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Human germ cell secreting factor nodal regulates sertoli cell functions
P-659 Wednesday, October 22, 2014 OVEREREXPRESSION OF X-LINKED EIF2S3X CAN SUBSTITUTE FOR THE LOSS OF Y-LINKED EIF2S3Y AND ALLOWS FOR SPERMATOGONIAL PROLIFERATION AND DIFFERENTIATION IN Y. Yamauchi,a J. M. Riel,a THE MOUSE. V. A. Ruthig,a M. J. Mitchell,b M. A. Ward.a aInstitute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, University of Hawai’i, John A. Burns School of Medicine, Honolulu, HI; bMedical Genetics and Functional Genomics, INSERM, Marseille, Codex 05, France. OBJECTIVE: Mice with a single X chromosome transgenic for Sry (XOSry) develop testes populated with spermatogonia. Without other Y chromosome genes these spermatogonia undergo proliferation arrest, and meiotic and post-meiotic stages of spermatogenesis are absent. Spermatogonial proliferation block can be overcome by transgenic addition of Y chromosome gene Eif2s3y. In XOSry,Eif2s3y males spermatogenesis progresses but with meiotic and postmeiotic arrest allowing only for occasional appearance of round spermatids, and never sperm. Eif2s3x is an X chromosome gene with high homology to Eif2s3y but whether it has similar functional potential remains unknown. We aimed to determine if overexpression of Eif3s3x can substitute for lack of Eif2s3y and allow for spermatogonial proliferation and differentiation in XOSry mice. DESIGN: Research study. MATERIALS AND METHODS: Mice transgenic for Eif2s3x were generated and the transgene placed in the context XOSry by breeding. Quantitative analysis of spermatogenesis progression was performed on staged histological sections from XOSry,Eif2s3x males (n¼11), XOSry,Eif2s3y (n¼3), XOSry (n¼3) and XY (n¼3). For each male 10 tubules were examined per stage category, the number of spermatogonia (sg), round spermatids (rs), and Sertoli cells (sc) counted, and the data expressed as germ cell/Sertoli cell ratio. Counts and categorization of cellular abnormalities were also performed. RESULTS: Spermatogonial proliferation was similar in XOSry,Eif2s3x and XOSry,Eif2s3y males (sg/sc: 0.54 .03 vs. 0.410.09, P¼NS); both male types had fewer spermatogonia than XY controls (0.790.08, P<0.05). Meiotic and post-meiotic arrests were present in both XOSry,Eif2s3x and XOSry,Eif2s3y, with rare development to round spermatids. The efficiency of spermatid production in XOSry,Eif2s3x was about 10-fold lower than in XOSry,Eif2s3y (rs/sc: 0.090.04 vs. 0.920.15, P<0.001) and >80-fold lower than in XY (7.880.50; P<0.001). Abnormalities of seminiferous epithelium were observed in both XOSry,Eif2s3y and XOSry,Eif2s3x males, with the latter specifically exhibiting a large population of apoptotic cells at meiotic metaphase. CONCLUSION: Eif2s3x, when present in sufficient levels, can replace the function of Eif2s3y and act as the initiator of spermatogonial proliferation but with decreased progression to post-meiotic stages. Supported by: HCF13ADVC-60314, NIH HD072380 & RR024206 grants to MAW.
