Prolactin activation of the long form of its cognate receptor causes increased visceral fat and obesity in males as shown in transgenic mice expressing only this receptor subtype.

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.

Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors.

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.

Involvement of SOCS-1, the suppressor of cytokine signaling, in the prevention of prolactin-responsive gene expression in decidual cells.

The cells forming the rat decidua produce PRL and PRL-related proteins and express both the long and short forms of the PRL receptor. Yet, only a defined subpopulation, the mesometrial cells, express the PRL-dependent alpha2-macroglobulin gene. This gene is silenced in vivo in the antimesometrial cells and in the GG-AD cell line, derived from antimesometrial cells. To examine whether the lack of alpha2-macroglobulin expression is due to defective components in the PRL signaling pathway, we compared the relative expression of Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 a and b (Stat5 a and b), suppressor of cytokine signaling-1 (SOCS-1), and the tyrosine phosphatase SHP-2 mRNA in mesometrial and antimesometrial decidua on days 12 and 13 of pseudopregnancy, the time of maximal alpha2-macroglobulin expression. We found no significant differences in the relative expression of either Jak2, Stat5 (a and b), or SHP-2 in the two cell populations. However, we discovered a profound difference in the expression of SOCS-1, an inhibitor of the Jak/Stat pathway. This gene was highly expressed in the antimesometrial cells and in the GG-AD cells, which do not produce alpha2-macroglobulin. Immunoprecipitation experiments with GG-AD cells revealed that although Jak2 and Stat5 coprecipitate in response to PRL stimulation, no phosphorylation of Jak2 and Stat5 could be observed. To examine whether SOCS-1 plays a role in silencing the alpha2-macroglobulin gene, we cultured GG-AD cells in the presence of either a SOCS-1 antisense oligonucleotide or an irrelevant oligonucleotide for 4, 12, and 28 h. Cells were also treated with PRL. Within 4 h of SOCS-1 antisense treatment, alpha2-macroglobulin mRNA expression was initiated. After 28 h, only cells treated with PRL and SOCS-1 antisense oligonucleotide retained the ability to express the alpha2-macroglobulin gene. In summary, results of this study reveal that constitutive expression of SOCS-1 can prevent PRL signaling and that the lack of PRL-induced expression of alpha2-macroglobulin in a defined decidual cell population is largely due to SOCS-1 expression in these cells.

PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase.

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERalpha expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERalpha expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERalpha mRNA levels, suggesting that PRL signaling to the ERalpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERalpha promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERalpha promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERalpha expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.

Janus kinase 2 (JAK2) regulates prolactin-mediated chloride transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na+-K+-2Cl- cotransporter.

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.

Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription.

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.

Isolation, culture, and characterization of the two cell subpopulations forming the rat decidua: differential gene expression for activin, follistatin, and decidual prolactin-related protein.

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)

Intraovarian localization of luteinizing hormone/human chorionic gonadotropin stimulation of testosterone and estradiol synthesis in the pregnant rat.

