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Prolactin is a hormone that is essential for normal reproduction and signals through two types of receptors. Not only is the classical long form of the prolactin receptor identified, but so are many short form receptors in rodents and human tissues. Mouse mutagenesis studies have offered insight into the biology of prolactin family, providing compelling evidence that the different isoforms have independent biological activity. The possibility that short forms mediate cell proliferation is important for a variety of tissues including mammary gland and ovarian follicles. This review summarizes our current knowledge about prolactin signaling and its role in reproduction through either long or short isoform receptors.

To date the best defined function of prolactin (PRL) is its action on the ovary and mammary gland, although it has also been shown to have an effect on lipid metabolism. Using mice engineered to express only the long form of the prolactin receptor (PRL-RL), we demonstrate that PRL acting through PRL-RL alone causes severe adipose accumulation in visceral fat of males at 6 months of age. The increase in visceral fat accumulation is attributed to loss of adipose-derived leptin, which results in diminished lipolysis. The reduction in leptin also corresponds to decreased activation of AMP-activated protein kinase (AMPK), which further results in diminished fatty acid oxidation and increased fatty acid synthesis. Interestingly, the blunted AMPK response was only observed in adipose tissue and not in liver suggesting that this PRL mediated effect is tissue specific. A glucose tolerance study inferred that PRL-RL mice may suffer from insulin resistance or a reduction in insulin production that is not due to aberrant expression of glucose transporter 4 (Glut4). Collectively, our findings demonstrate that PRL signaling through the long form receptor causes reduced fatty acid oxidation, increased lipid storage, glucose intolerance, and obesity. These findings are of great importance towards understanding the etiology of obesity associated with hyperprolactinemia in humans as well as the role of PRL as a metabolic regulator in adipose tissue.

Prolactin (PRL) is essential for normal reproduction and signals through two types of receptors, the short (PRL-RS) and long (PRL-RL) form. We have previously shown that transgenic mice expressing only PRL-RS (PRLR−/−RS) display abnormal follicular development and premature ovarian failure. Here, we report that MAPK, essential for normal follicular development, is critically inhibited by PRL in reproductive tissues of PRLR−/−RS mice. Consequently, the phosphorylation of MAPK downstream targets are also markedly inhibited by PRL without affecting immediate upstream kinases, suggesting involvement of MAPK specific phosphatase(s) in this inhibition. Similar results are obtained in a PRL-responsive ovary-derived cell line (GG-CL) that expresses only PRL-RS. However, we found the expression/activation of several known MAPK phosphatases not to be affected by PRL, suggesting a role of unidentified phosphatase(s). We detected a 27-kDa protein that binds to the intracellular domain of PRL-RS and identified it as dual specific phosphatase DUPD1. PRL does not induce expression of DUDP1 but represses its phosphorylation on Thr-155. We also show a physical association of this phosphatase with ERK1/2 and p38 MAPK. Using an in vitro phosphatase assay and overexpression studies, we established that DUPD1 is a MAPK phosphatase. Dual specific phosphatase inhibitors as well as siRNA to DUPD1, completely prevent PRL-mediated MAPK inhibition in ovarian cells. Our results strongly suggest that deactivation of MAPK by PRL/PRL-RS contributes to the severe ovarian defect in PRLR−/−RS mice and demonstrate the novel association of PRL-RS with DUPD1 and a role for this phosphatase in MAPK deactivation.

Our laboratory has previously cloned and purified a protein named PRAP (prolactin receptor-associated protein) that was shown to be a novel 17beta-hydroxysteroid dehydrogenase (HSD) enzyme with dual activity. This enzyme, renamed HSD17B7 or PRAP/17beta-HSD7, converts estrone to estradiol and is also involved in cholesterol biosynthesis. The major site of its expression is the corpus luteum of a great number of species including rodents and humans. To examine the functional significance of HSD17B7 in pregnancy, we generated a knockout mouse model with targeted deletions of exons 1-4 of this gene. We anticipated a mouse with a severe fertility defect due to its inability to regulate estrogen levels during pregnancy. The heterozygous mutant mice are normal in their development and gross anatomy. The females cycle normally, and both male and female are fertile with normal litter size. To our surprise, the breeding of heterozygous mice yielded no viable HSD17B7 null mice. However, we found HSD17B7 null embryo alive in utero on d 8.5 and d 9.5. By d 10.5, the fetuses grow and suffer from severe brain malformation and heart defect. Because the brain depends on in situ cholesterol biosynthesis for its development beginning at d 10, the major cause of fetal death appears to be due to the cholesterol synthetic activity of this enzyme. By ablating HSD17B7 function, we have uncovered, in vivo, an important requirement for this enzyme during fetal development.

Prolactin receptor-associated protein (PRAP) originally cloned in our laboratory was shown to be a novel, luteal isoform of 17β hydroxysteroid dehydrogenase 7 (17βHSD7). In this study, we cloned the promoter region of rat PRAP/17βHSD7 and investigated the mechanisms regulating both basal activity and LH-induced repression of this promoter. Truncated and site-specific mutants of PRAP/17βHSD7 promoter identified two enhancer regions that contained highly conserved Sp1 binding site and bound Sp1 from nuclear extracts of both corpora lutea and a rat luteal cell line. Repression of PRAP/17βHSD7 expression and promoter activity by human chorionic gonadotropin/forskolin was localized to a −52-bp proximal segment of the promoter. This region contained a conserved CCAAT site and bound nuclear factor Y; binding of this transcription factor was inhibited by human chorionic gonadotropin in vivo. Furthermore, mutation of the nuclear factor Y site in the −52-bp promoter-reporter construct abolished forskolin-mediated inhibition of the promoter in a rat luteal cell line. In summary, we have identified the promoter elements involved in the basal expression of PRAP/17βHSD7. We have also found that LH-mediated repression of this gene is at the level of transcription and involves inhibition of nuclear factor YA binding to the CCAAT site within the proximal promoter.

Nuclear factor kappa B (NFkappaB) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NFkappaB in regulating ovarian gene expression, we examined in the rat: (i) whether NFkappaB is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NFkappaB complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NFkappaB is maintained in the cytoplasm bound to IkappaB, whose phosphorylation allows NFkappaB translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of IkappaBalpha. Western analysis revealed that IkappaBalpha was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NFkappaB consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NFkappaB binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NFkappaB DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NFkappaB/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NFkappaB/p65 in luteal cells resulted in inhibition of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD) promoter activity as well as endogenous 20alphaHSD mRNA expression. In summary, we have shown that: (i) NFkappaB is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NFkappaB activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NFkappaB/p65 binding to an NFkappaB DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NFkappaB in luteal cells inhibits 20alphaHSD gene expression. The results further support a role for NFkappaB as a survival factor in the CL.

