scholarly journals TRIM71 deficiency causes germ cell loss during mouse embryogenesis and promotes human male infertility

2021 ◽  
Author(s):  
Lucia A. Torres-Fernández ◽  
Jana Emich ◽  
Yasmine Port ◽  
Sibylle Mitschka ◽  
Marius Wöste ◽  
...  
Keyword(s):  
Male Infertility ◽  
Germ Cell ◽  
Germ Cells ◽  
Cell Loss ◽  
Seminiferous Tubules ◽  
Obstructive Azoospermia ◽  
Sequencing Analysis ◽  
Mouse Embryogenesis ◽  
Human Male

AbstractMutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling the complex molecular network orchestrating germ cell development. TRIM71 is a stem cell-specific factor essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype, which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of developing germ cells in the testes’ seminiferous tubules. Infertility originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, in vitro growth competition assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells, a human GCT-derived cell line which we used as a surrogate model for proliferating PGCs, showed that TRIM71 promotes NCCIT cell proliferation and survival. Our data collectively suggest that germ cell loss in cKO mice results from combined defects during the specification and maintenance of PGCs prior to their sex determination in the genital ridges. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with SCO phenotype. Our work reveals for the first time an association of TRIM71 variants with human male infertility, and uncovers further developmental roles for TRIM71 in the generation and maintenance of germ cells during mouse embryogenesis.

scholarly journals TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility

2021 ◽  
Vol 9 ◽  
Author(s):  
Lucia A. Torres-Fernández ◽  
Jana Emich ◽  
Yasmine Port ◽  
Sibylle Mitschka ◽  
Marius Wöste ◽  
...  
Keyword(s):  
Male Infertility ◽  
Germ Cell ◽  
Cell Line ◽  
Rna Binding ◽  
Seminiferous Tubules ◽  
Obstructive Azoospermia ◽  
Sequencing Analysis ◽  
Mouse Embryogenesis ◽  
Human Male

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.

Differentiation of Human Male Germ Cells From Human Umbilical Cord-Derived Mesenchymal Stem Cells in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4581-4581
Author(s):  
Lian Ma ◽  
Xiaoying Wu ◽  
Limin Lin ◽  
Guangyu Lin ◽  
Qiuling Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Umbilical Cord ◽  
Germ Cells ◽  
Seminiferous Tubules ◽  
Human Umbilical Cord ◽  
Cell Markers ◽  
Human Male

Abstract Abstract 4581 Introduction Infertility affects 15% of couples, about 50% infertility caused by male and growing evidence suggested an increasing problems in male reproductive. Recent studies have demostrated that adult stem cells have more flexible potentials than expected, and possessed the plasticity and capacity to transdifferentiate into mutilineage cells, including germ cells. Human umbilical cord-derived mesenchymal stem cells (HUCMSCs) possess stem cell properties. In this study, we cultured HUCMSCs, and assessed the possibility of HUCMSCs differentiated into human male germ cells in vivo and in vitro, and find a new source of cells for the transplantation to the male infertility. Methods The ethics committee of our institution approved this study. HUCMSCs were isolated from the Wharton's jelly of the umbilical cord, clonally expanded. To investigate the capacity of differentiation in vitro, HUCMSCs were treated with human menopausal gonadotropinn (HMG) and retinoid acid (RA) in vitro. While investigate the capacity of differentiation in vivo, HUCMSCs were transplanted into the seminiferous tubules of busulfan-treated mice testes after labeled with pIRES2-EGFP or bromodeoxyuridine (BrdU). After induction in vitro, the morphologic changes of the differentiated cells were detected by phase contrast microscopy?Aelectron microscopy and transmission electron microscope?Gthe male germ cell markers were detected by immunohistochemistry, Western-blot and PT-PCR. HUCMSCs were also transplanted into the seminiferous tubules of the busulfan-treated mice by microinjection. To assess the fate of HUCMSCs in the testis, the survival?Amigration and germ cell markers of the HUCMSCs in the infertility mice testis were detected by immunohistochemistry?A immunofluorescence. Results HUCMSCs can express some some molecular markers of germ cells after induction. Immunohistochemistry revealed that HUCMSCs can survive in the mice testis at least 120 days, and they can migrate from the lumens to the basement membrane then to lumens again. Immunofluorescence showed that HUCMSCs can go further differentiation in the mice favorable testicular environment, and express the germ cell markers. Conclusions These suggested that HUCMSCs may differentiate into male germ cell-like cells after induced by HMG and RA in vitro; and it could survive also in a favorable testicular environment, may differentiate into germ cell lineages. This finding may provide a new strategy for the treatment of male infertility. Disclosures: No relevant conflicts of interest to declare.

