Glycodelin mRNA is expressed in the genital tract of male and female rats (Rattus norvegicus).

In the present study we demonstrate for the first time the expression of glycodelin mRNA in the female and male genital tracts of rats using non-radioactive in situ hybridisation. Glycodelin fragment 1 (+41 to +141) shares 100% homology with the human gene sequence. In the ovary, glycodelin mRNA was restricted to granulosa cells. In the uterus, glycodelin mRNA was expressed in all epithelial cells of the endometrium. In the male reproductive tract, glycodelin mRNA was distributed in all epithelial cells of the epididymis, the prostate and the seminal vesicle. However, in the testis, glycodelin mRNA was predominantly found in spermatogonia and in spermatocytes of the seminiferous epithelium. The expression in several reproductive organs of rats offers an excellent tool to study further the physiological role of glycodelin, which is so far thought to act as an immunosuppressive factor.     

Testicular growth hormone (GH): GH expression in spermatogonia and primary spermatocytes

Growth hormone (GH) gene expression is not restricted to pituitary somatotrophs and has recently been demonstrated in a variety of extrapituitary sites in mammals and the domestic chicken. The possibility that GH gene expression occurs in the male reproductive system of chickens was therefore examined, since GH has established roles in male reproductive function and GH immunoreactivity is present in the chicken testis. Using RT-PCR and oligonucleotide primers for pituitary GH cDNA, GH mRNA was shown to be present in the testes and vas deferens of adult cockerels. Although testicular GH mRNA was of low abundance (not detectable by Northern blotting), a 690 bp fragment of the amplified testicular GH cDNA was cloned and had a nucleotide sequence 99.6% homologous with pituitary GH cDNA. GH mRNA was localized by in situ hybridization in spermatogonia and primary spermatocytes of the seminiferous tubules, but unlike testicular GH-immunoreactivity, GH mRNA was not present in secondary spermatocytes, spermatids or spermatozoa. The presence of Pit-1 mRNA in the male reproductive tract may indicate Pit-1 involvement in GH expression in these tissues. The presence of GH receptor mRNA in the testis and vas deferens also suggests they are target sites for GH action. These results demonstrate, for the first time, expression of the pituitary GH gene in the testis, in which GH mRNA was discretely localized in primary spermatocytes. The local expression of the GH gene in these cells suggests autocrine or paracrine actions of GH during spermatogenesis.     

Developmentally regulated expression of cyclin D3 and its potential in vivo interacting proteins during murine gametogenesis.

To begin to assess the function of the cell cycle regulator cyclin D3 during gametogenesis, the present study examined its expression, interacting partners, and associated kinase activity in the murine testis and ovary. In the early stages of postnatal testicular development, cyclin D3 protein was detected in spermatogonia and Leydig cells. In the adult testis, cyclin D3 was also expressed in terminally differentiating spermatids. In the embryonic ovary, detection of cyclin D3 was limited to somatic cells. In the postnatal ovary, its localization was predominantly in the nuclei of oocytes in primordial and small follicles, a localization that diminished with oocyte growth. Cdk4 and p27 were expressed in a similar subset of testicular and ovarian cells, suggesting that they may regulate cyclin D3 function during testicular and ovarian development in a cell type-specific manner. Cyclin D3-associated kinase activity was detected in immature, but not adult, testes and ovaries. These observations suggest unique roles for cyclin D3 in the control of cell division and differentiation in the germ line and the differential regulation of mitotic and meiotic cell cycles during male and female gametogenesis.     

Growth hormone in the male reproductive tract of the chicken: heterogeneity and changes during ontogeny and maturation