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.

Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens. activin, apoptosis, decidua, prolactin

Progesterone production by the corpus luteum (CL) is essential for preparation of the endometrium for implantation and for the maintenance of gestation. Progesterone modulates its own production and opposes functional luteal regression induced by exogenous agents, such as prostaglandin F2α. In the present study, we evaluated whether progesterone is also capable of interfering with the process of structural luteal regression, which is characterized by a decrease in weight and size of the gland because of programmed cell death (i.e., apoptosis). We have found that a low number of luteal cells undergo apoptosis throughout gestation. On the day of parturition, but following the initial decline in endogenous progesterone production, a small increase in the number of luteal cells undergoing cell death was observed. This increase in apoptotic cells continued postpartum, reaching dramatic levels by Day 4 postpartum, and was accompanied by a marked decrease in average luteal weight. We have established th...

Androgens, in concert with lactogenic hormones, contribute to the maintenance of function of the corpus luteum (CL) in pregnant rats. Whereas some of the androgenic actions in the CL are clearly mediated by intracrine conversion to estrogen, pure androgenic effects are also implicated in the regulation of this transient endocrine gland. In this report, we have established, to our knowledge for the first time, the expression of androgen receptor (AR) mRNA and protein throughout gestation in the rat CL. We have found that the AR remains expressed in the CL of gestation on Day 4 postpartum and becomes expressed in the newly formed CL after postpartum ovulation. An AR immunoreactive protein was identified in the CL of pregnancy as well as in prostate and epididymis, which were used as positive controls. The luteal AR protein had mainly nuclear localization, yet some diffuse cytoplasmic staining was also observed. Moreover, we have established that androstenedione, the main circulating androgen in pregnant rats, significantly reduces the decline in luteal weight observed during postpartum structural regression. This effect was correlated with a decrease in the number of cells undergoing apoptosis and with enhanced levels of circulating progesterone. In addition, in vivo administration of androstenedione delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that the interference with apoptosis in vitro elicited by androstenedione is accompanied by an increased capacity of the CL to secrete progesterone. In summary, the results of this study have established that the rat CL expresses AR throughout pregnancy and after parturition, and they have defined a potential role for androstenedione in opposing postpartum luteal regression through inhibition of apoptosis and stimulation of progesterone production. androgen receptor, apoptosis, corpus luteum, corpus luteum function, progesterone

Cell cycle progression of granulosa cells is critical for ovarian function, especially follicular maturation. During follicular maturation, FSH induces cyclin D2, which promotes G1 progression by activating cyclin-dependent kinase-4 (Cdk4). Because cyclin D2-deficient mice exhibit a block in follicular growth, cyclin D2/Cdk4 has been hypothesized to be required for FSH-dependent proliferation of granulosa cells. Here we investigate ovarian function in Cdk4-knockout mice we recently generated. Cdk4(-/-) females were sterile, but the morphology of their ovaries appeared normal before sexual maturation. The number of preovulatory follicles and the ovulation efficiency were modestly reduced in gonadotropin-treated Cdk4(-/-) mice. However, unlike cyclin D2-deficient mice, Cdk4(-/-) mice showed no obvious defect in FSH-induced proliferation of granulosa cells. Cdk4(-/-) ovaries displayed normal preovulatory expression of aromatase, PR, and cyclooxygenase-2. Postovulatory progesterone secretion was markedly impaired in Cdk4(-/-) mice, although granulosa cells initiated luteinization with induction of p450 side-chain cleavage cytochrome and p27(Kip1). Progesterone treatment rescued implantation and restored fertility in Cdk4(-/-) mice. Serum PRL levels after mating were significantly reduced in Cdk4(-/-) mice, suggesting the involvement of perturbed PRL regulation in luteal failure. Thus, Cdk4 is critical for luteal function, and some redundant protein(s) can compensate for the absence of Cdk4 in proliferation of granulosa cells.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

It is well established that prolactin (PRL) sustains, while prostaglandin F(2 alpha) (PGF(2 alpha)) curtails, progesterone production by the rat corpus luteum (CL). We have previously shown that the actions of both molecules converge on the 20 alpha-HSD gene and control its expression in a dramatically opposed manner. In this investigation, we have found twelve more genes that are inversely regulated by PRL and PGF(2 alpha). In addition to 20 alpha-HSD, PGF(2 alpha) stimulated and PRL inhibited PGF(2 alpha)-receptor, phospholipase C delta(1) and TGF beta(1) expression. In contrast PRL stimulated and PGF(2 alpha) inhibited the LH receptor, 11 beta-HSD2, sterol carrier protein 2, mitochondrial glutathione S-transferase (GST), GST mu(2), inhibitory DNA-binding proteins 1, 2, and 3, and calcium binding protein 2. We have also identified new target genes for PRL and PGF(2 alpha). PGF(2 alpha) stimulated the expression of genes involved in cell signaling such as cell adhesion kinase-beta, ERK3, FRA2, IL-2 receptor, and 14-3-3 proteins. PGF(2 alpha) also up-regulated the expression of the sodium channel beta(1), Na/K ATPase, annexin IV, GST7pi, and P450 reductase. In contrast PGF(2 alpha) inhibited the expression of two genes involved in cell cycle: cyclin D2 and retinoblastoma related protein (Rb2/p130). It also inhibited genes involved in estradiol (P-450(AROM)) and cholesterol biosynthesis (HMG-CoA synthase), as well as genes involved in tissue remodeling: VEGF and TIMP3. PRL had a profound inhibitory effect on the expression of genes encoding the ADP-ribosylation factor 3, annexin V and c-jun, yet increased the expression of P450scc, 3beta-HSD, and SR-B1 (HDL-receptor), all genes involved in steroidogenesis. PRL also stimulated the expression of beta(2)-microglobulin, TIMP2, cytochrome c oxidase IV, cathepsin H and L, and copper-zinc superoxide dismutase as well as elongation factor SIII, heat shock protein-60 and mitochondrial ATP synthase-D. In conclusion, this investigation has revealed a "yin-yang" relationship between PRL and PGF(2 alpha) in regulating certain critical genes in the rodent CL, and has demonstrated novel regulation by these factors of other important genes involved in luteal function.

