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.

Bradykinin stimulates prepubertal rat germ cell proliferation in vitro

1998 ◽  
Vol 40 (3) ◽  
pp. 173-178 ◽  
Author(s):  
Nina Atanassova ◽  
Ludmila Kancheva ◽  
Boris Somlev
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Proliferation In Vitro ◽  
Germ Cell Proliferation

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.

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.

Stem cell factor protects germ cells from apoptosis in vitro

2000 ◽  
Vol 113 (1) ◽  
pp. 161-168 ◽  
Author(s):  
W. Yan ◽  
J. Suominen ◽  
J. Toppari
Keyword(s):  
Stem Cell ◽  
Germ Cells ◽  
Stem Cell Factor ◽  
Primordial Germ Cells ◽  
Survival Function ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Dna Laddering ◽  
Survival Effect

Stem cell factor (SCF) plays an important role in migration, adhesion, proliferation, and survival of primordial germ cells and spermatogonia during testicular development. However, the function of SCF in the adult testis is poorly described. We have previously shown that, in the presence of SCF, there were more type A spermatogonia incorporating thymidine at stage XII of rat seminiferous tubules cultured in vitro than in the absence of SCF, implying that the increased DNA synthesis might result from enhanced survival of spermatogonia. To explore the potential pro-survival function of SCF during spermatogenesis, the seminiferous tubules from stage XII were cultured in the presence or absence of SCF (100 ng/ml) for 8, 24, 48, and 72 hours, respectively, and apoptosis was analyzed by DNA laddering and in situ 3′-end labeling (ISEL) staining. Surprisingly, not only spermatogonia, but also spermatocytes and spermatids, were protected from apoptosis in the presence of SCF. Apoptosis took place much later and was less severe in the SCF-treated tubules than in the controls. Based on previous studies showing that FSH prevents germ cells from undergoing apoptosis in vitro, and that SCF level is increased dramatically in response to FSH stimulation, we also tested if the pro-survival effect of FSH is mediated through SCF by using a function-blocking monoclonal antibody, ACK-2, to block SCF/c-kit interaction. After 24 hours of blockade, the protective effect of FSH was partially abolished, as manifested by DNA laddering and ISEL analyses. The present study demonstrates that SCF acts as an important survival factor for germ cells in the adult rat testis and FSH pro-survival effect on germ cells is mediated partially through the SCF/c-kit pathway.

scholarly journals Overexpression of Bcl-w in the Testis Disrupts Spermatogenesis: Revelation of a Role of BCL-W in Male Germ Cell Cycle Control

2003 ◽  
Vol 17 (9) ◽  
pp. 1868-1879 ◽  
Author(s):  
Wei Yan ◽  
Jun-Xing Huang ◽  
Anna-Stina Lax ◽  
Lauri Pelliniemi ◽  
Eeva Salminen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germ Cell ◽  
Sertoli Cell ◽  
Germ Cells ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Testicular Development ◽  
Dna Ladder

Abstract To explore physiological roles of BCL-W, a prosurvival member of the BCL-2 protein family, we generated transgenic (TG) mice overexpressing Bcl-w driven by a chicken β-actin promoter. Male Bcl-w TG mice developed normally but were infertile. The adult TG testes displayed disrupted spermatogenesis with various severities ranging from thin seminiferous epithelium containing less germ cells to Sertoli cell-only appearance. No overpopulation of any type of germ cells was observed during testicular development. In contrast, the developing TG testes displayed decreased number of spermatogonia, degeneration, and detachment of spermatocytes and Sertoli cell vacuolization. The proliferative activity of germ cells was significantly reduced during testicular development and spermatogenesis, as determined by in vivo and in vitro 5′-bromo-2′deoxyuridine incorporation assays. Sertoli cells were structurally and functionally normal. The degenerating germ cells were TUNEL-negative and no typical apoptotic DNA ladder was detected. Our data suggest that regulated spatial and temporal expression of BCL-W is required for normal testicular development and spermatogenesis, and overexpression of BCL-W inhibits germ cell cycle entry and/or cell cycle progression leading to disrupted spermatogenesis.

