The C-X-C signalling system in the rodent vs primate testis: impact on germ cell niche interaction

In zebrafish, action of the chemokine Cxcl12 is mediated through its G-protein-coupled seven-transmembrane domain receptor Cxcr4 and the atypical receptor Cxcr7. Employing this animal model, it was revealed that this Cxcl12 signalling system plays a crucial role for directed migration of primordial germ cells (PGC) during early testicular development. Importantly, subsequent studies indicated that this regulatory mechanism is evolutionarily conserved also in mice. What is more, the functional role of the CXCL12 system does not seem to be limited to early phases of testicular development. Data from mouse studies rather demonstrate that CXCL12 and its receptors are also involved in the homing process of gonocytes into their niches at the basal membrane of the seminiferous tubules. Intriguingly, even the spermatogonial stem cells (SSCs) present in the adult mouse testis appear to maintain the ability to migrate towards a CXCL12 gradient as demonstrated by functional in vitro migration assays and in vivo germ cell transplantation assays. These findings not only indicate a role of the CXCL12 system throughout male germ cell development in mice but also suggest that this system may be evolutionarily conserved. In this review, we take into account the available literature focusing on the localization patterns of the CXCL12 system not only in rodents but also in primates, including the human. Based on these data, we discuss whether the CXCL12 system is also conserved between rodents and primates and discuss the known and potential functional consequences.

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Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00114.2018 ◽

2018 ◽

Vol 315 (5) ◽

pp. E924-E948 ◽

Cited By ~ 15

Author(s):

Qing Wen ◽

Elizabeth I. Tang ◽

Wing-yee Lui ◽

Will M. Lee ◽

Chris K. C. Wong ◽

...

Keyword(s):

Germ Cell ◽

Heavy Chain ◽

Sertoli Cells ◽

Cytoplasmic Dynein ◽

Seminiferous Epithelium ◽

Organelle Transport ◽

Cellular Events

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

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

Molecular Endocrinology ◽

10.1210/me.2002-0389 ◽

2003 ◽

Vol 17 (9) ◽

pp. 1868-1879 ◽

Cited By ~ 22

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.

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Xenopus LSm Proteins Bind U8 snoRNA via an Internal Evolutionarily Conserved Octamer Sequence

Molecular and Cellular Biology ◽

10.1128/mcb.22.12.4101-4112.2002 ◽

2002 ◽

Vol 22 (12) ◽

pp. 4101-4112 ◽

Cited By ~ 33

Author(s):

Nenad Tomasevic ◽

Brenda A. Peculis

Keyword(s):

Ribosome Biogenesis ◽

Rna Binding ◽

Ribosomal Subunit ◽

High Specificity ◽

Sm Proteins ◽

U6 Snrna ◽

Evolutionarily Conserved

ABSTRACT U8 snoRNA plays a unique role in ribosome biogenesis: it is the only snoRNA essential for maturation of the large ribosomal subunit RNAs, 5.8S and 28S. To learn the mechanisms behind the in vivo role of U8 snoRNA, we have purified to near homogeneity and characterized a set of proteins responsible for the formation of a specific U8 RNA-binding complex. This 75-kDa complex is stable in the absence of added RNA and binds U8 with high specificity, requiring the conserved octamer sequence present in all U8 homologues. At least two proteins in this complex can be cross-linked directly to U8 RNA. We have identified the proteins as Xenopus homologues of the LSm (like Sm) proteins, which were previously reported to be involved in cytoplasmic degradation of mRNA and nuclear stabilization of U6 snRNA. We have identified LSm2, -3, -4, -6, -7, and -8 in our purified complex and found that this complex associates with U8 RNA in vivo. This purified complex can bind U6 snRNA in vitro but does not bind U3 or U14 snoRNA in vitro, demonstrating that the LSm complex specifically recognizes U8 RNA.

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

Cells Tissues Organs ◽

10.1159/000440949 ◽

2016 ◽

Vol 201 (1) ◽

pp. 1-13 ◽

Cited By ~ 12

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.