During gestation, the uterus undergoes severe changes to accommodate and protect the developing conceptus. In particular, stromal endometrial cells proliferate and differentiate to form the decidual tissue, which produces PRL. Once the conceptus begins to grow, extensive regression by apoptosis take place in the decidua coincident with the loss of the PRL receptor in this tissue. In this report we have established for the first time that PRL, acting through the long form of the PRL receptor and the PI3K pathway, exerts an antiapoptotic effect in rat decidua. We have also shown that protein kinase B phosphorylation on serine 473 as well as its nuclear translocation are stimulated by PRL in decidual cells. Moreover, we have found that caspase-3, a well known effector of apoptosis, becomes expressed and active in the rat decidua just at a time when this tissue undergoes extensive apoptosis. PRL was able to down-regulate both caspase-3 mRNA levels as well as activity. Furthermore, using a protein kinase B dominant-negative expression vector, we provide evidence that PRL inhibition of caspase-3 requires an intact protein kinase B pathway. Finally, we have also found that rat placental lactogen I and II dose-dependently inhibit caspase-3 mRNA, suggesting multiple sources of PRL in the hormonal control of rat decidual regression. In summary, the results of this study have defined an important role for decidual PRL in the normal progress of pregnancy, specifically in the regression and reorganization of the decidua.

Epithelial chloride (Cl-) transport is achieved by the coordinated action of symporters such as the Na+-K+-2Cl- cotransporter (NKCC1) and chloride channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). As a secretory tissue, mammary epithelial cells are obvious candidates for such mechanisms, but Cl- transport and its hormonal regulation have been poorly delineated in mammary epithelial cells. We determined whether the mammary epithelial cell line, HC11, transports chloride and whether this was regulated by PRL, a hormone known to stimulate ion transport. HC11 cells express both CFTR and NKCC1. Exposure to PRL or PGE1 increased Cl- transport in HC11 cells. This was inhibited by the NKCC1 blocker, furosemide, and by the Cl- channel inhibitor, diphenylamine 2-carboxylate. Dose and time course of PRL action indicate that PRL had maximal effect on Cl- transport at 1 microg/ml and at 10 min of stimulation. Examination of the signaling pathways suggests that the PRL effect on Cl- transport does not involve an increase in [Ca2+]i or MAP kinase activity. RT-PCR analyses indicate that HC11 cells express mRNA for Janus kinase 1 (JAK1), JAK2, and signal transducer and activator of transcription 5 (STAT5) but not for JAK3. PRL treatment of HC11 cells increased phosphorylation of STAT5. The JAK2 inhibitor AG490 blocked phosphorylation of STAT5 and PRL-induced, but not PGE1-induced, Cl- transport. NKCC1, but not CFTR, is tyrosine phosphorylated in HC11 cells. PRL enhanced tyrosine phosphorylation of NKCC1, and this effect was attenuated by the JAK2 inhibitor AG490. These results are the first demonstrations of a role for tyrosine phosphorylation of NKCC1 and of the PRL-JAK2 cascade in the regulation of Cl- transport.

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogenbinding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), a and b. In this investigation, we analyzed the expression of decidual ERa and ERb, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERb to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERa and ERb by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERb messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERa expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERa and had no synergistic effect on ERb expression. The use of GG-AD cells, which express only ERb, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERb is expressed in addition to ERa in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERa, but concomitantly increases the expression of a functional ERb that mediates estradiol up-regulation of the decidual progesterone receptor. (Endocrinology 141: 3842‐3851, 2000)

Establishment and maintenance of pregnancy require the activity of a highly specialized maternal tissue, the decidua. It is well established that the human decidua synthesizes and releases prolactin. However, in the rat, no study has been able to demonstrate the production of prolactin by the decidua. In this report, we established for the first time using Northern blot analysis and reverse transcription-polymerase chain reaction, Western blot analysis, immunocytochemistry, and enzyme-linked immunosorbent assay, that a defined cell population located in the rat antimesometrial decidua expresses prolactin mRNA, as well as synthesizes and secretes this hormone. By reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, we cloned a full-length cDNA for rat decidua prolactin, whose sequence was identical to that of pituitary prolactin. Our results also showed that pituitary prolactin appeared to down-regulate decidual prolactin levels. Under these circumstances, inhibition of pituitary prolactin secretion led to a rise in both decidual prolactin mRNA and protein expression. Moreover, addition of exogenous prolactin to primary decidual cells in culture also caused a marked decrease in decidual prolactin mRNA expression. Finally, treatment of primary decidual cells with steroid hormones or 8-bromo-cAMP revealed a differential regulation of decidual prolactin expression from that of pituitary suggesting a tissue-specific regulation of prolactin gene expression, possibly through the use of an alternative promoter in rat decidua.

PRL is known to be a major secretory product of the human decidua. However, the physiological role of decidual PRL during the menstrual cycle and pregnancy has not been fully defined, primarily due to the lack of an appropriate nonhuman primate model for in vivo studies. Therefore, this initial study examined the expression of PRL and its receptor in the baboon uterus during the cycle and pregnancy. PRL and PRL receptor messenger ribonucleic acid expression were detected by semiquantitative RT-PCR, and protein was localized by immunocytochemistry. PRL was shown to be expressed in myometrial smooth muscle during the follicular phase by both RT-PCR and immunocytochemistry. Expression of PRL messenger ribonucleic acid and protein was first observed in the epithelial cells of the deep basal glands during the late luteal phase. With the onset of pregnancy, PRL expression increased steadily and was evident primarily in the decidual tissue. In contrast to PRL, its receptor was expressed at constant levels in both the myometrium and endometrium during the cycle. An increase in receptor expression was evident in both the decidua and placenta throughout pregnancy. In summary, these results demonstrate that the baboon uterus is a site of both PRL production and action during the cycle and pregnancy. These studies establish the baboon as a nonhuman primate model to investigate the potential roles of PRL in implantation and maintenance of pregnancy.

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor \g=b\mRNA contents were determined by semiquantitative RT\p=n-\PCR.The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor \g=b\.Furthermore, whereas IGF-I upregulated oestrogen receptor \g=b\ expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor \g=b\.Luteal cells with increased oestrogen receptor \g=b\can respond fully to oestradiol, leading to cell hypertrophy.

The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,ZnSOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1a (IL-1a), IL-b, IL-6, and tumor necrosis factor a. LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,ZnSOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed ...

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.