VEGF/VEGFR2 Signaling Regulates Germ Cell Proliferation in vitro and Promotes Mouse Testicular Regeneration in vivo

2016 ◽  
Vol 201 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Ruhui Tian ◽  
Shi Yang ◽  
Yong Zhu ◽  
Shasha Zou ◽  
Peng Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Male Germ Cells ◽  
Vegfr2 Signaling ◽  
Proliferation In Vitro ◽  
Germ Cell Proliferation

Vascular endothelial growth factor (VEGF) plays fundamental roles in testicular development; however, its function on testicular regeneration remains unknown. The objective of this study was to explore the roles VEGF/VEGFR2 signaling plays in mouse germ cells and in mouse testicular regeneration. VEGF and the VEGFR2 antagonist SU5416 were added to culture medium to evaluate their effects on spermatogonial stem cell line (C18-4 cells) proliferation. Testicular cells obtained from newborn male ICR mice were grafted into the dorsal region of male BALB/c nude mice. VEGF and SU5416 were injected into the graft sites to assess the effects of the VEGF and VEGFR2 signaling pathways on testicular reconstitution. The grafts were analyzed after 8 weeks. We found that VEGF promoted C18-4 proliferation in vitro, indicating its role in germ cell survival. HE staining revealed that seminiferous tubules were reconstituted and male germ cells from spermatogonia to spermatids could be observed in testis-like tissues 8 weeks after grafting. A few advantaged male germ cells, including spermatocytes and spermatids, were found in SU5416-treated grafts. Moreover, VEGF enhanced the expression of genes specific for male germ cells and vascularization in 8-week grafts, whereas SU5416 decreased the expression of these genes. SU5416-treated grafts had a lower expression of MVH and CD31, indicating that blockade of VEGF/VEGFR2 signaling reduces the efficiency of seminiferous tubule reconstitution. Collectively, these data suggest that VEGF/VEGFR2 signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization.

scholarly journals Long non-coding RNAs (lncRNAs) in spermatogenesis and male infertility

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Meghali Joshi ◽  
Singh Rajender
Keyword(s):  
Male Infertility ◽  
Germ Cell ◽  
Germ Cells ◽  
Motile Sperm ◽  
Rna Seq ◽  
Small Noncoding Rnas ◽  
Cellular Processes ◽  
Human Male ◽  
Non Coding Rnas

