scholarly journals Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1813-1819 ◽  
Author(s):  
Eri Shiraishi ◽  
Norifumi Yoshinaga ◽  
Takeshi Miura ◽  
Hayato Yokoi ◽  
Yuko Wakamatsu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sex Differentiation ◽  
Somatic Cells ◽  
Oryzias Latipes ◽  
Cell Number ◽  
Gonadal Differentiation ◽  
Loss Of Function ◽  
Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

scholarly journals Mutations that Affect Channel Opening of Innexin Hemichannels in the C. elegans Gonad

2020 ◽  
Author(s):  
Todd Starich ◽  
David Greenstein
Keyword(s):  
Cell Proliferation ◽  
Gap Junctions ◽  
Germ Cell ◽  
Structural Model ◽  
Loss Of Function ◽  
C Elegans ◽  
Somatic Gonad ◽  
Channel Opening ◽  
Germline Proliferation ◽  
Germ Cell Proliferation

In C. elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. We previously characterized a strong loss-of-function mutation (T239I) affecting the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. Here we describe the characterization of mutations that restore germ cell proliferation in the T239I EL2 mutant background. We recovered seven intragenic mutations located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky hemichannels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three other suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. These latter mutations may be useful to probe interactions with the biochemical pathways that produce the molecules transiting through soma-germline gap junctions.

scholarly journals Testes of DAZL null sheep lack spermatogonia and maintain normal somatic cells

2019 ◽  
Author(s):  
Zachariah McLean ◽  
Sarah Jane Appleby ◽  
Jingwei Wei ◽  
Russell Grant Snell ◽  
Björn Oback
Keyword(s):  
Germ Cell ◽  
Somatic Cell ◽  
Germ Cells ◽  
Genetic Improvement ◽  
Rna Binding ◽  
Somatic Cells ◽  
Marker Genes ◽  
Specific Marker ◽  
Loss Of Function ◽  
Fetal Fibroblasts

AbstractMultiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem and produce chimaeras with absolute donor germline transmission, we have disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9 mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single-cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL-null male neonatal sheep lacked germ cells. Somatic cells within their testes were morphologically intact and expressed normal levels of somatic cell-specific marker genes, indicating that the germ cell niche remained intact. This extends the DAZL-mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute transmitters in rapid livestock improvement.

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 A Limited and Diverse Set of Suppressor Mutations Restore Function to INX-8 Mutant Hemichannels in the Caenorhabditis elegans Somatic Gonad

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1655
Author(s):  
Todd Starich ◽  
David Greenstein
Keyword(s):  
Cell Proliferation ◽  
Caenorhabditis Elegans ◽  
Gap Junctions ◽  
Germ Cell ◽  
Structural Model ◽  
Loss Of Function ◽  
Suppressor Mutations ◽  
Somatic Gonad ◽  
Germline Proliferation ◽  
Germ Cell Proliferation

In Caenorhabditis elegans, gap junctions couple cells of the somatic gonad with the germline to support germ cell proliferation and gametogenesis. A strong loss-of-function mutation (T239I) affects the second extracellular loop (EL2) of the somatic INX-8 hemichannel subunit. These mutant hemichannels form non-functional gap junctions with germline-expressed innexins. We conducted a genetic screen for suppressor mutations that restore germ cell proliferation in the T239I mutant background and isolated seven intragenic mutations, located in diverse domains of INX-8 but not the EL domains. These second-site mutations compensate for the original channel defect to varying degrees, from nearly complete wild-type rescue, to partial rescue of germline proliferation. One suppressor mutation (E350K) supports the innexin cryo-EM structural model that the channel pore opening is surrounded by a cytoplasmic dome. Two suppressor mutations (S9L and I36N) may form leaky channels that support germline proliferation but cause the demise of somatic sheath cells. Phenotypic analyses of three of the suppressors reveal an equivalency in the rescue of germline proliferation and comparable delays in gametogenesis but a graded rescue of fertility. The mutations described here may be useful for elucidating the biochemical pathways that produce the active biomolecules transiting through soma–germline gap junctions.

scholarly journals Exposure to Diethylstilbestrol During Embryonic and Larval Stages of Medaka Fish (Oryzias latipes) Leads to Sex Reversal in Genetic Males and Reduced Gonad Weight in Genetic Females