In this study, we investigated whether progesterone exerts an intraluteal action despite the lack of progesterone receptors (PR) in the rat corpus luteum and whether progesterone acts through the glucocorticoid receptor (GR) to enhance its own levels by down-regulating the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). We first established that the corpus luteum constitutively expresses the GR throughout pregnancy and after parturition. We also generated a temperature sensitive SV-40 transformed luteal cell line (GG-CL) that expresses the GR and 20alpha-HSD but lacks the PR. Treatment with different doses of either progesterone or dexamethasone caused a dose-related decrease in 20alpha-HSD mRNA in both cultured corpora lutea and in the luteal cell line. RU486, a PR/GR antagonist, completely blocked both the progesterone and the dexamethasone mediated inhibition of 20alpha-HSD expression in GG-CL cells. In summary, this report provides the first evidence that despite the absence of the PR in the rat corpus luteum, progesterone can act through the GR to down-regulate the expression of 20alpha-HSD, an enzyme that catabolizes progesterone and reduces progesterone secretion by the corpus luteum.

We have recently identified, characterized, and cloned a luteal microsomal 32-kDa phosphoprotein that we named PRAP (for PRL-receptor associated protein), and we have demonstrated that PRAP binds to the intracellular domain of the short but not the long form of the PRL receptor. In this study, we used PRAP cDNA to examine the tissue specificity, the developmental expression, and the hormonal regulation of PRAP gene expression. Northern blot analysis revealed that in the corpus luteum, PRAP cDNA hybridized to multiple transcripts (5.5 kb, 4.3 kb, and 1.8 kb), with the smallest transcript (1.8 kb) corresponding to the size of the cDNA clone. However, none of these transcripts were detected in any other tissues examined. PRAP appears to be tightly regulated by steroids and PRL. When pregnant rats were treated with aminoglutethimide, a steroid synthesis inhibitor, all three PRAP transcripts became barely detectable. Similar results were obtained when all luteotropic support was removed by hypophysectomy and hysterectomy. Estradiol up-regulated PRAP expression and, more specifically, the two lower transcripts. PRL had no stimulatory effect on PRAP messenger RNA (mRNA) expression but caused a substantial increase in the level of PRAP protein when administered to hypophysectomized pregnant rat, suggesting that PRL may stabilize this protein. Similar dissociation between levels of mRNA and protein were observed during luteal development. Although both PRAP mRNA and protein were barely detectable in early pregnancy, their expression increased abruptly from midpregnancy; however, whereas levels of PRAP mRNA declined from day 18, those of the protein remained elevated until parturition. In summary, results of this study have defined the tissue specificity and developmental expression of PRAP mRNA during pregnancy. The data have also revealed that the gene expression of this protein is up-regulated by estradiol, suggesting a pivotal role for PRAP in the synergistic action of estradiol and PRL on the function of the rat corpus luteum.

The enzyme, rat ovarian 20α-hydroxysteroid dehydrogenase (20αHSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20α-hydroxypregn-4-ene-3-one (20α-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20αHSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20αHSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20αHSD expressed as glutathione-S-transferase-20αHSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatogr...

Decidualization of the endometrial stroma in the rat gives rise to two different cell populations, located either mesometrially or antimesometrially in the uterus. We have previously shown that the rat decidua is the site of production and action of a PRL-like hormone. In this investigation we examined, using reverse transcription-PCR, whether and which type of PRL receptor (PRL-R) messenger RNA (mRNA) is expressed in the decidua, whether the receptor is confined only to one cell population, and whether the PRL-R expression is regulated by decidua-derived factors. The results indicate that the uterus of pseudopregnant rats does not express the PRL-R and that decidualization does not trigger a rapid appearance of PRL-R mRNA. It is only 3 days after the induction of decidualization that the long form of the PRL-R was first expressed. Thereafter, mRNAs for both the short (PRL-RS) and the long (PRL-RL) form became detectable in both antimesometrial and mesometrial decidua, although PRL-RL mRNA was much more abundant than PRL-RS. As development proceeded, PRL-R mRNA decreased and disappeared specifically from the antimesometrial decidua, whereas the mesometrial decidua continued to express this receptor mRNA. Concomitant with down-regulation of the PRL-R in the antimesometrial tissue was a rather abrupt expression of activin A. In contrast, the mesometrial tissue that maintained high levels of PRL-R mRNA expressed little activin A, but produced an activin-binding protein, alpha(2)-macroglobulin (alpha(2)MG). To determine whether activin A and alpha(2)MG regulate PRL-R expression, antimesometrial and mesometrial cells were separated by elutriation and maintained in culture in the presence or absence of activin A, alpha(2)MG, or follistatin. Just after cell separation, both cell populations expressed PRL-R, but not activin A. Within 6 h, activin A mRNA and protein became highly expressed in the mesometrial cells, whereas PRL-RL mRNA became undetectable. In contrast, activin A mRNA was at very low levels in the antimesometrial cells, and no activin A protein could be detected in the medium for at least 12 h. In these cells PRL-RL mRNA remained elevated. Addition of activin A to antimesometrial cells caused a marked down-regulation of PRL-RL mRNA expression, whereas addition of alpha(2)MG and follistatin to mesometrial cells prevented the disappearance of PRL-R. In summary, the results of this investigation 1) indicate that decidualization of the endometrial stroma induces the appearance of both forms of the PRL-R mRNAs; 2) show differential expression of the PRL-R mRNA in the two-cell population forming the decidua; 3) establish that this differential expression is due to two key decidual molecules, activin A and alpha(2) macroglobulin; and 4) demonstrate that activin A can cause the decidual cells to lose the PRL-R and that the disappearance of the decidual PRL-R can be prevented by addition to the culture of two activin binding proteins, follistatin and alpha(2) MG.