P-660 Wednesday, October 22, 2014 EXPOSURE TO ELEVATED TEMPERATURES RESTORES FERTILITY IN ‘‘JUVENILE SPERMATOGONIAL DEPLETION’’ MUTANTS. M. A. Ward,a P. B. Comish,b L. Y. Liang,b Y. Yamauchi,a C. C. Weng,b G. Shetty,b K. A. Naff,b M. Meistrich.b aInstitute for Biogenesis Research, University of Hawaii, Honolulu, HI; bUniversity of Texas, M.D. Anderson Cancer Center, Houston, TX. OBJECTIVE: ‘‘Juvenile spermatogonial depletion’’ (jsd) mice are homozygous for the mutated Utp14b gene and have spermatogenic arrest. Several waves of spermatogenesis proceed in the immature jsd mice, but at 6 wks of age spermatogonial differentiation arrests and spermatocyte numbers decline, resulting in the only germ cells being type A spermatogonia by 10 wks. We previously observed that suppression of testosterone or elevation of testicular temperature by cryptorchidization restored spermatogonial and spermatocyte differentiation. In this study we tested if maintaining jsd mice at higher temperature enables spermatogenesis to proceed. DESIGN: Research study. MATERIALS AND METHODS: Jsd mice were placed in humidified incubators at elevated ambient temperatures. This treatment resulted in spermatogonia differentiation to the spermatocyte stage, but it inhibited
FERTILITY & STERILITYÒ
spermiogenesis and sperm production. The effects of elevated ambient temperatures on spermiogenesis were therefore tested in wild-type mice and revealed that sperm production was only affected at 34 C and above. Based on these results, a 24-day induction period in 35 C incubators was used to induce spermatocyte formation in jsd mice, and then the mice were transferred to 32 C incubators for 24 days to allow spermiogenesis and sperm production to proceed. RESULTS: Incubation of jsd mice at 35 C increased the percentage of tubules with spermatocytes from 0% in untreated mice to over 80%. Subsequent maintenance at 32 C resulted in progression of spermatid differentiation so that up to 42% of tubules had late spermatids. About half of the mutant mice treated with the induction/progression regimen had sperm in testicular cell suspensions. These sperm were used for intracytoplasmic sperm injection (ICSI) and viable, healthy, fertile offspring were obtained. CONCLUSION: Maintenance of jsd mice at elevated temperatures with a regime compatible with both spermatogonia differentiation and spermatogenesis progression overcomes their infertility and allows them to reproduce with the help of assisted fertilization. In humans, one of the UTP14 genes, UTP14C, is expressed exclusively in germ cells and its mutations result in azoospermia or severe oligospermia. If a standard TESE-ICSI treatment of azoospermic patients with the UTP14C mutation have failed, those patients might benefit from a second round of TESE-ICSI following a mild testicular warming regime. Supported by: NIH HD40397 to MM and NIH HD072380 to MAW.
P-661 Wednesday, October 22, 2014 HUMAN GERM CELL SECRETING FACTOR NODAL REGULATES SERTOLI CELL FUNCTIONS. R. Tian,a S. Yang,b Z. Zhu,c J. Wang,d Z. He,e Z. Li.f aUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; bUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; cUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; dUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; eStem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; fUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. OBJECTIVE: To explore the regulatory effects of germ cells and germ cells secreting factor Nodal on the function of Sertoli cells derived from obstructive azoozpermia and non-obstructive azoozpermia patients. DESIGN: Comparative and controlled study. MATERIALS AND METHODS: Human Sertoli cells and germ cells were isolated using two-steps enzymatic digestions from the testes of azoozpermia patients. Expressions of Nodal signaling components in Sertoli cells and germ cells were identified by PCR and immunochemistry. Human germ cells and Sertoli cells were cocultured in vitro to evaluate their effects on Sertoli cells. Human recombinant nodal and its receptor inhibitor SB431542 were added in the Sertoli cells culture medium to study their effects on cells functions. CCK8 measurement was used to evaluate the proliferative activity. Q-PCR and western blot were applied to assess the expression of functional Sertoli cell genes. RESULTS: Human germ cells down-regulated blood-testis-barrier associated genes (CLDN11, OCLN) expressions of Sertoli cells in coculture system. Nodal was expressed in germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ActR-IIB were detected on Sertoli cells, which indicated Nodal signaling pathway play roles in the regulation of germ cells to Sertoli cells. Nodal could promote the proliferation of human Sertoli cells, while the proliferative activity was inhibited by SB431542. Nodal could enhance the expressions of functional Sertoli cell genes (GDNF, SCF, BMP4, and ABP), while SB431542 decreased their expressions. In contrast, Nodal decreased the expression of blood-testis-barrier associated genes (CLDN11, OCLN), while SB431542 increase their expressions. CONCLUSION: Human Sertoli cell functions could be regulated by germ cells via paracrine pathway. Human germ cells secrete Nodal which could regulate Sertoli cell functions. Supported by: This work was Supported by National Science Foundation of China (31201109) and China National Key Project (2010CB945200).