Growth hormone (GH) gene expression is not confined to pituitary somatotrophs and occurs in many extrapituitary tissues. In this study, we describe the presence of GH moieties in the chicken testis. GH-immunoreactivity (GH-IR), determined by ELISA, was found in the testis of immature and mature chickens, but at concentrations <1% of those in the pituitary gland. The immunoassayable GH concentration in the testis was unchanged between 4 and 66 weeks of age, and approximately 10-fold higher than that at 1-week of age and 25-fold higher than that in 1-day-old chicks and perinatal (embryonic day 18) embryos. This immunoreactivity was associated with several proteins of different molecular size, as in the pituitary gland, when analyzed by SDS-PAGE under reducing conditions. However, while most of the GH-IR in the pituitary ( approximately 40 and 15%, respectively) is associated with monomer (26 kDa) or dimer (52 kDa) GH moieties GH-IR in the testis is primarily (30-50%) associated with a 17 kDa moiety. GH bands between 32 and 45 kDa are also relatively more abundant in the testis than in the pituitary. During ontogeny the relative abundance of a 14 kDa GH and 40 kDa GH moieties in the testis significantly declined, whereas the relative abundance of the 17 and 45 kDa moieties increased with advancing age. In adult birds, GH-IR was widespread and intense in the seminiferous tubules. Although the GH-IR was not present in the basal compartment of Sertoli cells, nor in spermatogonia and primary spermatocytes, it was abundantly present in secondary spermatocytes and spermatids in the luminal compartments of the tubules as well as in some surrounding myocytes and interstitial cells. In summary, immunoreactive GH moieties are present in the chicken testis but at concentrations far less than in the pituitary. Age-related changes in the relative abundance of testicular GH variants may be related to local (autocrine/paracrine) actions of testicular GH. The localization of GH in spermatocytes and spermatids suggests hitherto unsuspected roles in gamete development.     

Immunopresence and enzymatic activity of nitric oxide synthases, cyclooxygenases and PGE2-9-ketoreductase and in vitro production of PGF2α, PGE2 and testosterone in the testis of adult and prepubertal alpaca (Lama pacos)

This study presents the first evidence for differences in COXs, PGE2-9-ketoreductase and NOSs immunopresence and enzyme activity, and prostaglandin and testosterone production between the testes of adult and prepubertal alpacas. The prepubertal testis immunohistochemical data revealed that COX1 was expressed in spermatogonia and endothelial cells whereas COX2 was present only in the stromal cells. In adult animals, COX2 immunosignals were evidenced in germ cells, as well as both COX1 and -2 in Leydig and Sertoli cells. In adult testes, the spermatogonia, spermatocytes and round spermatids had expression of e- and n-NOS only, whereas elongated spermatids exhibited immunopositivity for i- and e-NOS and Sertoli cells expressed only n-NOS. In prepubertal alpacas, i-NOS was localized in spermatogonia, e-NOS in Sertoli cells and all three NOS isoforms in Leydig cells. PGE2-9-ketoreductase immunopresence was observed in spermatogonia nuclei and cytoplasm of prepubertal testis whereas they were localized in spermatid acrosomal vesicle of adult. The enzymatic data indicated that COX1 activity was higher than COX2 in adult alpaca testis whereas the activity of COX2 was greater than that of COX1 in prepubertal animals. Total NOS and PGE2-9-ketoreductase activities were more extensive in adult alpacas. In vitro hormone production results showed that prepubertal testes released lower amounts of testosterone and PGF2α while PGE2 synthesis was six times more elevated than in in vitro incubated adult testes. Taken together, the data on COX2, i-NOS and PGE2 led us to hypothesize that development in prepubertal male reproductive tissues utilizes a mechanism similar to that of inflammation.     

Dependence of testicular germ cells on hormones: a quantitative study in hypophysectomized testosterone-treated rats

Quantitative analysis of spermatogenesis was used to investigate the dependence of testicular germ cells on hormones in Sprague-Dawley rats. Adult hypophysectomized rats were given daily injections of testosterone propionate (TP) for 30 days. Intact and hypophysectomized control rats received vehicle only. Spermatogonia were classified as undifferentiated or differentiated, using established criteria suitable for morphological identification on periodic acid-Schiff's-haematoxylin stained sections of testis. Data on cell counts showed that the undifferentiated spermatogonia may be partially dependent on TP and/or pituitary hormones. The group of differentiated spermatogonia were dependent on pituitary hormones, and TP only partially restored their number by partially protecting them from spontaneous degeneration in stages XIV to II (A3-to-Intermediate). The maturation and division of B type spermatogonia and maturation of preleptotene spermatocytes to the zygotene stage appeared to be independent of hormones. Maturation of pachytene spermatocytes was hormone dependent, and TP completely supported their development, meiotic division and spermiogenesis in the complete absence of pituitary hormones.     

Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse

AIM: The aim of this study was to determine the action of bone morphogenetic proteins (BMPs) on testicular cell proliferation during early postnatal life, a definite developmental time at which crucial changes in germ cell and Sertoli cell maturation occur. METHODS: We investigated the effect of BMP2 and BMP7, two factors which belong to the relatively distant decapentaplegic (DPP) and 60 A classes of the large BMP family, upon spermatogonial and Sertoli cell proliferation, and we examined the expression of activin/BMP type II and type I receptors. We used in vitro cultured testis fragments from 7-day-old mice, highly purified populations of somatic and germ cells and total testes from mice of different ages. Cell proliferation was assessed by BrdU labelling and [3H]-thymidine incorporation. Ribonuclease protection assays and Northern blotting were performed to analyse receptor expression. RESULTS AND CONCLUSIONS: We have demonstrated a stimulatory action of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation respectively. ActRIIB is the type II receptor expressed most in spermatogonia, whereas Sertoli cells specifically expressed BMPRIIB, in addition to ActRIIB. By contrast, the presence of ActRIIA was undetectable in either germ or somatic cells. The type I receptors ActRIA, ActRIB and BMPRIA were all found in both cell types, indicating that the observed effect of BMP2 and BMP7 on testicular cell proliferation may be mediated by a number of combinatorial interactions in the receptor complexes. These findings suggest that BMPs are involved in physiological paracrine signalling during the first wave of spermatogenesis.     

Expression of pro-opiomelanocortin-like gene in the testis and epididymis.

Adrenocorticotropin (ACTH), beta-endorphin, and the melanocyte-stimulating hormones (MSHs), which are products of a common precursor, pro-opiomelanocortin (POMC), are present in a variety of tissues other than pituitary. The recent detection of immunoreactive POMC-derived peptides in the male reproductive tract raised the possibility that these hormones might regulate reproductive function. To determine whether the low concentrations of POMC-derived peptides in the male reproductive tract are synthesized locally and are not contaminants from blood, we have demonstrated POMC-like gene expression in both testis and epididymis. The identification of cells in testis capable of synthesizing POMC mRNA was established by showing the presence of this mRNA in mouse Leydig cell lines (TM3 and I10A). The hybridizing species of POMC-like mRNA in the testis, epididymis, and Leydig cell lines (TM3 and I10A) were approximately 150 bases shorter than those in the pituitary or hypothalamus but were similar in size to that in the amygdaloid nucleus of rat brain. The concentration of POMC-like mRNA in the testis is almost as high as that in the hypothalamus. This finding is quite unexpected because the concentrations of POMC-derived peptides in the testis were 2-3 orders of magnitude lower than those in the hypothalamus. The demonstration of a POMC-like gene expression in male reproductive tissues suggests that POMC-derived peptides are synthesized in Leydig cells and epididymis. These observations are consistent with the postulate that POMC-derived peptides may exert paracrine and/or autocrine effects in these organs.     

Expression and regulation of proopiomelanocortin-like gene in the ovary and placenta: comparison with the testis

Proopiomelanocortin (POMC), a precursor protein for ACTH, beta-endorphin, and the MSHs, has been identified in the reproductive tracts of both male and female. With rat pituitary POMC complementary DNA (cDNA) as a hybridization probe, POMC-like messenger RNA (mRNA) was identified in the ovaries of rat, mouse, and monkey. The molecular size of POMC-like mRNA in the ovary was 150-200 bases smaller than in the pituitary and hypothalamus but identical to that in the testis and epididymis. The size heterogeneity of POMC mRNA observed in various tissues is not due to differences in the lengths of the poly(A) tail, as measured by RNase H digestion. S1 nuclease mapping analysis revealed that POMC mRNAs isolated from pituitary, testis, or ovary share the nucleotide sequences coding for ACTH, beta-lipotropin, and the 3'-untranslated region. The regulation of ovarian POMC-like mRNA was also investigated. Treatment of 25-day-old immature female rats with PMSG resulted in profound increases in the ovarian content of total RNA, poly(A) RNA, and POMC-like mRNA. The concentration of ovarian POMC-like mRNA during pregnancy increased increased to 3-4 times that in immature or normally cycling animals. POMC-derived peptides are present in the human placenta and are synthesized de novo in cultured placental cells. In this report we also demonstrate POMC-like mRNA in the placenta of rat, mouse, and human. The size of POMC-like mRNA in the placenta was similar to that observed in the testis, epididymis, and ovary and different from that found in the pituitary or hypothalamus. The concentration of placental POMC-like mRNA did not change throughout pregnancy. In conclusion, we have demonstrated that 1) POMC-like mRNA is present in the ovary and placenta of rodents and primates; 2) the size of POMC-like mRNA in the ovary and placenta, like that in the testis and epididymis, is smaller than that in the pituitary and hypothalamus, probably owing to a shortening of the 5'-ends; and 3) the expression of this gene is regulated by gonadotropins in the ovary but probably not in the placenta.     

Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.