The decidual tissue of the pregnant rat is formed primarily by two markedly different decidual cell populations located in the mesometrial and antimesometrial sites of the uterus. The antimesometrial decidua functions as an endocrine organ, secreting PRL-like hormones and expressing activin and follistatin. In contrast, the mesometrial decidua has no apparent endocrine activity, but secretes abundant alpha 2-macroglobulin. To determine whether the profound difference in morphology and gene expression is inherent to each cell population or whether it results from the position of these cells in the uterus and their subjection to regionally restricted information, we isolated the giant antimesometrial decidual cells from the small mesometrial decidual cells by elutriation. We examined, using Northern and reverse transcription-polymerase chain reaction analysis, the expression of PRL-related protein (DPRP), follistatin, activin, and alpha 2-macroglobulin in the two cell populations just after cell separation and after primary culture. We also examined, using coculture, the possible cross-talk between the two cell populations. In culture, the cells maintained the morphological appearance seen in the decidua; the mesometrial cells remained less differentiated, whereas the antimesometrial cells formed giant cells. In addition, the antimesometrial cells, which have been shown previously to be the only site of DPRP messenger RNA (mRNA) expression, remained, after several days of culture, the only cells able to express this gene. The coculture experiment suggested that the absence of DPRP mRNA in the mesometrial cells is not due to inhibitory signals from the antimesometrial cells, but, rather, to the inherent inability of the mesometrial decidual cells to express the DPRP gene. The cellular expression pattern of follistatin was also not changed by cell separation. However, in sharp contrast to that of DPRP and follistatin, the cell-specific expression of activin-beta A and alpha 2-macroglobulin was markedly affected by cell separation. A remarkable switch in activin-beta A cell expression was observed when mesometrial cells were separated from antimesometrial cells and maintained in culture. Activin-beta A mRNA, which was barely detectable in these cells in situ, became expressed in higher levels than in antimesometrial cells. The isolated antimesometrial cells also acquired the ability to express the alpha 2-macroglobulin gene, whose expression is highly restricted to the mesometrial cells in vivo. No down-regulation of antimesometrial alpha 2-macroglobulin mRNA could be induced by mesometrial cells in a coculture experiment. However, alpha 2-macroglobulin mRNA levels in mesometrial cells were clearly up-regulated by antimesometrial cell signal(s).(ABSTRACT TRUNCATED AT 400 WORDS)

Prolactin (PRL), a pleiotropic hormone essential for maintenance of corpus luteum (CL) function and pregnancy, transduces its signal through two types of receptors, a short form (PRLR-S) and a long form (PRLR-L). Both types of receptors are expressed in the CL, yet their individual roles are not well defined. We have shown previously that female transgenic mice expressing only PRLR-S display total infertility characterized by defective follicular development and early degeneration of CL, suggesting that expression of PRLR-L is a prerequisite for normal follicular development and maintenance of CL. To determine whether PRLR-L alone is the sole receptor required to maintain normal CL formation, differentiation, and progesterone secretion, we generated two transgenic mice which express only PRLR-L, either ubiquitously (Tg-RL) or in a CL-specific manner (CL-RL). To generate CL-specific expression, we used the HSD17B7 promoter. We found both transgenic mice models cycled normally, displayed no apparent defect in follicular development, and had normal ovulation rates. The STAT5 signaling pathway, considered essential for luteinization and progesterone production, was activated by PRL in both transgenic mice models. However, soon after mating, Tg-RL and CL-RL mice showed early regression of CL, lack of progesterone production, and implantation failure that rendered them totally infertile. Embryo transfer studies demonstrated no embryo abnormalities, and supplementation with progesterone rescued implantation failure in these mice. Close observation revealed lack of luteinization and reduced expression of proteins involved in progesterone biosynthesis despite normal levels of LHCGR (LH-R), ESR1 (ER-alpha), CEBPB (C/EBP-beta) and CDKN1B (p27), proteins essential for luteinization. However, we found VEGFA, a key regulator of angiogenesis and vascularization, to be dramatically reduced in both Tg-RL and CL-RL mice. We also found collagen IV, a marker for the basal lamina of endothelial cells, aberrantly expressed and a discordant organization of endothelial cells in CL. Although luteinization did not occur in vivo, granulosa cells isolated from these mice luteinized in culture. Taken together, these results suggest that a vascularization defect in the CL may be responsible for lack of luteinization, progesterone production, and infertility in mice expressing only PRLR-L. This investigation therefore demonstrates that in contrast to earlier presumptions that PRLR-L alone is able to support normal CL formation and function, both isoforms of the PRL receptor are required in the CL for normal female fertility.

A M(r) 100,000 phosphoprotein in the corpus luteum was identified as elongation factor 2 (EF-2). Since prolactin (PRL) is necessary for optimal luteal development and protein synthesis, we determined whether this hormone affects the content and/or phosphorylation of EF-2 in the corpus luteum. PRL treatment enhanced the Ca2+/calmodulin (CaM)-dependent phosphorylation of endogenous EF-2 in luteal cytoplasmic extracts. Immunoblot analysis revealed that PRL had no effect on EF-2 levels, but examination of luteal EF-2 by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis showed that PRL increased the relative amount of the most basic dephosphorylated forms of EF-2. This suggests that PRL induces net dephosphorylation of the protein in vivo. Since EF-2 phosphorylation is regulated by both Ca2+/CaM-dependent kinase III (CaM kinase III) and protein phosphatase 2A, we examined the effect of PRL on both enzymes. Paradoxically, PRL enhanced the in vitro activity of CaM kinase III, possibly reflecting increased kinase levels, but had no effect on phosphatase activity. These results suggest that PRL maintains luteal EF-2 in a relatively dephosphorylated state in vivo by limiting the availability of Ca2+ and/or CaM to CaM kinase III. These data provide strong evidence for a role of the EF-2/CaM kinase III system in PRL action in the corpus luteum.

Our laboratory has previously cloned and purified an ovarian protein found to be a novel 17β-hydroxysteroid dehydrogenase type 7 enzyme (HSD17B7) (formerly prolactin receptor-associated protein) that converts the weak estrogen, estrone, to the highly potent estradiol. The regulation of this enzyme has not yet been explored. In this report, we show high expression of HSD17B7 in human ductal carcinoma and breast cancer cell lines and present evidence for a strong up-regulation of this enzyme by estradiol at the level of mRNA, protein expression, and promoter activity in MCF-7 cells. The effect of estradiol is mediated by estrogen receptor (ER)α, whereas ERβ prevents this stimulation. ER antagonists, ICI 182,780 and 4-hydroxytamoxifen, prevent estradiol-induced stimulation of the endogenously expressed HSD17B7, suggesting that these inhibitors not only block estradiol action but also its production. We have identified a -185-bp region of the hsd17b7 promoter that is highly conserved among rat, mouse, and human and confers regulation by estradiol in MCF-7 cells. This region is devoid of a classical estradiol-response element but contains a nuclear factor 1 (NF1) site that is essential for estradiol action. We found that estradiol stimulates the recruitment and DNA binding of NF1 to this region of the hsd17b7 promoter. Furthermore, knockdown of NF1 family members, NF1B, NF1A, and NF1X, completely prevents induction of this gene by estradiol. In summary, our findings demonstrate that estradiol stimulates HSD17B7 transcriptional activity in breast cancer cells through a novel mechanism requiring NF1 and strongly suggest a positive feedback mechanism to increase local estradiol synthesis causing growth of estrogen-dependent breast cancers.

