Cellular localization and age dependent changes in mRNA for glutathione S-transferase-P in rat testicular cells

Using Northern blotting techniques we report that mRNA for Glutathione S-transferase-P (GST-P or GST 7-7) is present in rat testis. GST-P mRNA was detected in cultured Sertoli cells, cultured peritubular cells, as well as in transplantable Leydig cell tumor. However, no GST-P mRNA was detected in rat germ cell fractions. There was a marked increase in mRNA for GST-P from day 5 to day 20 in rats, after which a decrease was seen. The decreased level of mRNA for GST-P in the testis after 20 days of age, coincided in time with the exponential increase in germ cells, and accompanying relative decrease in somatic cells. The results show that mRNA for GST-P is primarily present in somatic cells of the rat testis.     

Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.     

RNA binding protein Musashi1 is expressed in sertoli cells in the rat testis from fetal life to adulthood.

The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed silver grains within the seminiferous epithelium. Immunohistochemical studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.     

Developmental regulation of calmodulin, actin, and tubulin RNAs during rat testis differentiation

Postnatal testis differentiation involves transition through neonatal, pre-meiotic, meiotic, haploid, and mature stages. We have examined the qualitative and quantitative changes in rat testis RNAs that specifically hybridize to cDNAs encoding the cytoskeletal proteins, calmodulin, beta-actin, alpha- and beta-tubulin at ages corresponding to each of these developmental periods. We compared the species and relative levels of specific RNAs from testes of animals engaged in normal spermatogenesis with RNA from germ cell-depleted, Sertoli cell-enriched (SCE) testis. Distinct developmental patterns of expression of the specific RNAs were found with each of the cDNAs in the two animal models. A 2.2 kb (kilobase) actin RNA and a 2.7 kb beta-tubulin RNA are maximal at 5-10 days of age, suggesting these RNAs are required by somatic and germ cells in the postnatal phase prior to puberty. Between 19 and 29 days, when pachytene spermatocytes appear in significant numbers, there is a slight increase in the 2.2-kb actin RNA, but a 4- to 10-fold increase in RNAs hybridizing to cDNAs for calmodulin, alpha- and beta-tubulin. These changes are much less pronounced in the SCE testis than in the normal testis, indicating increases in these RNAs are related to germinal cell maturation. The germ cell-related increase in 1.8-kb beta-tubulin RNA appears to reflect a developmental "switch" in the gene from which the RNA is derived. This hypothesis is based on the observation that the ratio of hybridization of a chicken brain beta-tubulin cDNA versus a rat spleen beta-tubulin cDNA to the 1.8-kb RNA band increases more than 40-fold between 5 and 29 days of age in normal testis, but is constant in SCE testis. These data suggest that a specific beta-tubulin gene is activated in maturing germ cells. Analogously, a 2.1-kb alpha-tubulin RNA is found only in maturing normal testis and increases as spermatids are produced. A 2.0-kb beta-tubulin RNA, not found in normal testes, is maximal in maturing SCE testes, suggesting this RNA is of somatic cell origin. All of the RNA species studied, except the 2.0-kb beta-tubulin RNA, decrease between 5 and 19 days in SCE testes, as Sertoli cell mitotic activity wanes, indicating that their levels may be regulated by the developmental signals that influence mitosis.     

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells.     

The role of Sertoli cells in the differentiation and exclusion of rat testicular germ cells in primary culture

We have previously established a primary co-culture of spermatogenic and somatic cells of the rat testis, in which spermatogenic cells differentiate to some extent in terms of the occurrence of testis-specific gene expression. In the present study, the interaction between spermatogenic and somatic Sertoli cells was investigated in this culture system. Spermatogenic differentiation did not proceed when these two cell types were placed on opposite sides of a permeable membrane, thus avoiding direct contact. Further, a significant proportion of spermatogenic cells died by apoptosis during culture, and Sertoli cells engulfed and digested the degenerating spermatogenic cells. These results indicate that Sertoli cells participate both in the differentiation of spermatogenic cells and in the exclusion of degenerating spermatogenic cells, by directly attaching to those cells.     

Germ cell deficiency causes testis cord differentiation in reconstituted mouse fetal ovaries

Sex-reversal in fetal ovaries was studied by using a dissociation-reconstitution technique. Gonads of 12.5 gestation-day male and female mouse fetuses were dissociated into single cells. To eliminate germ cells, the dissociated cells were cultured for 14 h, and then somatic cells attached to culture dishes were harvested and aggregated by gyratory culture for 24 h. The aggregates were then transplanted into ovarian bursa in ovary-ectomized nude mice. The recovered explants were examined histologically. Male somatic cells developed into testes containing Sertoli cells, Leidig cells, and tunica albuginea. Female somatic cells formed testis cords and differentiated into Sertoli cells, but they did not differentiate into other testis components or ovarian tissues. However, aggregates consisting of both female and male somatic cells differentiated into well-developed testes containing Leidig cells and tunica albuginea as well as Sertoli cells. Enzyme marker analysis showed significant contributions of female cells in these organized testes. In contrast, aggregates containing both female germ cells and somatic cells developed into ovaries and did not differentiate into any testicular tissues. The results indicate that female somatic cells in fetal gonads at 12.5 gestation day have the potency to form testis cords and differentiate into Sertoli cells. The subsequent steps in testis development require the contributions of male cells. The present study also suggests that testicular differentiation is independent of germ cells but ovarian development involves the interaction between germ cells and somatic cells.