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Testicular cells composed mostly of germ cells and immature Sertoli cells from neonatal mice 2 and 5 days old were cultured to investigate germ-cell proliferation mediated by the c-kit receptor. The addition of antibody to block the interaction of the c-kit receptor with its ligand inhibited the proliferation of cultured spermatogonia from 5-day-old mice in a dose-dependent manner, but not from that of 2-day-old mice. The addition of anti-c-kit ACK2 monoclonal antibody also inhibited the proliferation of spermatogonia from 5-day-old mutant Sld/Sld mice but not of 5-day-old mutant Wo/Wo mice. The results indicate that c-kit-positive type A spermatogonia in the testes of 5-day-old mice require steel factor (kit ligand) for their proliferation, whereas self-renewal and differentiation of c-kit-negative primitive type A spermatogonia in the testes of 2-day-old mice do not.
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Summary. Primordial germ cells (PGCs), collected from the blood of 2-day-old chick embryos, were concentrated by Ficoll density centrifugation. The blood contained 0·048% PGCs and the concentrated fraction contained 3·9% PGCs in blood cells. The PGCs were picked up with a fine glass pipette, and one hundred were then injected into the terminal sinuses of 2-day-old Japanese quail embryos (24 somites); bubbles were then inserted to prevent haemorrhage. The embryos were further incubated at 38°C for 24 h, and then fixed. Serial sections were stained with the periodic acid–Schiff reagent (PAS) to demonstrate chicken PGCs and with Feulgen stain to identify quail cells. On the basis of the differences in staining properties, 63·6 ± 5·3 chick PGCs were detected in the quail embryo in the area where the gonads develop. Furthermore, 39·3 ± 4·5 chick PGCs were incorporated into the quail germinal epithelium within 24 h of the injection. A similar percentage of the host (quail) PGCs had also migrated to the germinal epithelium at the same stage of development. This technique for obtaining germ-line chimaeras will facilitate research on avian germ-line differentiation.
Keywords: primordial germ cells; migration; germ-line chimaera; chick; quail; transplantation
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Regulation of steel factor (SF) production in Sertoli cells from postnatal mouse testes was studied in a mast cell–Sertoli cell coculture system. Treatment of Sertoli cells with dibutyryl cAMP (50–1000 μmol l−1), forskolin (1–25 mmol l−1), and cholera toxin (10 μg ml−1) increased SF production, whereas FSH and theophylline had no significant effect. Furthermore, growth factors and testosterone, which would play some roles in spermatogenesis, were also tested, but none of these stimulated SF production. The constitutive production of SF may, rather, reflect the physiological condition of Sertoli cells in vivo.
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Conservation of genetic material in chickens was attempted by preserving primordial germ cells in liquid nitrogen. Primordial germ cells collected from the blood of embryos at stage 13–15 of White Leghorn and Barred Plymouth Rock breeds were concentrated by Ficoll density gradient centrifugation. The primordial germ cells were then suspended in a freezing medium containing 10% dimethyl sulfoxide. The temperature of the cell suspension was decreased by 1°C min−1 to −80°C; the suspension was then placed in liquid nitrogen (−196°C) and stored for 4–5 months. The cell suspension was thawed by taking it out of liquid nitrogen and placing it in water at 4°C. The viability of the frozen–thawed primordial germ cells was 94.2%. One hundred frozen–thawed cells were injected into the bloodstream of recipient embryos (stage 14–15) from the other breed, from which blood had been drawn before the injection. These embryos were cultured in recipient eggshells until hatching. Viable offspring derived from the frozen–thawed primordial germ cells were obtained by mating male and female germline chimaeras or by mating the chimaeras with Barred Plymouth Rock chickens, and the offspring showed normal reproductive performance. This technique for cryopreservation of primordial germ cells giving rise to viable offspring makes it possible to conserve genetic material in avian species.
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Summary. The effects of Steel–Dickie (Sld) mutations on testicular germ cell differentiation were investigated using experimental cryptorchidism and its surgical reversal in mutant, C57BL/6-Sld/+ and wild-type C57BL/6- +/+ mice. In Sld/+ cryptorchid testes the maintenance of undifferentiated type-A spermatogonia was impaired and their numbers decreased. In contrast, the proliferative activity of type-A spermatogonia in the cryptorchid testis of mutant mice appeared normal as judged by their progression through the cell cycle. Surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells in +/+ testes. However, the regenerative differentiation of type-A spermatogonia which remained in Sld/+ cryptorchid testes was strongly impaired, particularly at two steps of cellular differentiation, from type-A spermatogonia to intermediate or type-B spermatogonia and at meiotic division. Furthermore, in mutant mice, no significant recovery of testicular weight was observed after surgical reversal compared with +/+ mice.
Keywords: Sld mutation; testis; germ cell; Sertoli cell; cryptorchidism; mouse
