Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat testis

Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.     

Cellular localization of mRNAs for retinoic acid receptor-alpha, cellular retinol-binding protein, and cellular retinoic acid-binding protein in rat testis: evidence for germ cell-specific mRNAs

In the present study we have examined the cellular localization and developmental changes of mRNAs for retinoid-binding proteins in rat testis. We demonstrate that mRNA (0.7 kb) for cellular retinol-binding protein (CRBP) is expressed only in Sertoli cells and peritubular cells. The mRNA for CRBP could not be detected in other testicular cells. In contrast, mRNA for cellular retinoic acid-binding protein (CRABP) was detected primarily in germ cells and to a small extent in tumor Leydig cells. The mRNA for CRABP in germ cells revealed distinct size heterogeneity and three distinct mRNA species were observed (1.0, 1.8, and 1.9 kb), in contrast to previous data for somatic cells where only the 1.0-kb mRNA has been reported. Messenger RNAs for retinoic acid receptor-alpha (RAR alpha) were detected in both somatic and haploid germ cells. The highest level of RAR alpha was seen in Sertoli cells, round spermatids, and tumor Leydig cells. Lower, but distinct, levels were observed in peritubular cells. Furthermore, we observed germ cell-specific species of RAR alpha mRNA (4 kb and approximately 7 kb). The smallest mRNA for RAR alpha (2.7 kb) in somatic cells was absent in germ cells. The levels of mRNAs for the various retinoid-binding proteins in whole testis obtained from rats of various ages confirmed this cellular localization. The mRNAs for CRBP, the small molecular size (2.7 kb) mRNA for RAR alpha (localized to somatic cells), and the 1-kb mRNA for CRABP showed an age-dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Hsf1, Hsf2, and Phlda1 in cells undergoing cryptorchid-induced apoptosis in rat testes

Surgery‐induced cryptorchidism, in which the testes are prevented from descending into the scrotal sac, results in testicular germ cell death, and it is commonly used as an experimental tool in the study of spermatogenesis. However, the molecular events underlying the activation of germ cell death remain poorly understood. In the present study, we investigate selective cell loss from cryptorchid rat testis by using DNA flow cytometry and by determining protein and mRNA expression of Hsf1, Hsf2, and Phlda1. The hypo‐haploid cell fraction is significantly decreased as early as 3 days after surgical induction of cryptorchidism (from 42.01 ± 5.74% to 15.98 ± 3.88%), followed by a significant decrease in the haploid cell fraction at Day 7. At the latter time point, an apoptotic peak of spermatocytes appears in DNA histograms just before the tetraploid peak; the percentage of aneuploid cells between diploid and tetraploid rises as high as 14.05 ± 2.98% of the total cells in 7‐day cryptorchid testis, suggesting that a large number of spermatocytes are undergoing apoptosis. The expression of Phlda1 mRNA is significantly elevated 3 days after induction of cryptorchidism. After 7 days of cryptorchidism, Hsf1 and Phlda1 are strongly expressed in the nucleus and cytoplasm, respectively, of primary spermatocytes. Numerous apoptotic spermatocytes are also observed at this time point. These results suggest that the Hsf1/Phlda1 pathway plays an important role in the apoptosis of primary spermatocytes in cryptorchid testis. We present evidence suggesting that Hsf2 is also involved in germ cell removal in cryptorchid testis. Mol. Reprod. Dev. 78:283–291, 2011. © 2011 Wiley‐Liss, Inc.     

Expression of the E3 SUMO-1 ligases PIASx and PIAS1 during spermatogenesis in the rat

PIASx and PIAS1, two members of the conserved protein inhibitor of activated STAT (PIAS) family, are able to interact with and modulate activities of several distinct nuclear proteins, including androgen receptor (AR). PIASx and PIAS1 also function as E3-type ligases in small ubiquitin-related modifier 1 modifications. To gain better insight into the physiological roles of PIASx and PIAS1 in vivo, their cell-type specific expression and regulation were analyzed during testicular development and spermatogenesis in the rat. The expression of both PIASx and PIAS1 was low or undetectable in newborn rats. PIASx mRNA started to accumulate after day 20 of postnatal life, whereas expression of PIAS1 mRNA increased around day 30 after birth. In the adult rat testis, both PIASx and PIAS1 mRNA were present in Sertoli cells and in germ cells in the seminiferous epithelium at all stages. However, PIASx mRNA was more abundant in spermatocytes than in other cell types, whereas higher levels of PIAS1 mRNA were detected in late spermatocytes and round spermatids than in early spermatocytes. Since PIASx and PIAS1 accumulate in developing male germ cells, their regulatory functions are not only restricted to AR in Sertoli cells, but they also participate in molecular processes during meiosis.     

Developmental and hormonal regulation of the monocarboxylate transporter 2 (MCT2) expression in the mouse germ cells