Prolactin (PRL) is essential for normal reproduction and signals through two types of receptors, the short (PRL-RS) and long (PRL-RL) form. We have previously shown that transgenic mice expressing only PRL-RS (PRLR(-/-)RS) display abnormal follicular development and premature ovarian failure. Here, we report that MAPK, essential for normal follicular development, is critically inhibited by PRL in reproductive tissues of PRLR(-/-)RS mice. Consequently, the phosphorylation of MAPK downstream targets are also markedly inhibited by PRL without affecting immediate upstream kinases, suggesting involvement of MAPK specific phosphatase(s) in this inhibition. Similar results are obtained in a PRL-responsive ovary-derived cell line (GG-CL) that expresses only PRL-RS. However, we found the expression/activation of several known MAPK phosphatases not to be affected by PRL, suggesting a role of unidentified phosphatase(s). We detected a 27-kDa protein that binds to the intracellular domain of PRL-RS and identified it as dual specific phosphatase DUPD1. PRL does not induce expression of DUDP1 but represses its phosphorylation on Thr-155. We also show a physical association of this phosphatase with ERK1/2 and p38 MAPK. Using an in vitro phosphatase assay and overexpression studies, we established that DUPD1 is a MAPK phosphatase. Dual specific phosphatase inhibitors as well as siRNA to DUPD1, completely prevent PRL-mediated MAPK inhibition in ovarian cells. Our results strongly suggest that deactivation of MAPK by PRL/PRL-RS contributes to the severe ovarian defect in PRLR(-/-)RS mice and demonstrate the novel association of PRL-RS with DUPD1 and a role for this phosphatase in MAPK deactivation.

Corpora lutea of rats, like those of many other species, contain two sub-populations of luteal cells. In this report we sought to determine whether the luteinizing hormone (LH)- and β-adrenergic cAMP signal transduction pathways known to be present in rat corpora lutea were segregated into separate luteal cell types. Results showed that large rat luteal cells, obtained on day 3 of pregnancy, exhibited elevated LH- and most notably epinephrine-stimulated adenylyl cyclase activities but equivalent cAMP-dependent catalytic protein kinase and total regulatory subunit cAMP binding activities compared to small luteal cells. Progesterone production by the large cell was greater than that by the small cell but both cells were equally sensitive to stimulation of progesterone by LH. However, neither the large nor the small rat luteal cell produced significant progesterone in response to epinephrine despite a marked epinephrine-stimulated adenylyl cyclase in both cell populations. The LH-stimulated progesterone synthetic response of the two sub-populations of rat luteal cells is more similar to that of the developing monkey corpus luteum and contrasts sharply with that of ruminants.

Prolactin (PRL) affects the development and function of the reproductive system by binding to two types of receptors, which differ by the size of their intracellular domain in rodents. Whereas the signaling pathway through the long form of the receptor (PRL-RL) is well characterized, signaling through the short form (PRL-RS) remains obscure. In this investigation, we examined transcription factors regulated by PRL in the ovary and decidua of mice expressing only PRL-RS in a PRL receptor null background. These mice provide a powerful in vivo model to study the selective signaling mechanism of PRL through PRL-RS independent of PRL-RL. We also examined the regulation of transcription factors in ovarian and uterine cell lines stably transfected with PRL-RS or PRL-RL. We focused our investigation on transcription factors similarly regulated in both these tissues and clearly established that signaling through PRL-RS does not activate the JaK/Stat in vivo but leads to severe down-regulation of Sp1 expression, DNA binding activity, and nuclear localization, events that appear to involve the calmodulin-dependent protein kinase pathway. Our in vivo and in culture data demonstrate that the PRL-RS activates a signaling pathway distinct from that of the PRL-RL.

International audience; Bachelot A, Beaufaron J, Servel N, Kedzia C, Monget P, Kelly PA, Gibori G, Binart N. Prolactin independent rescue of mouse corpus luteum life span: identification of prolactin and luteinizing hormone target genes. Am J Physiol Endocrinol Metab 297: E676-E684, 2009. First published June 16, 2009; doi: 10.1152/ajpendo.91020.2008.-The corpus luteum (CL) plays a central role in the maintenance of pregnancy in rodents, mainly by secreting progesterone. Female mice lacking prolactin (PRL) receptor (R) are sterile due to a failure of embryo implantation, which is a consequence of decreased luteinizing hormone (LH) receptor expression in the CL and inadequate levels of progesterone. We attempted to treat PRLR(-/-) females with human chorionic gonadotropin (hCG) and showed a de novo expression of LHR mRNA in the corpora lutea. Binding analysis confirmed that the LHR in hCG-treated PRLR(-/-) animals was functional. This was accompanied with increased expression of steroidogenic enzymes involved in progesterone synthesis. Despite these effects, no embryo implantation was observed because of high expression of 20 alpha-hydroxy-steroid dehydrogenase. To better appreciate the molecular mechanisms underlying maintenance of the CL, a series of mRNA expression-profiling experiments was performed on isolated corpora lutea of PRLR(-/-) and hCG-treated PRLR(-/-) mice. This approach revealed several novel candidate genes with potentially pivotal roles in ovarian function, among them, p27, VE-cadherin, Pten, and sFRP-4, a member of the Wnt/frizzled family. This study showed the differential role of PRL and LH in CL function and identified new targets of these hormones in luteal cells.

Although it is well established that PRL is essential for luteal cell steroidogenesis and growth in the rat, its exact mechanism of action remains unknown. Whereas PRL stimulates growth and induces the content of specific proteins in several target tissues, its effect on proteins in the corpus luteum has not been examined. To determine whether PRL affects the synthesis of specific luteal protein(s), corpora lutea were obtained from rats hypophysectomized on day 3 of pregnancy and then treated with or without PRL (125 micrograms x 2/day) for 4 days. In this rat model, corpora lutea are exquisitely responsive to PRL and produce high levels of progesterone in response to PRL stimulation. Analysis of cytosolic proteins on one- and two-dimensional gel electrophoresis revealed a dramatic inhibitory effect of PRL on a 37,000 mol wt (MW) protein. This PRL-regulated protein (PRP) resolved into five separate isoelectric variants (pIs 5.5, 5.6, 6.15, 6.6, and 6.85). Since differences in phosphorylation state can alter the isoelectric point of proteins, we examined the possibility that the 37,000 MW PRP is a substrate for phosphorylation. Luteal homogenates were incubated with [32P]ATP in the presence or absence of Ca2+, Ca2+/calmodulin, Ca2+/phospholipid, or cAMP. Phosphorylation of PRP was not affected by PRL treatment or the addition of specific cofactors. The least abundant isoelectric species (pI 6.85) was identified by Western blot analysis as annexin I, a known regulator of phospholipase A2 and prostaglandin synthesis. To determine whether the other four isoelectric species represent variants of the same protein, we developed a polyclonal antibody to the 6.15 isoelectric species which is remarkably regulated by PRL. In the absence of PRL, the 6.15 isoelectric species (37K) is the major protein of the PRP band. Conversely, PRL treatment resulted in the virtual disappearance of this luteal protein. The antibody recognized the 37K alone, indicating that the other 37,000 MW isoelectric species were distinct proteins. To examine the time course of PRL action on the expression of the 37K, luteal cells from day 3 pregnant rats were cultured with different doses of PRL from 6 h to 5 days. Western blot analysis of luteal cellular proteins indicated that PRL caused a decrease in the expression of the 37K within 6 h of treatment. Although it is well known that estradiol together with PRL is required for optimal growth of the rat corpus luteum, estradiol alone had no inhibitory action on the 37K nor did it affect the inhibitory action of PRL.(ABSTRACT TRUNCATED AT 400 WORDS)

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.