Abstract Background Long non-coding RNAs (lncRNAs) have a size of more than 200 bp and are known to regulate a host of crucial cellular processes like proliferation, differentiation and apoptosis by regulating gene expression. While small noncoding RNAs (ncRNAs) such as miRNAs, siRNAs, Piwi-interacting RNAs have been extensively studied in male germ cell development, the role of lncRNAs in spermatogenesis remains largely unknown. Objective In this article, we have reviewed the biology and role of lncRNAs in spermatogenesis along with the tools available for data analysis. Results and conclusions Till date, three microarray and four RNA-seq studies have been undertaken to identify lncRNAs in mouse testes or germ cells. These studies were done on pre-natal, post-natal, adult testis, and different germ cells to identify lncRNAs regulating spermatogenesis. In case of humans, five RNA-seq studies on different germ cell populations, including two on sperm, were undertaken. We compared three studies on human germ cells to identify common lncRNAs and found 15 lncRNAs (LINC00635, LINC00521, LINC00174, LINC00654, LINC00710, LINC00226, LINC00326, LINC00494, LINC00535, LINC00616, LINC00662, LINC00668, LINC00467, LINC00608, and LINC00658) to show consistent differential expression across these studies. Some of the targets of these lncRNAs included CENPB, FAM98B, GOLGA6 family, RPGR, TPM2, GNB5, KCNQ10T1, TAZ, LIN28A, CDKN2B, CDKN2A, CDKN1A, CDKN1B, CDKN1C, EZH2, SUZ12, VEGFA genes. A lone study on human male infertility identified 9879 differentially expressed lncRNAs with three (lnc32058, lnc09522, and lnc98497) of them showing specific and high expression in immotile sperm in comparison to normal motile sperm. A few lncRNAs (Mrhl, Drm, Spga-lncRNAs, NLC1-C, HongrES2, Tsx, LncRNA-tcam1, Tug1, Tesra, AK015322, Gm2044, and LncRNA033862) have been functionally validated for their roles in spermatogenesis. Apart from rodents and humans, studies on sheep and bull have also identified lncRNAs potentially important for spermatogenesis. A number of these non-coding RNAs are strong candidates for further research on their roles in spermatogenesis.

scholarly journals Preservation of fertility in nature and ART

Reproduction ◽  
2002 ◽  
pp. 3-11 ◽  
Author(s):  
R Gosden ◽  
M Nagano
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Reproductive Technologies ◽  
Spermatogonial Stem Cells ◽  
Seminiferous Tubules ◽  
Natural Fertility ◽  
Fertilization In Vitro ◽  
Gonadal Failure

Individuals may regard reproduction as optional but sufficient number of them must be productive to perpetuate the species. The reproductive system is surprisingly vulnerable and depends, among other things, on a limited endowment of oocytes, controlled proliferation of spermatogonial stem cells and the genetic integrity of both. The developmental competence of oocytes and spermatogonial stem cells is maintained by evolved mechanisms for cellular detoxification and genomic stability, and excess or damaged cells are eliminated by apoptosis. Gonadal failure as a result of germ cell depletion can occur at any age, and from the effects of chemical cytotoxicity, disease and infection as well as genetic predisposition. Among extrinsic factors, alkylating agents and ionizing radiation are important causes of iatrogenic gonadal failure in young women and men. In animal models, there is evidence that hormonal manipulation, deletion of genes involved in apoptotic pathways and dietary manipulation can protect against natural and induced germ cell loss, but evidence in humans is absent or unclear. Assisted reproductive technologies (ARTs) provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Semen cryopreservation was the first technology for preserving male fertility, but this cannot serve prepubertal boys, for whom banking of testicular biopsies may provide a future option. In sterilized rodents, cryopreserved spermatogonial stem cells can recolonize seminiferous tubules and reinitiate spermatogenesis, and subcutaneous implantation of intact tubules can generate spermatozoa for fertilization in vitro by intracytoplasmic sperm injection. Transplantation of frozen-banked ovarian tissue is well-established for restoring cyclicity and fertility and is currently undergoing clinical evaluation for cancer patients. When restoration of natural fertility is unnecessary or reimplantation is unsafe, it is desirable to culture the germ cells from thawed tissue in vitro until they reach the stage at which they can be fertilized. Low temperature banking of immature germ cells is potentially very versatile, but storage of embryos and, to a lesser extent, mature oocytes is already practised in a number of species, including humans, and is likely to remain a mainstay for fertility preservation.