The objective of this investigation was to determine whether LH acts directly on luteal cells to stimulate testosterone and estradiol synthesis or whether it stimulates follicular and/or interstitial production of androgen and thus provides androgen substrate for luteal cell production of estradiol. Pregnant rats were injected with 1.5 IU human CG (hCG) twice daily sc between days 12 and 14. On day 14, blood was obtained from both the jugular and ovarian vein. Corpora lutea (CL), follicles, and interstitium were isolated and incubated at 37 C for 4 h. Estradiol, testosterone, and progesterone levels were measured in the peripheral circulation, ovarian vein plasma, tissues, and medium. After hCG treatment, no ovulation occurred, rats remained pregnant, and progesterone levels in the serum and in the ovarian vein plasma remained unchanged. In contrast, estradiol and testosterone levels in the ovarian vein increased from 0.7 +/- 0.1 and 0.6 +/- 3.4 ng/ml, respectively, in vehicle-treated rats to 12.5 +/- 3.5 and 4.8 +/- 1.4 ng/ml in hCG-treated animals. In vivo treatment with hCG dramatically increased the in vitro capacity of luteal cells to synthesize de novo both testosterone and estradiol but had no stimulatory effect on progesterone synthesis. Testosterone synthesis by CL increased from 21 +/- 4 to 255 +/- 114 pg/CL whereas estradiol synthesis rose from 30 +/- 7 to 4481 +/- 641 pg/CL. hCG also increased follicular synthesis of both estradiol and testosterone. The interstitium responded to the hCG challenge with a 50-fold increase in testosterone synthesis but with no change in estradiol production. To determine whether hCG rapidly stimulates ovarian production of testosterone, both in vivo and in vitro approaches were used. In the in vitro experiments, CL follicles, or interstitium obtained from day 14 pregnant rats were incubated with or without 3 IU hCG. In the in vivo experiments, day 14 pregnant rats were injected with 3 IU hCG iv and were bled from the jugular and ovarian veins 0.5 and 2 h later. No increase in testosterone and estradiol production was observed after a short challenge with hCG. In summary, this study demonstrates that in the pregnant rat a sustained increase in serum hCG activity stimulates ovarian secretion of both testosterone and estradiol. We conclude that LH can act to induce the synthesis and/or activation of enzyme(s) involved in the conversion of progesterone to androgen in luteal tissue. The results also demonstrate that LH stimulates the synthesis of androgens but not of estradiol in interstitial tissue and confirms the finding that LH stimulates follicular production of both testosterone and estradiol.

The luteotrophic action of decidual tissue: the stimulating effect of decidualization on the serum progesterone level of pseudopregnant rats.

On day 5 (4th diestrous day), pseudopregnant rats were arranged in pairs, one member of which was subjected to hysterectomy (H-ect) and the other to uterine scratching to induce formation of decidual tissue (DT). Blood samples were collected from each pair of rats immediately before and at various intervals after operation until day 13, and with very few exceptions were assayed for progesterone by CPB in the same assay. From 21 such pairs, the following findings emerged: During the period from day 7-day 11 inclusive, a DT-bearing rat's serum progesterone level was higher than that of its H-ect paired control in 79% of the paired comparisons; the mean serum progesterone level of the DT-bearing rats was higher than that of the H-ect rats on each day during this period; and the progesterone level group mean for all the DT-bearing rats during this period was at least 25% (P less than 0.05) higher than that of the H-ect rats. On days 6, 12, and 13, however, no significant differences were found between the 2 groups by any basis of comparison. Ordinary pseudopregnant rats (subjected only to 1aparotomy on day 5) and rats subjected to uterine scratching which failed to form DT had progesterone levels below or similar to, respectively, those of the H-ect members in the pairs. The findings were interpreted as evidence that DT increases the rate of progesteron secretion.

Prolactin action on luteal protein expression in the corpus luteum.

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)

Role of intraluteal estrogen in the regulation of the rat corpus luteum during pregnancy.

The pronounced aromatizing ability of the rat corpus luteum and the ability of estradiol to maintain luteal function. To examine this hypothesis, rats were treated with either estradiol (100 microgram/day), high or low levels of testosterone via Silastic capsules (20 cm or 1 cm in length), or dihydrotestosterone (20-cm capsule) after hypophysectomy and hysterectomy on day 12 of pregnancy. Hypophysectomy and hysterectomy caused serum progesterone and androgen levels, estradiol concentrations in the corpora lutea, and the content of estradiol receptor in luteal cell nuclei to decrease significantly, and caused the cessation of luteal growth. The daily administration of 100 microgram estradiol or of high levels of testosterone via the 20-cm Silastic capsule increased the estradiol concentration in the corpora lutea dramatically, maintained serum progesterone from day 12 through day 15 at concentrations similar to those in pregnant, sham-operated animals, and increased the nuclear content of estradiol receptor in the corpora lutea. Treatment with the small testosterone capsule maintained the serum androgen and progesterone levels, estradiol concentrations in the corpora lutea, and luteal growth at levels observed in pregnant, sham-operated animals. Treatment with the 20-cm dihydrotestosterone capsule did not sustain progesterone secretion and luteal growth. These results suggest that estrogen formed by aromatization within the corpora lutea may play an important role in the regulation of luteal function during pregnancy in the rat.