Estradiol assumes a major role in the regulation of growth, vascularization, and progesterone synthesis in the midpregnant rat corpus luteum. To explore whether molecular events triggered by estradiol could be mediated, at least in part, by protein phosphorylation, we investigated whether estradiol treatment in vivo affects endogenous luteal protein phosphorylation systems detectable in vitro. Luteal nuclear, mitochondrial, and microsomal fractions were obtained by differential centrifugation from rats hypophysectomized and hysterectomized on day 12 of pregnancy and treated with or without estradiol for 72 h. Using [gamma-32P]ATP as phosphate donor, proteins were phosphorylated in the presence or absence of either calcium (Ca), Ca plus calmodulin, or Ca plus phospholipid. Phosphoproteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. The Coomassie blue stained proteins and phosphoprotein profiles were markedly different in the various fractions. Estradiol treatment in vivo caused an increase in the basal endogenous phosphorylation of several proteins in vitro. It also substantially enhanced the protein kinase C (PKC) and Ca-calmodulin kinase-dependent phosphorylation of selected proteins in subcellular fractions. The Ca-calmodulin kinase catalyzed phosphorylation of microsomal 56 and 60 kilodalton (kDa) proteins was remarkably increased by estradiol. Proteins (56 and 60 kDa) were also phosphorylated when Ca-calmodulin was added to the nuclear fraction, however, this phosphorylation did not appear to be affected by estradiol treatment. A major PKC substrate in the nuclear fraction was an 80 kDa protein whose phosphorylation was increased remarkably by estradiol treatment. In the mitochondrial fraction the most striking effect of estradiol was a marked increase in PKC-mediated phosphate transfer into a 76 kDa substrate. To determine whether estradiol action on protein phosphorylation was related to its tropic effect in the corpus luteum, the hormone was administered to day 10 hypophysectomized and hysterectomized pregnant rats. In this rat model, where estradiol has no stimulatory effect on either luteal steroidogenesis or growth, neither endogenous nor kinase-mediated phosphorylation was affected by this steroid. In summary, the present investigation has revealed that in vivo treatment with estradiol affects the PKC and the Ca-calmodulin dependent in vitro phosphorylation of selected proteins localized in different subcellular compartments and further suggests that phosphorylation systems are potential control points for estradiol regulation of rat corpus luteum function.

Prolactin (PRL) is a hormone with over 300 biological activities. Although the signaling pathway downstream of the long form of its receptor (RL) has been well characterized, little is known about PRL actions upon activation of the short form (RS). Here, we show that mice expressing only RS exhibit an ovarian phenotype of accelerated follicular recruitment followed by massive follicular death leading to premature ovarian failure. Consequently, RS-expressing ovaries of young adults are depleted of functional follicles and formed mostly by interstitium. We also show that activation of RS represses the expression of the transcription factor Forkhead box O3 (FOXO3) and that of the enzyme galactose-1-phosphate uridyltransferase (Galt), two proteins known to be essential for normal follicular development. Our finding that FOXO3 regulates the expression of Galt and enhances its transcriptional activity indicates that it is the repression of FOXO3 by PRL acting through RS that prevents Galt expression in the ovary and causes follicular death. Coexpression of RL with RS prevents PRL inhibition of Galt, and the ovarian defect is no longer seen in RS transgenic mice that coexpress RL, suggesting that RL prevents RS-induced ovarian impairment. In summary, we show that PRL signals through RS and causes, in the absence of RL, a severe ovarian pathology by repressing the expression of FOXO3 and that of its target gene Galt. We also provide evidence of a link between the premature ovarian failure seen in mice expressing RS and in mice with FOXO3 gene deletion as well as in human with Galt mutation.

Interleukin 11 receptor alpha (Il11ra) null mice are infertile due to defective decidualization and abnormal trophoblast invasion. We have previously shown in these mice that downregulation of decidual proteinase inhibitors plays a role in uncontrolled trophoblast invasion. However, the decidua is abnormally smaller in pseudopregnant Il11ra null mice, where trophoblast invasion is not a factor. Here, we examined whether defective decidualization is due to dysregulation of key molecules involved in decidual cell growth and differentiation. We found a dramatic downregulation of cyclin D3 in Il11ra null mice. We also found that IL11 robustly stimulates the expression of cyclin D3 in cell culture. CDK4 and CDK6, known partners of cyclin D3, are not affected. Immunolocalization studies show absence of cyclin D3 in the mesometrial site and absence of differentiated polyploid cells in the antimesometrial site of Il11ra null mice. We also examined the expression of cell differentiation factors CDKN1A (p21) and CDKN1B (p27), and found that in both in vivo and cell culture the expression of CDKN1A (p21) but not CDKN1B (p27) is under the control of IL11. Another clear target of IL11 in the decidua is BIRC5 (Survivin), whose expression is repressed in the decidua of Il11ra null mice and stimulated by IL11 in cell culture. Taken together, these results provide, at least in part, an explanation for the defective small decidua of mice lacking the Il11ra gene, and reveal for the first time that cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) are targets of IL11 in the decidua.