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Candesartan Mediated Amelioration of Cisplatin-Induced Testicular Damage Is Associated with Alterations in Expression Patterns of Nephrin and Podocin

BioMed Research International ◽

10.1155/2015/273784 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 2

Author(s):

Noritoshi Enatsu ◽

Hideaki Miyake ◽

Koji Chiba ◽

Masato Fujisawa

Keyword(s):

Angiotensin Ii ◽

Marked Decrease ◽

Expression Patterns ◽

Basal Membrane ◽

Seminiferous Tubules ◽

Angiotensin Ii Receptor Blocker ◽

Testicular Damage ◽

Angiotensin Ii Receptor

Nephrin and podocin are known to be closely related to the pharmacological effects of angiotensin-II receptor blocker (ARB). The objectives of this study were to investigate the role of nephrin and podocin using cisplatin-induced testicular damage and to evaluate the effect of ARB. At first, we evaluated the effects of cisplatin either alone or in combination with ARB candesartan on changes in expression patterns of nephrin and podocin in the rat testes. We then conductedin vitrostudies to investigate the effects of angiotensin using cultured Sertoli cells, line TM4. As a result, the expression of nephrin and podocin was shown to localize around the basal membrane of seminiferous tubules. Treatment with cisplatin resulted in a marked decrease in the expression of nephrin and podocin and induced a shift of both proteins from linear to granular expression patterns, accompanying the increased apoptotic index in the testes; these changes were partially restored by the additional administration of candesartan.In vitrostudies with TM4 revealed the angiotensin-II mediated expression changes of nephrin and podocin. These findings suggest that candesartan can prevent cisplatin-induced testicular damage by regulating expression patterns of the nephrin-podocin complex in the testes.

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Genetic and structural analysis of the in vivo functional redundancy between murine NANOS2 and NANOS3

Development ◽

10.1242/dev.191916 ◽

2020 ◽

pp. dev.191916

Author(s):

Danelle Wright ◽

Makoto Kiso ◽

Yumiko Saga

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Double Knockout ◽

Germ Cell Differentiation ◽

Evolutionarily Conserved ◽

Male Specific

NANOS2 and NANOS3 are evolutionarily conserved RNA-binding proteins involved in murine germ cell development. NANOS3 is required for protection from apoptosis during migration and gonadal colonization in both sexes, whereas NANOS2 is male-specific and required for the male-type differentiation of germ cells. Ectopic NANOS2 rescues the functions of NANOS3, but NANOS3 cannot rescue NANOS2 function even though its expression is up-regulated in Nanos2-null conditions. It is unknown why NANOS3 cannot rescue NANOS2 function and it is unclear whether NANOS3 plays any role in male germ cell differentiation. To address these questions, we made conditional Nanos3/Nanos2 knockout mice and chimeric mice expressing chimeric NANOS proteins. Conditional double knockout of Nanos2 and 3 led to the rapid loss of germ cells, and in vivo and in vitro experiments revealed that DND1 and NANOS2 binding is dependent on the specific NANOS2 zinc finger structure. Moreover, murine NANOS3 failed to bind CNOT1, an interactor of NANOS2 at its N-terminal. Collectively, our study suggests that the inability of NANOS3 to rescue NANOS2 function is due to poor DND1 recruitment and CNOT1 binding.

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AN ASSESSMENT OF THE POSSIBLE ROLE OF 17α,20α-DIHYDROXY-4-PREGNEN-3-ONE IN THE REGULATION OF TESTOSTERONE SYNTHESIS BY RAT AND RABBIT TESTIS

Journal of Endocrinology ◽

10.1677/joe.0.0610401 ◽

1974 ◽

Vol 61 (3) ◽

pp. 401-410 ◽

Cited By ~ 7

Author(s):

H. W. A. de BRUIJN ◽

H. J. van der MOLEN

Keyword(s):

