. DNA methylation of retrotransposon genes is erased and re-established during male germ cell development. Both piRNA-dependent and piRNA-independent mechanisms are active during the re-establishment process, with the piRNA-independent mechanism occurring first. In this study, we analyzed the role of PIWIL4/MIWI2 in the modification of histone H3 and subsequent piRNA-dependent DNA methylation. Dimethylation at H3K4 is highly enriched at piRNA-dependent methylated regions and anti-correlated with de novo DNA methylation during the phase of piRNA-independent DNA methylation. In addition, PIWIL4, which binds the H3K4 demethylases KDM1A and KDM5B, is required for removing H3K4me2 marks. These data show that PIWIL4 plays important roles in histone modification and piRNA-dependent DNA methylation. Graphical Abstract Highlights • Dimethylation of H3K4 is enriched at piRNA-dependent genomic regions • H3K4me2 is subsequently erased in a PIWIL4-dependent manner • PIWIL4 functions through an interaction with KDM1A and KDM5B Nagamori et al. report that H3K4me2 is enriched at piRNA-dependent regions, such as LINE1s/L1s, in embryonic germ cells. H3K4me2 is subsequently erased in a PIWIL4-dependent manner. They postulate that preferential interaction of PIWIL4 with KDM1A and KDM5B plays a critical role in PIWIL4-dependent methylation erasure. [ABSTRACT FROM AUTHOR]

During neonatal testis development, centrally located gonocytes migrate to basement membrane of the seminiferous cords, where physical contact with a niche established by Sertoli cells is essential for transition of gonocytes into spermatogonial stem cells (SSCs). To provide structural support and signaling stimuli for the gonocyte-to-SSC transition that occurs at a specific location during a finite phase, temporal-spatial establishment of the niche is critical. To date, the factors that guide Sertoli cells to establish the initial stem cell niche remain largely unknown. Using the Sertoli cell-specific Arid4b knockout ( Arid4bSCKO) mice, we demonstrated that ablation of AT-rich interaction domain 4B (ARID4B) resulted in abnormal detachment of Sertoli cells from the basement membrane of seminiferous cords during the gonocyte-to-SSC transition phase, suggesting failure to establish a niche for the SSC formation. Without support by a niche environment, gonocytes showed disarranged cell distribution in the Arid4bSCKO testes and underwent apoptosis. The commitment of gonocytes to differentiate into the spermatogonial lineage was broken and the capability of SSCs to self-renew and differentiate was also impaired. Gene expression profiling revealed the molecular mechanisms responsible for the phenotypic changes in the Arid4bSCKO testes, by identifying genes important for stem cell niche function as downstream effectors of ARID4B, including genes that encode gap junction protein alpha-1, KIT ligand, anti-Müllerian hormone, Glial cell-line derived neurotrophic factor, inhibin alpha, inhibin beta, and cytochrome P450 family 26 subfamily b polypeptide 1. Our results identified ARID4B as a master regulator of a signaling network that governs the establishment of a niche during the critical gonocyte-to-SSC transition phase to control the fate of gonocytes and SSCs. S tem C ells 2017;35:1554-1565 [ABSTRACT FROM AUTHOR]

Enrichment and propagation of gonocytes or spermatogonial stem cells (SSCs) from cryopreserved testicular tissue is essential to apply SSCs-related techniques in large domestic animals. We previously reported the cryopreservation of adult bovine testicular tissue. Here, we conducted the enrichment and culture of putative gonocytes from cryopreserved testicular tissues of post-natal 1-day-old bulls. The testicular structure was well maintained after freezing and thawing. Higher mRNA levels of gonocyte/SSCs markers (PLZF, GFRα1, and UCHL-1) than those of pluripotency genes (Oct4, Sox2, and Nanog) were detected in the frozen–thawed sex cords. GFRα1 was specifically detected in the membrane and cytoplasm of gonocytes by immunostaining. Differential plating provided 40–50% enrichment of putative gonocytes. They were single, paired-, aligned-cells, or grape cluster-like colonies in minimum essential medium (MEM) containing 2.5% FBS + 2 mM glutamine + 100 IU/mL penicillin-streptomycin + 40 μg/mL gentamycin + 15 mM HEPES + 10 mM β-mercaptoethanol + 0.1 mM non-essential amino acids + 1 mM sodium pyruvate. On day 3, gonocyte progeny increased and the contaminated somatic cells spread and concurrently divided slowly. On day 5, gonocyte progeny proliferated continuously and typical intercellular bridges formed by incomplete cytokinesis in paired-cells or aligned-cysts were observed. Immunochemically, they were still GFRα1 and PLZF positive. These cells expressed significantly higher gonocyte/SSCs marker mRNAs than pluripotency gene mRNAs, concomitant with a higher level of differentiated spermatogonia marker c-kit. With time, gonocyte progeny colonies appeared in varied sizes and expanded dramatically on day 7. After cultured for 9–10 days, however, large colonies collapsed and dispersed as some single cells and small syncytial cysts. Together, MEM containing 10% dimethyl sulfoxide + 2.5% newborn calf serum provides efficient cryoprotection for the testicular tissue from 1-day-old neonatal bulls. Putative gonocytes enriched from these nascent tissues present robust proliferation capacity, conserved gonocyte/SSCs markers, and SSCs-like in vitro features. [ABSTRACT FROM AUTHOR]