Opposite effect of prolactin and prostaglandin F(2 alpha) on the expression of luteal genes as revealed by rat cDNA expression array.

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.

The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy.

Pregnant rats, hypophysectomized-hysterectomized on day 12 (day 1=insemination), secreted progesterone (P) at about 50% of the day 12 level for 3-5 days. Daily treatment with estradiol 100 microng (E100) but not with E25 or E50, from day 12 to day 20, of such rats, restored P secretion until day 16 to that of intact pregnant rats; on day 17 a drastic permanent fall occurred. E100 had no effect after ovariectomy, and did not change the metabolic clearance rate of P. This E100 effect was absent in decidual tissue (DT)-bearing, or hysterectomized pseudopregnant (PSP) rats, and in pregnant ones before day 11. When pregnant rats were hypophysectomized-hysterectomized on day 10, and were treated with rat placental luteotrophin (rPL) (in the form of day 12 pregnant rat serum: "PRS-12") on days 10 and 11, however, E100 increased P secretion above that found with either PRS-12 or E100 alone. DT-bearing PSP rats, similarly operated on on day 12 and treated with PRS-12 on day 12, responded in the same way to E100. In these rats also the E100 effect lasted for four days. In the day 12 hypophysectomized-hysterectomized pregnant rat, the E100 effect could not be prolonged by single treatments with PRS-12 on either days 13, 14 or 16, but when PRS-12 was given daily from day 12 to 19, P was secreted until day 20 only slightly below the day 12 level; this treatment plus E100 raised P secretion, prolonged it to day 18, and led to a marked fall by day 20 similar to that of the intact pregnant rat at term. The marked increase in P secretion between days 12 and 15 of normal pregnancy may thus be a response to intraluteal estrogen; the pattern of P secretion from day 12 to term may reflect the effects of both estrogen and rPL. rPL probably induces this effect by generating luteal estrogen and LH receptors. The placenta may also secrete an LH-like hormone (rCG?) which, through the LH receptors, could stimulate intraluteal estrogen production.

Progesterone inhibits 20alpha-hydroxysteroid dehydrogenase expression in the rat corpus luteum through the glucocorticoid receptor.

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.

The rat follistatin gene is highly expressed in decidual tissue.

Follistatin was originally described as an ovarian polypeptide hormone able to suppress pituitary follicle-stimulating hormone (FSH) secretion in in vitro systems. We have isolated rat ovarian follistatin cDNA clones and used these to investigate the possibility that decidual tissue expresses the follistatin gene. We found that the follistatin gene is highly expressed during early pregnancy in tissue derived from the site of blastocyst implantation. Follistatin mRNA was observed in tissue obtained from the implantation site at days 6, 8 and 10 of gestation, but was not found in the placenta at later stages of gestation. This same pattern of follistatin expression was observed in pseudopregnant animals in which decidualization had been artificially induced, indicating that decidual tissue is the primary source of follistatin mRNA. In situ hybridization was used to examine follistatin gene expression in decidua in greater detail. The mRNA is preferentially found in antimesometrial tissue, and is strictly confined to the endometrial-derived decidual cells. These results suggest that follistatin might function as an important endocrine hormone during implantation of the fetus, perhaps by acting to appropriately modulate maternal FSH secretion during early pregnancy.

Differential action and secretion of rat placental lactogens.