During spermatogenesis, postmeiotic germ cells utilize lactate produced by Sertoli cells as an energy metabolite. While the hormonal regulation of lactate production in Sertoli cells has been relatively well established, the transport of this energy substrate to the germ cells, particularly via the monocarboxylate transporters (MCTs), as well as the potential endocrine control of such a process remain to be characterized. Here, we report the developmentally and hormonally regulated expression of MCT2 in the testis. At Day 18, MCT2 starts to be expressed in germ cells as detected by Northern blot. The mRNA are translated into protein (40 kDa) in elongating spermatids. Ultrastructural analysis demonstrated that MCT2 protein is localized to the outer face of the cell membrane of spermatid tails. MCT2 mRNA levels are under the control of the endocrine, specifically follicle-stimulating hormone (FSH) and testosterone, and paracrine systems. Indeed, a 35-day-old rat hypophysectomy resulted in an 8-fold increase in testicular MCT2 mRNA levels. Conversely, FSH and LH administration to the hypophysectomized rats reduced MCT2 mRNA levels to the basal levels observed in intact animals. The decrease in MCT2 mRNA levels was confirmed in vitro using isolated seminiferous tubules incubated with FSH or testosterone. FSH or testosterone inhibited in a dose-dependent manner MCT2 mRNA levels with maximal inhibitory doses of 2.2 ng/ml and 55.5 ng/ml for FSH and testosterone, respectively. In addition to the endocrine control, TNFalpha and TGFbeta also exerted an inhibitory effect on MCT2 mRNA levels with a maximal effect at 10 ng/ml and 6.6 ng/ml for TGFbeta and TNFalpha, respectively. Together with previous studies, the present data reinforce the concept that among the key functions of the endocrine/paracrine systems in the testis is the control of the energy metabolism occurring in the context of Sertoli cell-germ cell metabolic cooperation where lactate is produced in somatic cells and transported to germ cells via, at least, MCT2.     

The effect of testosterone withdrawal and subsequent germ cell depletion on transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels in the adult rat testis.

We have examined the effects of decreasing intratesticular testosterone concentration and of decreasing germ cell number on levels of transferrin mRNA and sulfated glycoprotein (SGP)-2 mRNA in the adult rat testis. Intact rats received implants of testosterone- and estradiol-filled capsules to suppress LH secretion from the pituitary, thereby suppressing Leydig cell testosterone production. The levels of intratesticular testosterone declined 70% to 20 ng/ml within 3 days, were reduced further to approximately 15 ng/ml by 14 days, and subsequently reached a minimum of about 10 ng/ml. In contrast, the number of elongated spermatids per testis remained unchanged through 14 days, then declined to fewer than 20% of normal between 14 and 28 days, and reached zero by 56 days postimplantation. Likewise, both pachytene spermatocytes and round spermatids declined only after 14 days postimplantation. Northern blots of testicular RNA showed that Sertoli cell transferrin mRNA per testis decreased markedly between 14 and 28 days postimplantation. However, SGP-2 mRNA per testis was unchanged over the time course of the experiment. The decrease in transferrin mRNA, concomitant with germ cell loss, suggests that this mRNA is regulated by the number of germ cells in the testis and not directly by testosterone. In contrast, the constant level of SGP-2 mRNA in the face of reduced intratesticular testosterone and the subsequent loss of germ cells suggests that this mRNA is constitutively maintained in the adult rat testis.     

Cellular distribution, developmental changes and effects of cryptorchidism on uridine kinase in the rat testis

High specific activity of uridine kinase was found in cultured peritubular cells (3.0 nmol/min per mg protein) which was more than 3-fold higher than that found in cultured Sertoli cells (0.79 nmol/min per mg protein). In the various classes of germ cells a decrease in specific uridine kinase activity was associated with increased maturity of the cells, primary spermatocytes, round spermatids and spermatozoa showing 1.3, 0.65 and 0.16 nmol/min per mg protein, respectively. A relationship between uridine kinase activity and the rate of RNA synthesis in these cells is suggested. A decrease in specific uridine kinase activity in testis with increasing age supports the finding of lower uridine kinase in mature germ cells than in earlier germ cells and somatic cells. This finding is further supported by the observation that cryptorchidism, which is associated with a time-dependent depletion of germ cells, resulted in an increase in specific uridine kinase activity. The results indicate that pyrimidine salvage is important in earlier germ cells, as well as in somatic cells in the testis, to produce substrates for nucleic acid synthesis.     

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.     

Expression of tumor necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in rat testis during development

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-alpha family of cytokines that is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated in the rat testis during development. TRAIL and its receptors were immunolocalized to the different testicular cell types. TRAIL and its receptors were also identified in the rat testis in terms of protein and mRNA. Our immunohistochemical studies indicate that TRAIL, DR5/TRAIL-R2, and DcR2-TRAIL-R4 are detected in Leydig cells, whereas ligand and all receptors are localized in germ cells. TRAIL was permanently immunodetected in germ cells from the fetal stage to adulthood, whereas its receptors were immunolocalized exclusively in postmeiotic germ cells. The expression of TRAIL and receptor mRNAs was consistent with the immunodetection of TRAIL and receptor proteins. Indeed, TRAIL ligand mRNA was also identified in the rat testis from the fetal stage to adulthood. The mRNAs of the death receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2, were weakly detected during the perinatal period and increased from the pubertal stage to adulthood. The mRNAs of the decoy receptors, DcR1 and DcR2, were present in the rat testis at all ages studied, but the DcR2/TRAIL-R4 mRNa level was higher from the pubertal period to adulthood. Together, the present findings demonstrate that 1) TRAIL and its receptors are expressed in the testis during normal development, and 2) TRAIL protein is present in the different germ cell types, whereas its receptors were predominantly detected in the postmeiotic germ 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.     

Requirement of serum components for the preservation of primordial germ cells in testis cords during early stages of testicular differentiation in vitro in the mouse

Mouse gonadal primordia were isolated from embryos on the 11th day of gestation and cultured in vitro. They developed into either testes or ovaries after 7 days of culture in Eagle's minimum essential medium (MEM) supplemented with horse serum, whereas they did not differentiate in MEM alone. We studied how serum components are required for testicular development in vitro. When gonadal primordia were cultured in MEM alone for the first 1-3 days and subsequently in MEM supplemented with serum, testis cords developed while germ cells disappeared or only a few remained in the testis cords. In contrast, when serum was present in the medium during the first day of culture and omitted thereafter, germ cells were retained within testis cords. These results suggested that some serum component(s) is specifically required by germ cells independent of testis cord organization. Of more than 10 serum components tested, low and very low density lipoprotein fractions increased the number of germ cells in testicular explants.