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P-660 Wednesday, October 22, 2014 EXPOSURE TO ELEVATED TEMPERATURES RESTORES FERTILITY IN ‘‘JUVENILE SPERMATOGONIAL DEPLETION’’ MUTANTS. M. A. Ward,a P. B. Comish,b L. Y. Liang,b Y. Yamauchi,a C. C. Weng,b G. Shetty,b K. A. Naff,b M. Meistrich.b aInstitute for Biogenesis Research, University of Hawaii, Honolulu, HI; bUniversity of Texas, M.D. Anderson Cancer Center, Houston, TX. OBJECTIVE: ‘‘Juvenile spermatogonial depletion’’ (jsd) mice are homozygous for the mutated Utp14b gene and have spermatogenic arrest. Several waves of spermatogenesis proceed in the immature jsd mice, but at 6 wks of age spermatogonial differentiation arrests and spermatocyte numbers decline, resulting in the only germ cells being type A spermatogonia by 10 wks. We previously observed that suppression of testosterone or elevation of testicular temperature by cryptorchidization restored spermatogonial and spermatocyte differentiation. In this study we tested if maintaining jsd mice at higher temperature enables spermatogenesis to proceed. DESIGN: Research study. MATERIALS AND METHODS: Jsd mice were placed in humidified incubators at elevated ambient temperatures. This treatment resulted in spermatogonia differentiation to the spermatocyte stage, but it inhibited
FERTILITY & STERILITYÒ
spermiogenesis and sperm production. The effects of elevated ambient temperatures on spermiogenesis were therefore tested in wild-type mice and revealed that sperm production was only affected at 34 C and above. Based on these results, a 24-day induction period in 35 C incubators was used to induce spermatocyte formation in jsd mice, and then the mice were transferred to 32 C incubators for 24 days to allow spermiogenesis and sperm production to proceed. RESULTS: Incubation of jsd mice at 35 C increased the percentage of tubules with spermatocytes from 0% in untreated mice to over 80%. Subsequent maintenance at 32 C resulted in progression of spermatid differentiation so that up to 42% of tubules had late spermatids. About half of the mutant mice treated with the induction/progression regimen had sperm in testicular cell suspensions. These sperm were used for intracytoplasmic sperm injection (ICSI) and viable, healthy, fertile offspring were obtained. CONCLUSION: Maintenance of jsd mice at elevated temperatures with a regime compatible with both spermatogonia differentiation and spermatogenesis progression overcomes their infertility and allows them to reproduce with the help of assisted fertilization. In humans, one of the UTP14 genes, UTP14C, is expressed exclusively in germ cells and its mutations result in azoospermia or severe oligospermia. If a standard TESE-ICSI treatment of azoospermic patients with the UTP14C mutation have failed, those patients might benefit from a second round of TESE-ICSI following a mild testicular warming regime. Supported by: NIH HD40397 to MM and NIH HD072380 to MAW.
P-661 Wednesday, October 22, 2014 HUMAN GERM CELL SECRETING FACTOR NODAL REGULATES SERTOLI CELL FUNCTIONS. R. Tian,a S. Yang,b Z. Zhu,c J. Wang,d Z. He,e Z. Li.f aUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; bUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; cUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; dUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; eStem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; fUrology, Shanghai Human Sperm Bank, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. OBJECTIVE: To explore the regulatory effects of germ cells and germ cells secreting factor Nodal on the function of Sertoli cells derived from obstructive azoozpermia and non-obstructive azoozpermia patients. DESIGN: Comparative and controlled study. MATERIALS AND METHODS: Human Sertoli cells and germ cells were isolated using two-steps enzymatic digestions from the testes of azoozpermia patients. Expressions of Nodal signaling components in Sertoli cells and germ cells were identified by PCR and immunochemistry. Human germ cells and Sertoli cells were cocultured in vitro to evaluate their effects on Sertoli cells. Human recombinant nodal and its receptor inhibitor SB431542 were added in the Sertoli cells culture medium to study their effects on cells functions. CCK8 measurement was used to evaluate the proliferative activity. Q-PCR and western blot were applied to assess the expression of functional Sertoli cell genes. RESULTS: Human germ cells down-regulated blood-testis-barrier associated genes (CLDN11, OCLN) expressions of Sertoli cells in coculture system. Nodal was expressed in germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ActR-IIB were detected on Sertoli cells, which indicated Nodal signaling pathway play roles in the regulation of germ cells to Sertoli cells. Nodal could promote the proliferation of human Sertoli cells, while the proliferative activity was inhibited by SB431542. Nodal could enhance the expressions of functional Sertoli cell genes (GDNF, SCF, BMP4, and ABP), while SB431542 decreased their expressions. In contrast, Nodal decreased the expression of blood-testis-barrier associated genes (CLDN11, OCLN), while SB431542 increase their expressions. CONCLUSION: Human Sertoli cell functions could be regulated by germ cells via paracrine pathway. Human germ cells secrete Nodal which could regulate Sertoli cell functions. Supported by: This work was Supported by National Science Foundation of China (31201109) and China National Key Project (2010CB945200).
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