The abilities of the two different mol wt forms of rat placental lactogen (rPL) to maintain luteal steroidogenesis in pregnant rats in the absence of PRL were determined. The two forms of rPL present in day 12 pregnant rat serum were separated by gel chromatography, lyophilized, and reconstituted in a volume of saline equal to the original volume of serum. Rats were injected with 0.4 mg ergocryptine (ECO) on day 6 of pregnancy to suppress PRL and were then treated with a preparation of the large mol wt (LMW) hormone, the small mol wt (SMW) hormone, or both molecules (2 ml/day). Control animals received saline. Twice daily injections of the amount of LMW hormone contained in 1 ml day 12 pregnant rat serum reversed the abortifacient effect of ECO. In contrast, administration of the SMW hormone did not maintain either pregnancy or progesterone levels. Administration of both mol wt forms of rPL was also capable of maintaining luteal function. Sera obtained from day 18 pregnant rats containing only the SMW hormone had no luteotropic activity when administered, yet treatment with sera of day 12 pregnant rats sustained progesterone synthesis and fetal survival after ECO treatment. rPL were measured in the peripheral circulation and in the uterine vein throughout pregnancy by radioreceptor assay (RRA) using particulate membranes from either rabbit mammary gland or rat ovaries. Two peaks of activity were observed in the peripheral circulation by the two RRAs: one between days 11-14 and another between days 17-21, with a decline in activity between days 14-16. In contrast, levels of rPL in the uterine vein remained elevated throughout pregnancy. Concentrations of the LMW placental luteotropin were 5-10 times higher by ovarian RRA (O-RRA) than by mammary gland RRA (MG-RRA) between days 11-13, but concentrations of the SMW placental lactogen were found by the two RRAs to be similar in the later stages of pregnancy. Gel filtration of day 12 pregnant rat serum revealed two peaks of PRL-like activity. The O-RRA detected 19 times more LMW placental luteotropin than did the MG-RRA in the first peak of activity, yet measured equivalent amounts of the SMW placental lactogen in the second peak. Similar to results found in the peripheral circulation, levels of LMW placental luetotropin in the uterine vein measured by MG-RRA were significantly lower than those determined by O-RRA until day 14 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of decidual prolactin-related protein in the rat decidua.

It is well established that rat decidual tissue produces a PRL-like hormone(s) that binds to the PRL receptor on both the corpus luteum and the decidual cells and initiates profound changes in the endocrine milieu required for the establishment of pregnancy. The recent cloning of a decidual PRL-related protein (dPRP) prompted us 1) to determine whether the expression of this gene is triggered by decidualization of the endometrial stromal cells, 2) to examine the temporal and cell-specific pattern of its expression, and 3) to examine the role of both decidual signals and PRL on levels of its messenger RNA (mRNA). Total RNA was isolated from uteri of either nonpseudopregnant rats or pseudopregnant rats with or without decidual tissue. A 1-kilobase mRNA species hybridizing strongly with the dPRP probe was present in decidualized uteri. No dPRP mRNA could be detected in uteri not subjected to decidualization. Developmental studies indicated a constant high level of dPRP mRNA in the decidual tissue until day 12 of pseudopregnancy, followed by a marked decline at a time when extensive cell death occurs in the decidua, suggesting that dPRP is constitutively expressed in this tissue. To examine the cell-specific expression of dPRP, antimesometrial decidua was separated from mesometrial decidua, and the large antimesometrial cell population was separated from the small mesometrial cells by elutriation. The results of Northern analysis revealed clearly that dPRP is abundantly and solely expressed in the large antimesometrial cells. No dPRP mRNA could be detected in the mesometrial cells and in numerous other endocrine and nonendocrine tissues. A faint signal was observed, however, in the trophoblast. Despite the very strong paracrine regulation between the antimesometrial and mesometrial cells and the high levels of PRL receptor expression in these cells, both in vivo and coculture experiments revealed no regulation of dPRP gene expression by either PRL or mesometrial cell signal, adding further support to the possibility that once induced, dPRP remains constitutively expressed. In summary, the results of this investigation revealed that the expression of dPRP in endometrial stromal cells is triggered by the induction of decidualization and that this gene is selectively and abundantly expressed in a defined cell population located in the anti-mesometrial region of the uterus. Thus, dPRP is not only a useful indicator of decidualization, but is also an excellent marker for the differentiated antimesometrial cells.

Evidence for the secretion of decidual luteotropin: a prolactin-like hormone produced by rat decidual cells.

