MicroRNA-200a locally attenuates progesterone signaling in the cervix, preventing embryo implantation.

Although cervical pregnancy and placenta previa, in which the embryo and placenta embed in or adjacent to the cervix, are life-threatening complications that result in massive bleeding and poor pregnancy outcomes in women, the incidence of these aberrant conditions is uncommon. We hypothesized that a local molecular mechanism is normally in place to prevent embryo implantation in the cervix. The ovarian hormones progesterone (P(4)) and estrogen differentially direct differentiation and proliferation of endometrial cells, which confers the receptive state for implantation: P(4) dominance causes differentiation of the luminal epithelium but increases stromal cell proliferation in preparation of the uterus for implantation. In search for the cause of cervical nonresponsiveness to implantation, we found that the statuses of cell proliferation and differentiation between the uterus and cervix during early pregnancy are remarkably disparate under identical endocrine milieu in both mice and humans. We also found that cervical levels of progesterone receptor (PR) protein are low compared with uterine levels during this period, and the low PR protein levels are attributed to elevated levels of microRNA(miR)-200a in the cervix. These changes were associated with up-regulation of the P(4)-metabolizing enzyme 20α-hydroxysteroid dehydrogenase (200α-HSD) and down-regulation of its transcriptional repressor signal transducer and activator of transcription 5 in the cervix. The results provide evidence that elevated levels of miR-200a lead to down-regulation of P(4)-PR signaling and up-regulation of (200α-HSD) in the cervix, rendering it nonresponsive to implantation. These findings may point toward not only the physiological but also the pathological basis of the cervical milieu in embryo implantation.

Bovine luteal prolactin receptor expression: potential involvement in regulation of progesterone during the estrous cycle and pregnancy.

In the present study, we performed quantitative reverse-transcription PCR (qPCR) to examine changes in gene expression of prolactin receptor (long form: l-PRLR; short form: s-PRLR) and 20α-hydroxysteroid dehydrogenase (20α-HSD; EC 1.1.1.149) in the bovine corpus luteum (CL) throughout the estrous cycle and pregnancy. Western blotting was used to determine protein abundance. Bovine CL were collected and luteal stages (n = 6/stage) were classified by macroscopic observation as early (d 1 to 4 after ovulation), mid (d 5 to 10), late (d 11 to 17), and regressing (d 18 to 20). A CL of pregnancy (n = 6) was determined by the presence of conceptus (d 28 to term). The mRNA for both forms of PRLR were expressed at all the luteal stages. Expression of s-PRLR and l-PRLR mRNA was less (P < 0.01) during early and regressing luteal stages compared with mid and late stages. Expression of s-PRLR mRNA in CL of pregnancy was greater (P < 0.01) than early, mid, and regressing CL and did not differ from late luteal stage expression. A greater (P < 0.01) expression of l-PRLR mRNA was observed in pregnant vs. early and regressing CL. In addition, qPCR showed the presence of 20α-HSD mRNA during all luteal stages of the estrous cycle, with the greatest (P < 0.01) expression observed in the regressing luteal stage. Western blotting revealed protein abundance of both PRLR isoforms during all luteal stages and pregnancy, with a predominance of the s-PRLR protein. Densitometry analysis indicated that protein abundances of s-PRLR were greater (P < 0.05) than l-PRLR during early, mid, and late luteal stages and did not differ during the regressing luteal stage. Protein abundances of 20α-HSD were least (P < 0.05) during the early luteal stage. In conclusion, results of the current study suggest a possible involvement of PRLR, especially s-PRLR, in the regulation of progesterone secretion and metabolism during the bovine estrous cycle and pregnancy.

Alterations of folliculogenesis in women with polycystic ovary syndrome.

The objective of the present study was to examine some factors involved in follicular development of women with polycystic ovary syndrome (PCOS). Women with PCOS showed increased levels of serum luteinizing hormone (LH) but decreased follicular production of progesterone and estradiol by pre-ovulatory follicles. The mRNA expression corresponding to steroidogenic acute regulatory protein (StAR), and 20alpha-hydroxysteroid dehydrogenase (20α-HSD) was increased, while that corresponding to cytochrome P450 aromatase (P450arom) was decreased in PCOS follicles as compared to controls. No changes in the mRNA expression for 3beta-hydroxysteroid dehydrogenase 2 (3ß-HSD2), cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha hydroxylase/lyase (P450c17), cyclooxygenase 2 (COX2), and transcription factors (GATA-4 and GATA-6) were found. We conclude that despite the hyper-luteinized environment of PCOS follicles, these follicles produce lower levels of progesterone and estradiol, and that this is characterized by increased degradation of progesterone and decreased estradiol synthesis. Our data demonstrate that the synthesis of prostaglandin F2α (PGF2α) may be affected in PCOS-follicles and that the transcription factors GATA-4 and GATA-6 are present in PCOS-follicles but they are not involved in the abnormal transcription observed in the steroidogenic enzymes.

Galectins in the mouse ovary: concomitant expression of galectin-3 and progesterone degradation enzyme (20alpha-HSD) in the corpus luteum.

Galectin, an animal lectin that recognizes beta-galactosides of glycoconjugates, is involved in multiple biological functions such as cell growth, differentiation, apoptosis, and signal transduction. The present study using in situ hybridization revealed the predominant expression of galectin-1 and galectin-3 in the mouse ovary. Galectin-1 mRNA was diffusely expressed in the ovarian stroma, including the interstitial glands and theca interna, and intensely expressed in the corpus luteum (CL) at particular stages of regression. Transcripts of galectin-3 were restricted to CL and always coincident to the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a progesterone degradation enzyme. In the non-pregnant ovary, signals for both galectin-1 and -3 were intense in the old, regressing CL formed at previous estrous cycles. In the newly formed CL, the signal intensity of galectin-1 first increased at the starting point of regression followed by increasing galectin-3/20alpha-HSD expressions. Under gestation with active progesterone production, signals for both galectin-1 and -3 in CL completely disappeared. At the perinatal stage, intense expressions of galectin-3/20alpha-HSD recovered in the remaining CL of gestation with the temporal expression of galectin-1 and continued until weaning. These findings suggest that galectin-1 and -3 may mediate progesterone production and metabolism in luteal cells via different mechanisms.

Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5'-flanking region of the mouse 20alpha-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5'-flanking region, revealed that the region between -83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wild-type Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20alpha-HSD gene.

Effects of deletion of the prolactin receptor on ovarian gene expression.

Prolactin (PRL) exerts pleiotropic physiological effects in various cells and tissues, and is mainly considered as a regulator of reproduction and cell growth. Null mutation of the PRL receptor (R) gene leads to female sterility due to a complete failure of embryo implantation. Pre-implantatory egg development, implantation and decidualization in the mouse appear to be dependent on ovarian rather than uterine PRLR expression, since progesterone replacement permits the rescue of normal implantation and early pregnancy. To better understand PRL receptor deficiency, we analyzed in detail ovarian and corpora lutea development of PRLR-/- females. The present study demonstrates that the ovulation rate is not different between PRLR+/+ and PRLR-/- mice. The corpus luteum is formed but an elevated level of apoptosis and extensive inhibition of angiogenesis occur during the luteal transition in the absence of prolactin signaling. These modifications lead to the decrease of LH receptor expression and consequently to a loss of the enzymatic cascades necessary to produce adequate levels of progesterone which are required for the maintenance of pregnancy.