scholarly journals Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line

2020 ◽  
Vol 21 (8) ◽  
pp. 3009
Author(s):  
Damian M. Janecki ◽  
Erkut Ilaslan ◽  
Maciej J. Smialek ◽  
Marcin P. Sajek ◽  
Maciej Kotecki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germ Cell ◽  
Cell Line ◽  
Germ Cells ◽  
Male Germ Cell ◽  
Seminiferous Tubules ◽  
Sequencing Analysis ◽  
Germ Cell Line ◽  
Apoptotic Genes

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes.

scholarly journals Germ Cell Derivation from Pluripotent Stem Cells for Understanding In Vitro Gametogenesis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1889
Author(s):  
Tae-Kyung Hong ◽  
Jae-Hoon Song ◽  
So-Been Lee ◽  
Jeong-Tae Do
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Assisted Reproductive Technologies ◽  
Primordial Germ Cells ◽  
Reproductive Technologies ◽  
Obstructive Azoospermia ◽  
Female Germ Cell

Assisted reproductive technologies (ARTs) have developed considerably in recent years; however, they cannot rectify germ cell aplasia, such as non-obstructive azoospermia (NOA) and oocyte maturation failure syndrome. In vitro gametogenesis is a promising technology to overcome infertility, particularly germ cell aplasia. Early germ cells, such as primordial germ cells, can be relatively easily derived from pluripotent stem cells (PSCs); however, further progression to post-meiotic germ cells usually requires a gonadal niche and signals from gonadal somatic cells. Here, we review the recent advances in in vitro male and female germ cell derivation from PSCs and discuss how this technique is used to understand the biological mechanism of gamete development and gain insight into its application in infertility.

P–531 hCFTR p.G970D mutation causes Sertoli Cell-only Syndrome (SCOS) and Congenital bilateral absence of the vas deferens (CBAVD)

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
J Hou ◽  
X Li ◽  
L Wang ◽  
W Xu
Keyword(s):  
Cell Membrane ◽  
Male Infertility ◽  
Germ Cell ◽  
Cell Lines ◽  
Vas Deferens ◽  
Seminiferous Tubules ◽  
Homozygous Mutation ◽  
Obstructive Azoospermia ◽  
Bilateral Absence ◽  
Cftr Mutations

Abstract Study question Whether CFTR is a pathogenic gene for azoospermia? Summary answer CFTR p.G970D affects spermatogenesis and leads to male infertility by affecting the proliferation and survival of Germ Cell. What is known already Male infertility is a multifactorial and heterogeneous pathological condition affecting 7% of the general male population. However, up to now, only a relatively low number of genic factors have a clear relationship with spermatogenesis. Although, increased frequency of CFTR mutations or impaired CFTR expression in men with non-obstructive azoospermia or oligospermia as compared to the fertile men has been reported, but there is no direct evidence CFTR mutations cause azoospermia. Compared to F508Del mutations in Caucasians, p.G970D mutation is the most frequent CFTR mutation identified in Chinese CF patients. However, p.G970D has not been reported involved with male infertility. Study design, size, duration In this study, began in an infertile man suffering CBAVD and SCOS with no CF-like phenotype related symptoms up to now. By identifying the patient with CFTR p.G970D mutation, we further verified the function of the mutation in spermatogenesis in spermatogonia cell lines. Control testicular tissue sample was obtained from fertility man donors. Participants/materials, setting, methods WES was performed for probands and relatives and the mutation was confirmed by Sanger sequencing. Hematoxylin-eosin (HE) staining and immune fluorescence (IF) was performed on seminiferous tubules from the patient and control to characterize the structural anomalies present in the patient. GC2 mCFTRG965D cells was knocked in by the CRISPR/Cas9 gene editing system. The effects of mutations on the growth and proliferation of GC2 cells were detected by CCK8, IF, WB, BCECF staining and RT-PCR. Main results and the role of chance First, we identified the CBAVD and SCOS patient with homozygous missense mutations p.G970D in the CFTR gene, and his mutation inherited from both parents. The patient has normal general parameters and fertility parameters except for smaller testes, lower semen volume and pH. His testicular histopathology and co-location of CFTR and DDX4 which is the marker of spermatogonia likewise showed SCOS. Second. given that the amino acid sequence is conserved and the same expression and localization patterns of CFTR between human and mouse, we generated mouse derived cell lines model (mCFTRG965D) that carried a homozygous mutation equivalent to the CFTR variant in patients, using CRISPR/Cas9-mediated genome editing. mCFTRG965D affects the proliferation of Germ Cell, but has less effect on Sertoli cells, which is similar to the SCOS patient’s phenotype. Third, lower mature CFTR were observed in the GC2 mCFTRG965D groups cells compared to those in wild type groups, and CFTR protein is not evident in the GC2 mCFTRG965D groups’ cell membrane, which demonstrated the mutation affecting the anchoring of CFTR to the cell membrane. What’s more, the missense mutation will affect the function of CFTR in regulating pH, thus affecting cell homeostasis. Limitations, reasons for caution The low number of biological samples, we need more patients to confirm this mutation and azoospermia. We only validated at the cellular level, not in an animal model. It is noteworthy that, the CFTRF508del mice are fertility. Wider implications of the findings: Our study reveals that CFTR has a broader indication than just the absence of the vas deferens. We recommend to take further understanding of CFTR playing important role in spermatogenesis by affecting germ cell survival not just regulating cell volume during spermiogenesis. Trial registration number Not applicable