Endocrinology ◽  
2011 ◽  
Vol 152 (2) ◽  
pp. 707-717 ◽  
Author(s):  
Bindhu Paul-Prasanth ◽  
Yasushi Shibata ◽  
Ryo Horiguchi ◽  
Yoshitaka Nagahama
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Sexual Differentiation ◽  
Ovarian Development ◽  
Oryzias Latipes ◽  
Sex Reversal ◽  
Short Exposure ◽  
Specific Gene ◽  
Gonad Weight ◽  
Germ Cell Proliferation

Abstract Molecular and cellular mechanisms involved in artificially induced ovarian differentiation were analyzed by exposing embryos of medaka (Oryzias latipes) to a potent nonsteroidal estrogen, diethylstilbestrol (DES). Embryos were exposed for short-exposure (SE) [from 0 to 8 d postfertilization (dpf)] and long-exposure (LE) periods (from 0 to 18/28 dpf) to 1 ng/ml of DES, and status of sexual differentiation in somatic and germ cells of these gonads was analyzed at 8, 18, and 28 dpf by histology, cell proliferation assays, TUNEL assay, and in situ hybridization using sex-specific somatic and germ cell markers. Additionally, gonads of exposed fry were examined after withdrawal of DES to see whether effects of DES in exposed fish were reversible or not. DES induced germ cell proliferation and meiosis in XY fry of SE and LE groups. However, SE induced only a partial reduction in expression of gonadal soma-derived factor, the male-dominant somatic cell marker, and was not sufficient to induce ovarian development after withdrawal of DES. On the contrary, LE resulted in complete loss of such male-specific gene expression in somatic cells of XY gonads, and these gonads underwent sustained ovarian development even after withdrawal of DES. Importantly, LE to DES affected germ cell proliferation in XX gonads adversely during early stages of sexual differentiation, leading to reduced gonad weight in adulthood. Interestingly, apoptosis was not the cause for reduction in germ cell number. Taken together, these results indicated that DES exposure has long-lasting effects on the gonadal development in genetic males (sex reversal) and females (reduced gonad weight) of medaka.

170. TGFβ SIGNALING IN AN IN VITRO SEMINOMA MODEL

2009 ◽  
Vol 21 (9) ◽  
pp. 88
Author(s):  
J. C. Young ◽  
A. Jaiprakash ◽  
S. Mithraprabhu ◽  
C. Itman ◽  
S. Kitazawa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Testicular Cancer ◽  
Germ Cell ◽  
Germ Cells ◽  
Type I ◽  
Tgfβ Signaling ◽  
Cell Markers ◽  
Germ Cell Proliferation

Testicular cancer, the second most common malignancy in young men, has a 95% cure rate but can result in infertility or subfertility. Its incidence has increased significantly in recent decades (1). This cancer is thought to arise during embryogenesis, based on the persistence of embryonic germ cell markers such as Blimp1 (2), Oct3/4 (3) and Nanog (3) in adult seminoma cells. TCam2 cells are a recently characterised in vitro seminoma model (4). We show by Q-PCR and immunofluorescence that they also express these early germ cell markers. TGFβ signaling plays a key role during germ cell development, and is implicated in the development of testicular cancers (5, 6). To investigate this further, we first determined whether the pathway is active in TCam2 cells. By Q-PCR we demonstrate expression of the TGFβ downstream transcription factors Smad 2, 3 and 4, and Activin type I and II receptors. Importantly, ActRIIA, which is undetectable in adult testicular germ cells, but readily detected in human foetal germ cells (7) and clinical seminoma samples (6), is readily detectable at both the mRNA and protein level in TCam2 cells. Furthermore, 24 hour treatment with Activin (5 and 50ng/ml) or BMP4 (5 and 50ng/ml) induces a 3-4 fold increase in ActRIIA mRNA levels, but not ActRIA, ActRIB or ActRIIB. Strikingly, in TCam2 cells BMP4 and to a lesser extent retinoic acid, but not activin, support survival and proliferation of TCam2 cells in the absence of serum. This is consistent with known roles of BMP4 and retinoic acid in enhancing murine foetal germ cell proliferation/self-renewal and survival (8, 9), and activin inhibition of foetal murine germ cell proliferation (10). This study is the first to demonstrate a functional response in seminoma cells consistent with their foetal germ cell-like identity and forms the basis for future mechanistic analyses of the role of TGFβ signaling in human testicular cancer.

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