Venous Blood ◽

Competitive Inhibitor ◽

Testicular Tissue ◽

Seminiferous Tubules ◽

Interstitial Tissue ◽

Lyase Activity ◽

Testosterone Biosynthesis

SUMMARY 17α,20α-Dihydroxy-4-pregnen-3-one is a competitive inhibitor of C17,20-lyase activity in rat testicular tissue in vitro and the significance of this inhibition in vitro was evaluated for testosterone biosynthesis in rat and rabbit testis in vivo. It is concluded that 17α,20α-dihydroxy-4-pregnen-3-one is not involved in the regulation of C17,20-activity in vivo, because it was not possible to detect any 17α,20α-dihydroxy-4-pregnen-3-one in rat and rabbit testicular tissue or in testicular venous blood. If present, the levels are lower than 10 pmol/g testis. Levels of 17α-hydroxyprogester-one are in the order of 50 pmol/g testis. The C17,20-lyase has a higher affinity for 17α-hydroxyprogesterone than for 17α,20α-dihydroxy-4-pregnen-3-one and hence inhibition under in-vivo conditions is not favoured. In rat testes the 20α-hydroxysteroid dehydrogenase activity, which can convert 17α-hydroxyprogesterone to 17α,20α-dihydroxy-4-pregnen-3-one, was found to be mainly (97%) localized in the seminiferous tubules and not at the site of testosterone formation in the interstitial tissue.

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The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations

Reproduction ◽

10.1530/rep-15-0565 ◽

2016 ◽

Vol 151 (5) ◽

pp. 527-538 ◽

Cited By ~ 10

Author(s):

M Faure ◽

E Guibert ◽

S Alves ◽

B Pain ◽

C Ramé ◽

...

Keyword(s):

Germ Cells ◽

Sertoli Cells ◽

Energy Content ◽

Morphological Changes ◽

Seminiferous Tubules ◽

Reduce Food Intake ◽

Testicular Weight

Abstract Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of ‘protamine’, a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells. Reproduction (2016) 151 527–538

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Pathways involved in testicular germ cell apoptosis in immature rats after FSH suppression

Journal of Endocrinology ◽

10.1677/joe-07-0637 ◽

2008 ◽

Vol 197 (1) ◽

pp. 35-43 ◽

Cited By ~ 21

Author(s):

Saleela M Ruwanpura ◽

Robert I McLachlan ◽

Peter G Stanton ◽

Kate L Loveland ◽

Sarah J Meachem

Keyword(s):

Germ Cell ◽

Fold Increase ◽

Seminiferous Tubules ◽

Testicular Development ◽

Caspase 8 ◽

Caspase 9 ◽

Testicular Germ Cell ◽

Specific Proteins ◽

Apoptotic Pathways

FSH is a key regulator of testis function, required for the establishment of full complements of Sertoli and germ cells during postnatal testis development and for the maintenance of spermatogenesis in the adult. FSH plays an important role in germ cell survival rather than proliferation, in the window between 14 and 18 days of testicular development, which coincides with the cessation of Sertoli cell proliferation and the onset of germ cell meiosis during the first wave of spermatogenesis. This study aimed to identify the pathway(s) of apoptosis regulated by changes in FSH levels in 14 - to 18-day-old rats, using a model of in vivo FSH suppression by passive immunoneutralization with a rat anti-FSH antibody. Apoptotic pathways were identified by immunohistochemistry using pathway-specific proteins as markers of the intrinsic (activated caspase 9) and extrinsic (activated caspase 8) pathways, followed by quantification of cell numbers using stereological techniques. In addition, RT-PCR was used to assess the expression of pathway-specific genes. We previously reported a 2.5-fold increase in spermatogonial apoptosis in these samples after 4 days of FSH suppression, and now show that this increase correlates with a 9.8-fold (P<0.001) increase in the frequency of caspase 9-positive spermatogonia in the absence of caspase 8 immunoreactivity. By contrast, spermatocytes exhibited both increased caspase 9 (7.5-fold; P<0.001) and caspase 8 (5.7 fold; P<0.001) immunoreactivities after 4 days of FSH suppression. No significant change in the transcription levels of candidate genes required for either pathway was detected. This study demonstrates that, in the seminiferous tubules, FSH suppression induces spermatogonial apoptosis predominantly via the intrinsic pathway, while spermatocyte apoptosis occurs via both the intrinsic and extrinsic pathways.

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

Blood ◽

10.1182/blood.v114.22.4581.4581 ◽

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.

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