scholarly journals Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 333-341 ◽  
Author(s):  
Zuping He ◽  
Lixin Feng ◽  
Xiaodong Zhang ◽  
Yixun Geng ◽  
Daniela A Parodi ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Adult Mouse ◽  
Type A ◽  
Seminiferous Tubules ◽  
Positive Staining ◽  
Cellular Expression ◽  
Old Mice ◽  
Type A Spermatogonia

The objective of this study was to compare the expression of Col1a1, Col1a2, and procollagen I in the seminiferous tubules of immature and adult mice and to characterize the cellular expression pattern of procollagen I in germ cells during spermatogenesis in order to provide necessary groundwork for further functional studies in the process of spermatogenesis. Microarray analysis demonstrated that Col1a1 and Col1a2 were abundantly expressed in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice, and the expression levels of Col1a1 and Col1a2 mRNA were validated using a semi-quantitative RT-PCR assay. Western blot analysis further confirmed that procollagen I was expressed at a higher level in the seminiferous tubules of 6-day-old mice compared with 60-day-old mice. Immunohistochemical analysis revealed that type A spermatogonia were positive for procollagen I in the testis of 6-day-old mice, whereas Sertoli cells were negative for this protein. Thein vivoprocollagen I staining in type A spermatogonia was corroborated in spermatogonia exhibiting a high potential for proliferation and the ability to form germ cell colonies inin vitroculture. Moreover, procollagen I was also detected in type A spermatogonia, intermediate spermatogonia, type B spermatogonia, and preleptotene spermatocytes in the adult mouse testes, but positive staining disappeared in more differentiated germ cell lineages detaching from the basement membrane, including leptotene spermatocytes, pachytene spermatocytes, round spermatids and elongated spermatids. These data suggest that Col1a1, Col1a2 and procollagen I are associated with type A spermatogonia and play a potential role in mediating the detachment and migration of germ cells during spermatogenesis.

P–803 Novel culture conditions for the improvement of the in vitro expansion of human male fetal germ cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Martin ◽  
M Ferreira ◽  
J Taelman ◽  
C Eguizabal ◽  
S M Chuv. d. Sous. Lopes
Keyword(s):  
Growth Factors ◽  
Germ Cells ◽  
Activin A ◽  
Culture Conditions ◽  
Media Culture ◽  
Germline Stem Cell ◽  
Growth Media ◽  
Human Male ◽  
In Vitro Expansion

Abstract Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable

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