Molecular Biological Studies of the Mechanisms in Amphibian Spermatogenesis.

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I. Differentiation from secondary spermatogonia to primary spermatocytes by FSH in organ culture of testes fragments from the newt. Cynops pyrrhogaster : When testes fragments rich in secondary spermatogonia were cultured in the presence of follicle-stimulating hormone (FSH) alone, primary spermatocytes appeared in more than half of the cysts by the second week. These results indicate that FSH is indispensable and sufficient for the differentiation from spermatogonia to primary spermatocytes.II. How is the flagellar length of mature sperm determined ? Comparison of flagellar growth in round spermatids between Cynops and Xenopus : The flagellar length of Cynops sperm is 13 times longer than that of Xenopus sperm. The rate and period of flagellar growth in Cynops spermatids were 2 to 3-fold those in Xenopus. The period of flagellar elongation almost coincided with that of tubulin synthesis. The differences in the rate and period of tubulin synthesis between Cynops and Xenopus seems to be … More due to the difference in the stability of tubulin mRNAs between the two species.III. Synthesis of sperm-specific basic nuclear proteins (SPs) in spermatids and expression of SP4 mRNA in primary spermatocytes from Xenopus laevis : In synchronous culture of round spermatids, the synthesis of SP1, 3-5 began by day 3. CDNA clones for SP4 and 5 were isolated from a cDNA library from round spermatids. The Northern blots showed that SP4 and 5 mRNAs were present in primary spermatocytes as well as in round spermatids. This result shows that SP4 and 5 genes are transcribed in the primary spermatocyte stage but are translated at the round spermatid stage.IV. Role of manchette (microtubule bundles) in nuclear elongation in spermatids from Cynops pyrrhogaster : The role of microtubules in nuclear shaping of spermatids has yet to be determined. We found that manchette structure in newt elongate spermatids is resistant to the treatment of Ca^<2+> and low temperature. 1M NaCl Sup extracted from low temp-stable microtubules in testes promoted polymerization of tubulin at 37 C and resisted depolymerization at 0 C in a dose-dependent manner. These results indicate that 1M NaCl Sup contains MAPs (microtubule associated proteins) which promote tubulin polymerization and confer the resistance against tubulin depolymerization. Less