Although the main role of prolactin (PRL) in pregnant rodents is to sustain progesterone production by the corpus luteum, progesterone treatment of PRL or PRL receptor (PRL-R) null mice is unable to prevent fetal loss. We have previously shown that the rat decidua is a site of PRL production and action. In this report, we examined the hypothesis, using PRL null mice and rat decidual cell culture, that the absence of this hormone leads to the expression in the decidua of genes detrimental to pregnancy. The results show that decidual growth is normal in PRL null mice treated with PRL, progesterone, or their combination. However, the decidua of mice treated with progesterone starts expressing IL-6 and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), two proteins absent from the decidua of wild-type mice and involved, respectively, in inflammation and progesterone catabolism. The expression of both IL-6 and 20alpha-HSD is prevented by PRL treatment. Our results further suggest that PRL inhibition of 20alpha-HSD expression is at the level of transcription and that decidual PRL (dPRL) inhibits 20alpha-HSD promoter activity. Inhibitors of Janus kinase 2 (Jak2) but not other kinases prevent dPRL down-regulation of the 20alpha-HSD promoter. Furthermore, cotransfection of the 20alpha-HSD promoter with expression vectors of constitutively active PRL-R, Jak2, or signal transducer and activator of transcription 5b (Stat5b) leads to substantial inhibition of promoter activity. Taken together, our investigation provides an explanation for the inability of progesterone to sustain pregnancy in PRL null mice and suggests that dPRL plays an important role in pregnancy by repressing the expression of IL-6 and 20alpha-HSD in the decidua. The study also demonstrates that PRL signals through the Jak2/Stat5 pathway to down-regulate 20alpha-HSD expression in the decidua.

The corpus luteum (CL) is one of the few endocrine glands that forms from the remains of another organ and whose function and survival are limited in scope and time. The CL is the site of rapid remodeling, growth, differentiation, and death of cells originating from granulosa, theca, capillaries, and fibroblasts. The apparent raison d’etre of the CL is the production of progesterone, and all the structural and functional features of this gland are geared toward this end. Because of its unique importance for successful pregnancies, the mammals have evolved a complex series of checks and balances that maintains progesterone at appropriate levels throughout gestation. The formation, maintenance, regression, and steroidogenesis of the CL are among the most significant and closely regulated events in mammalian reproduction. During pregnancy, the fate of the CL depends on the interplay of ovarian, pituitary, and placental regulators. At the end of its life span, the CL undergoes a process of regression leading to its disappearance from the ovary and allowing the initiation of a new cycle. The generation of transgenic, knockout and knockin mice and the development of innovative technologies have revealed a novel role of several molecules in the reprogramming of granulosa cells into luteal cells and in the hormonal and molecular control of the function and demise of the CL. The current review highlights our knowledge on these key molecular events in rodents.

IL-11 expressed by endometrial stromal cells is crucial for normal pregnancy. IL-11 receptor alpha (IL-11Ralpha) null mice are infertile due to abnormal development of the placenta. In these mice, the mesometrial decidual tissue, which is the site of trophoblast invasion, thins and disappears at mid-pregnancy. Degeneration of the decidua is accompanied by uncontrolled trophoblast invasion. In this report, we show, using IL-11Ralpha null mice, that a defect in IL-11 signaling in the decidua leads to severe down-regulation of alpha(2)-macroglobulin (alpha(2)-MG), a metalloproteinase inhibitor crucial for limiting trophoblast invasion. We also present evidence, using uterine stromal cells that decidualize in culture, that IL-11 robustly stimulates the endogenous alpha(2)-MG expression and enhances alpha(2)-MG promoter activity. Serial 5' deletion and internal deletion of the promoter reveal two important signal transducer and activator of transcription (Stat) binding sites. Mutation of either one of these motifs decreases IL-11 stimulation, whereas double mutation prevents IL-11 action. We also found that IL-11 activates Janus kinase 2 (Jak2) and induces rapid phosphorylation, nuclear translocation, and promoter binding activity of Stat3 in decidual cells, whereas Jak1, Tyk2, and Stat5 activities are not affected. In addition, Jak2 inhibitor totally prevents alpha(2)-MG expression in decidual cells. Taken together, results of this investigation provide, at least in part, an explanation for the overinvasiveness of the trophoblast in IL-11Ralpha null mice and reveal, for the first time, that IL-11 signals through the Jak2/Stat3 pathway in decidual cells to stimulate the expression of alpha(2)-MG, a protease inhibitor essential for normal placentation in pregnancy.

Pregnancy requires profound reorganization of the different tissues forming the uterus. Growth and differentiation of the uterine endometrial cells give rise to the decidual tissue, a transitory organ, which plays a key role in fetal survival. In this chapter, we describe a technique for the dispersion and the separation of the two different decidual cell subpopulations with high yield and viability. We also detail a cell culture method, which allows the maintenance of the function and life span of these highly purified decidual cells when cultured either separately or in a co-culture system.

Pregnancy requires profound reorganization of the different tissues forming the uterus. Growth and differentiation of the uterine endometrial cells give rise to the decidual tissue, a transitory organ, which plays a key role in fetal survival. In this chapter, we describe a technique for the dispersion and the separation of the two different decidual cell subpopulations with high yield and viability. We also detail a cell culture method, which allows the maintenance of the function and life span of these highly purified decidual cells when cultured either separately or in a co-culture system.

In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2alpha) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2alpha in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2alpha sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2alpha are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2alpha. The results strongly suggest that the abortificient role of elevated levels of PGF2alpha after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.

In the last 10 years many exciting discoveries have been made regarding the development, function, and regression of the corpus luteum (CL) of pregnancy. Techniques such as microarray analysis have enhanced the speed with which new genes of interest are identified, and trans-genic technology has revealed the frequently surprising functions of known and novel factors. The field of angiogenesis has blossomed, and the remarkably intense and rapid angiogenesis that occurs in the CL has made it an organ of choice for studies of this process. The cloning of steroid ogenic acute regulatory protein (StAR) has changed the way we think about steroidogenesis in the CL. Although StAR is no longer a novelty, the mechanism of its action continues to be a controversial topic and an exciting field of research. Similarly, the cloning of 17β-hydrox-ysteroid dehydrogenase type 7 has filled in one of the missing pieces of the steroidogenic puzzle in the CL of the rodent; however, more questions remain unanswered. A luteotropic role for progesterone, proposed long ago, has now been supported by numerous studies, and our knowledge of the mechanisms by which this and other hormones act on the CL continues to expand. In this chapter, we summarize the current body of knowledge, in particular the new information of the last 10 years of research, to present a complete picture of the CL of pregnancy. This fascinating organ, with its rapid formation and destruction and its complex regulation, continues to be a fertile field for research.

OBJECTIVES: The uterus responds to an implanting blastocyst by undergoing extensive tIssue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. DESIGN: In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39 degrees C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33 degrees C. METHODS: We performed Northern blot analysis for bax, bcl-x(L) and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. RESULTS: We found an increase in the expression of bax when GG-AD cells were grown at 39 degrees C. We also showed apoptosis with Annexin V staining at 39 degrees C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. CONCLUSIONS: These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression.