Newt Testis Research Articles

Expression of Activin β Subunit Genes in Sertoli Cells of Newt Testes

From newt testes we cloned by reverse transcription-polymerase chain reaction (RT-PCR) activin βA and βB subunit genes, each encoding the mature region of the peptides. A single form for each of the subunit genes was isolated. Newt βA and βB share 80 and 98% protein sequence similarity with the human subunits, respectively. Northern blot analysis showed that βA subunit mRNA is approximately 7 kilobases in length and is expressed during spermatogenesis in stages from spermatogonial to spermatid, but activin βB subunit mRNA is barely detectable during these stages. We also isolated a cDNA clone containing the entire coding region of βA subunit and performed in situ hybridization with its cRNA probe. Newt activin βA subunit mRNA was detected in Sertoli cells, but not in germ cells or pericystic cells. These findings suggest that activin A plays an important role in newt spermatogenesis.

Differential expression of annexin V during spermatogenesis in the newt Cynops pyrrhogaster

In order to isolate genes whose expression is up-regulated after the initiation of meiosis, we screened a cDNA expression library of newt testes with antiserum against homogenates of testes derived from the spermatogonial and spermatocyte stages. We report the isolation of spermatocyte-specific cDNA clones encoding a newt homologue of the calcium-dependent phospholipid-binding protein, annexin V. Northern blot analysis showed that newt annexin V mRNA was 1.7 kb in length and was expressed strongly in testes, but weakly in other organs. In situ hybridization revealed that the expression of newt annexin mRNA was barely observed in spermatogonia, but increased significantly in leptotene-zygotene primary spermatocytes and reached a maximum level in pachytene spermatocytes and round spermatids. The newt annexin V cDNA predicted a 323-amino acid protein and had a 68% homology to human annexin V. The predicted amino acid sequence contained a conserved 4-fold internal repeat of approximately 70 residues like other annexin proteins. Immunoblot analysis using the monoclonal antibody against newt annexin V showed that the protein was expressed scarcely in spermatogonia but was abundantly expressed in stages from primary spermatocytes to spermatids; this pattern was consistent to that of the mRNA. Immunohistochemical analysis revealed that newt annexin V was localized in the cytoplasm of the spermatogenic cells, but not in somatic cells such as Sertoli cells or pericystic cells. These results indicate that the expression of newt annexin V is up-regulated in the spermatogenic cells after the initiation of meiosis and suggest that newt annexin V plays an important role in spermatogenesis.

Initiation and stimulation of spermatogenesis in vitro by mammalian follicle-stimulating hormone in the Japanese newt, Cynops pyrrhogaster.

In order to elucidate the molecular mechanisms by which spermatogenesis is regulated, especially the roles of hormones and somatic cells in the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments rich in secondary spermatogonia were cultured in control medium for three weeks, most of the testicular cysts still remained as secondary spermatogonia. On the other hand, in the medium supplemented with follicle-stimulating hormone (FSH) alone, DNA syntheses in secondary spermatogonia and Sertoli cells were stimulated and secondary spermatogonia differentiated into primary spermatocytes (zygotene-pachytene) in more than half of the cysts by the second week. When newt testes fragments rich in primary spermatocytes were cultured in a control medium for three weeks only round spermatids were observed at the most advanced stage. On the other hand, in the medium supplemented with FSH alone, elongated spermatids appeared by the second week. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5 alpha-dihydrotestosterone) to the control medium stimulated differentiation for either step. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH for either stage of the testes (secondary spermatogonia and primary spermatocytes). Preliminary results indicate that FSH does not bind to germ cells but to somatic cells (most probably Sertoli cells). These and our unpublished data suggest that FSH triggers proliferation and differentiation of spermatogonia into elongated spermatids by acting on Sertoli cells which in turn act on germ cells.

Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster.

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene-pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α-dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.

The cycle of foilicuiar and interstitial cells (Leydig cells) in the testis of the marbled newt, Triturus marmoratus (caudata, salamandridae)

Ultrastructural examination of the marbled newt (Triturus marmoratus) testis throughout the annual cycle revealed that during the period of testicular quiescence (November-February), primordial germ cells proliferate within cords of filament-rich epithelial cells that will become follicular cells (FCs). Fibroblast-like cells surround the FCs and form the lobule-boundary interstitial cells (ICs). During the period of germ cell development from primordial germ cells to round spermatids (March-June), the FCs surrounding the developing germ cells contain scanty cytoplasm with abundant rough endoplasmic reticulum and scarce filaments. With spermatid elongation (July-August), the FC size grows, its nucleus becomes irregularly outlined, and its cytoplasm displays abundant smooth endoplasmic reticulum, residual bodies, lipid droplets, and large vacuoles. After spermatozoon release by the FCs (August-September), the adjacent ICs increase their size and transform into Leydig cells with abundant smooth endoplasmic reticulum, mitochondria with tubular cristae, and lipid droplets. During the period of testicular quiescence (November-February), the Leydig cells undergo involution, eventually developing the morphological attributes of mesenchymal cells. Intermingled among these cells, cords of filament-rich cells are observed. During this period of the cycle, spermatozoon cysts supported by FCs are present. At the beginning of the germ cell proliferation period (March), these spermatozoa are released, and the adjacent ICs undergo a transformation into Leydig cells similar to those observed in August-September. Maturation and involution of ICs occur when testosterone levels are known to be rising and falling, respectively.

Adaptation of testicular follicle-stimulating hormone receptors to ambient temperatures in vertebrates: Equilibrium analysis

Gonadotrophin receptors of the rat, turtle, and newt testes had different optimal temperatures of affinity for rat FSH. The optimal temperature in the rat was around 37° and that in the newt was between 15 and 20°. The binding affinity in the turtle did not differ significantly at temperatures ranging between 0 and 40°. These optimal temperatures coincided well with the temperatures at which gonadotrophin acts in each species under natural conditions.

A possible role of cadaverine in the biosynthesis of polyamines in the japanese newt testis.

Polyamine content in testes of various vertebrates was studied extensively. Putrescine, spermidine and spermine were detected in all the animals examined, although the distribution pattern varied greatly from animal to animal. Cadaverine was detected only in amphibian testes; sym-homospermidine was found not only in testes but also in various other tissues of amphibians and of some reptiles. In the newt testis the concentration of cadaverine was lower than that of any other polyamines in summer, but there was a great increase in cadaverine content from autumn to winter. The testicular content of cadaverine was greater than that of other polyamines in winter. There was a gradual decrease in the cadaverine content in spring. The spermidine and spermine levels, which were rather low in winter, increased in spring and reached a peak in summer when spermatogenesis was active. The testicular concentration of putrescine that was much higher than that of spermidine or spermine throughout the year, increased only a little in summer. There was a significant negative correlation between the cadaverine levels and four other polyamine levels. Exogenous cadaverine decreased the testicular levels of putrescine. Mammalian gonadotropins decreased the cadaverine levels and increased the levels of other polyamines. A partially purified LH fraction from pituitaries of bullfrog, Rana catesbeiana, was also potent in depleting cadaverine of the testes of newts kept at 8 degrees C. These results suggest that testicular cadaverine suppresses the biosynthesis of polyamines, especially spermidine and spermine which are closely associated with spermatogenesis.

BEHAVIOR OF SECONDARY SPERMATOGONIA OF THE NEWT, CYNOPS PYRRHOGASTER, IN IN VITRO CULTURE.

Several cell types migrated cut from small pieces of newt testes cultivated in vitro. Flat fibroblastic cells migrated out within a few days. Then, secondary spermatogonia, identified by the presence of germ cell-specific substances and by the shape and appearance of their nucleus and subcellular organelles, migrated out over the sheet of fibroblastic cells. Sertoli cells co-migrated with secondary spermatogonia, maintaining a similar cellular arrangement to that of testicular cells in vivo. Mitosis of secondary spermatogonia both in clusters and as single cells was frequent from the third day until about 2 weeks after inoculation. During mitosis, active and periodic rotation of chromosomes was observed. Identification of the cell types and studies on their behavior were performed by electron microscopy and phase contrast microscopy.

HISTOCHEMICAL AND ELECTRON MICROSCOPIC OBSERVATIONS ON THE STEROID HORMONE-SECRETING CELLS IN THE TESTIS OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER.

The testis of the Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, caught in the middle of May was investigated in order to demonstrate the presence of steroid hormone (SH)-secreting cells, by means of histochemical and electron microscopic techniques. The newt testis is divided into two main parts of spermatogenetic stages. The anterior part is immature and consists of many seminal cysts containing spermatogonia and spermatocytes. The posterior part is mature and made up of many cysts containing mature spermatozoa and glandular tissue. Histochemical studies revealed Δ5 -3β-hydroxysteroid dehydrogenase (Δ5 -3β-HSD) activity in the pericystic cells of the mature part, weak in those before spermiation, then gradually becoming more intense in the pericystic cells just after spermiation. Activity was most pronounced in the outer layer of cells of the glandular tissue which originate from the pericystic cells. In contrast to this, the Δ5 -3β-HSD reaction was entirely negative in the pericystic cells of the immature part, the Sertoli cells, and the inner cells of the glandular tissue originating from the Sertoli cells. Electron microscopic observation of the pericystic cells and glandular tissue gave further information on the histochemical features. The pericystic cells in the immature part are fibroblast-like cells. The cell organelles of these cells are poorly developed except for rough-surfaced endoplasmic reticulum and rod-shaped mitochondria. In the mature part, the fibroblast-like cells are transformed into cells characteristic of SH-secreting cells. In the cytoplasm, the amount of smooth-surfaced endoplasmic reticulum increases and typical globular mitochondria with tubular cristae take the place of the rod-shaped mitochondria. In addition, a number of lipid droplets and lipofuscin granules appear. Most of the fine structures of the outer layer of the cells of the glandular tissue were found to be similar in appearance to those of the SH-secreting cells in mammals and other vertebrates. These results strongly suggest that, as in the other urodeles, the SH-secreting cells in the testis of the Japanese red-bellied newt are the pericystic cells in the mature part and in the outer layer of cells of the glandular tissue.

Cultivation in vitro of the testis cord of the newt, Triturus pyrrhogaster.

Testis cords of Triturus pyrrhogaster were cultivated in vitro on (a) medium with chick embryo extract and calf serum, (b) medium with newt gonad extract, (c) Trowell's medium T8 and (d) liquid synthetic medium 199. Of the four media utilized, medium 199 gave the best result for long-term maintenance of the normal histological structures of the testis cords. Addition of insulin (5 μ/ml) to medium 199 resulted in a remarkable improvement for the maintenance of the testis cord and the migration of columnar cells of the peritoneal epithelium into the primordial germinal tissue occurred as in the intact testis of this animal. Trowell's medium T8 was proved inadequate. Medium with chick embryo extract and calf serum retained most of the germ cells healthy but caused gradual decrease in height of the columnar cells. Testis cords cultivated on the same medium in combination with Xenopus testis maintained normal histological structure for 18 days, whereas, those kept in contact with Xenopus ovary showed involution within the same period. Newt testis extract brought about transformation of somatic elements of the germinal tissue into fibroblastlike cells which was followed by the disintegration of germ cells. Ovary extract did not cause selective destruction on the somatic or germinal elements.