scholarly journals 253.Follicle stimulating-hormone (FSH) withdrawal induces germ cell apoptosis in the mature rat testis in vitro

2004 ◽  
Vol 16 (9) ◽  
pp. 253
Author(s):  
S. M. Degen ◽  
P. G. Stanton ◽  
K. L. Loveland ◽  
S. J. Meachem
Keyword(s):  
Cell Proliferation ◽  
Germ Cell ◽  
Cell Development ◽  
Brdu Incorporation ◽  
Proliferating Cells ◽  
Germ Cell Development ◽  
Sperm Output ◽  
The Impact ◽  
Germ Cell Proliferation

FSH is a key determinant of adult sperm output influencing both Sertoli and germ cell development. The aim of this study was to assess the impact of FSH action on Sertoli and germ cell proliferation and survival in vitro, and to identify FSH-regulated genes that may underpin these responses. Testis fragments from 17-day-old rats were cultured with recombinant human FSH for 2 or 24 h and then labelled with bromodeoxyuridine (BrdU) to identify proliferating cells. The testis fragments were then processed for analysis of cell numbers by stereology, BrdU incorporation by immunohistochemistry, and apoptosis by TUNEL. The TUNEL assay revealed that without FSH, spermatogonial apoptosis was induced to 195% and 179% (P�<�0.05) compared to fragments with FSH after 2 and 24 h, respectively. No difference in apoptosis was observed in spermatocyte or Sertoli cell populations at these time points. No differences in Sertoli or germ cell proliferation were observed with or without FSH. To understand how FSH mediates spermatogonial apoptosis the response of 5 testicular genes of interest was examined. Expression of cyclin D2 (cell cycle, G1-S), N-cadherin (N-Cad; adhesion molecule), Bax (pro-apoptotic), Bcl-w (anti-apoptotic), and stem cell factor (SCF; pro-apoptotic and other functions) was elevated to 151%, 348%, 209%, 258%, and 198%, respectively (all P�<�0.001), in fragments cultured without FSH for 24 h, compared to fragments with FSH. No gene expression differences were observed at 2 h, except for SCF, which was elevated to 135% (P�<�0.01). In conclusion, these studies have examined apoptosis and proliferation activities simultaneously in testis fragments in vitro, and demonstrated that FSH withdrawal induces both spermatogonial apoptosis and expression of testicular genes known to be involved in cell survival. This model will now be used to further investigate FSH-mediation of Sertoli and germ cell development.

Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture

Development ◽  
2002 ◽  
Vol 129 (14) ◽  
pp. 3359-3365 ◽  
Author(s):  
Noriyoshi Sakai
Keyword(s):  
Cell Culture ◽  
In Vitro Fertilization ◽  
Germ Cell ◽  
Germ Cells ◽  
Regulatory Function ◽  
Feeder Cells ◽  
Male Germ Cells ◽  
Vitro Fertilization ◽  
Testicular Cells

Because cell culture systems are easily accessible for experimental genetic manipulation, male germ cell culture is of great usefulness in creating sperm vectors. This report describes that cultured male germ cells of zebrafish (Danio rerio) underwent mitosis and transmeiotic differentiation, including the entire process of meiosis, to develop into functional sperm. Enzymatically dissociated testicular cells containing germ cells were co-cultured on feeder cells derived from tumor-like testis, which exhibited features characteristic of Sertoli cells such as phagocytic activity and transcription of the Wilms’ tumor suppressor wt1 and sox9a genes. Germ cells formed a clump, divided by mitosis, and differentiated into flagellated sperm on the feeders. Expression of the germ cell marker gene vas was prolonged in co-culture with the feeders, compared with culture of dissociated testicular cells alone, indicating that the feeder cells stimulate proliferation of spermatogonia. When cultured germ cells/sperm with the feeders were used for in vitro fertilization, normal embryos were obtained. Addition of the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) into culture medium resulted in BrdU-positive sperm and four-cell stage embryos after in vitro fertilization. This culture system should prove useful not only in producing transfected functional sperm, but also in analyzing the regulatory function of testicular somatic cells on the mitosis and meiosis of male germ cells in vertebrates.

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