Rat decidual tissue contains a PRL-like hormone named decidual luteotropin. We have recently revealed some of its physiological and biochemical characteristics. However, because rat decidual tissue contains specific binding sites for PRL, it was important to demonstrate that the hormone found in the tissue is not locally stored and structurally transformed PRL but a hormone actively synthesized by the rat decidual tissue. Decidual explants or decidual cells obtained from day 9 pseudopregnant rats were incubated for different times under either static conditions or continuously perifused with medium at a rate of 1 ml/h. Levels of decidual luteotropin were measured by a specific radioreceptor assay using luteal membranes as source of receptors and [125I]iodo-ovine(o)PRL as a tracer. In the static incubation, no proof of hormone production was obtained; levels of decidual luteotropin in medium and tissue or cells at the end of the incubation were similar to levels found in either cells or tissue before incubation. In sharp contrast, decidual cells perifused with media secreted large amounts of hormone. This may suggest that an inhibitor of decidual luteotropin production was being removed from the culture by the perifusion. For the first 4 h of perifusion, no hormone was produced. However by the fifth hour, cells began to actively release decidual luteotropin. Secretion of the hormone increased with time and reached maximal values between 7-15 h of perifusion. During the 15 h of perifusion, decidual cells released approximately 1000 times more decidual luteotropin than the amount they originally contained. A dose-response increase in hormone secretion was obtained with increased concentrations of decidual cells. The net amount of decidual luteotropin released into the medium over an 18-h period was approximately 6.5 micrograms/30 X 10(6) cells, 3.5 micrograms/10 X 10(6) cells, and 0.5 micrograms/2 X 10(6) cells. A similar profile of decidual luteotropin release was obtained when decidual explants were perifused. However, in contrast to decidual cells which secreted no hormone for the first 4 h of culture, decidual explants immediately began to release decidual luteotropin in the medium. The secretion of decidual luteotropin in vitro was inhibited 75% by the protein synthesis inhibitor, cycloheximide. In summary, results of this investigation demonstrate for the first time that rat decidual cells secrete in vitro a PRL-like hormone, decidual luteotropin.

Does the rat corpus luteum express the progesterone receptor gene?

To determine whether the progesterone receptor (PR) gene is expressed in rat corpora lutea (CL), PR messenger RNA (mRNA) and protein levels were examined in CL of both cycling and pregnant rats using in situ hybridization, reverse transcription-polymerase chain reaction, and Western blotting analyses. During the estrous cycle, levels of luteal PR mRNA were below the sensitivity of in situ hybridization. Although no signal could be detected in luteal cells, granulosa cells of preovulatory follicles of the same ovary expressed PR mRNA at high levels during the evening of proestrus. Likewise, CL of pregnant rats expressed undetectable levels of PR mRNA, which remained unchanged throughout pregnancy, as determined by in situ hybridization and reverse transcription-polymerase chain reaction. In addition, no PR protein could be detected in rat CL by Western analysis, indicating that luteal expression of the PR gene, if any, is negligible. To determine whether the lack of PR mRNA in the rat CL is due to progesterone-induced down-regulation of PR mRNA or to low levels of estrogen, aminoglutethimide was used to block the synthesis of progesterone in the presence and absence of exogenous estrogen, and PR mRNA levels were examined in the CL as well as the placenta. Inhibition of progesterone synthesis did increase PR mRNA levels in the placenta, and additional estrogen treatment further increased PR mRNA levels in this tissue. In contrast, neither aminoglutethimide alone nor aminoglutethimide plus estrogen induced PR mRNA expression in CL of the same rats. The temperature-sensitive luteal cell line derived from the rat CL, which produces very low levels of progesterone, also did not express PR mRNA. These results indicate that the rat corpus luteum expresses undetectable levels of PR mRNA and protein, which is not attributable to progesterone-induced down-regulation of PR mRNA or to a lack of estrogen-induced up-regulation of PR mRNA in this tissue.

Mechanism for control of hydroxymethylglutaryl-coenzyme A reductase and cytochrome P-450 side chain cleavage message and enzyme in the corpus luteum.

The present study was undertaken to investigate the mechanism for control of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase and cytochrome P-450 side chain cleavage (P-450scc), enzymes involved in the synthesis and processing of cholesterol in the corpus luteum of pregnant rats. The role of sterol and estradiol were investigated by administering 4-aminopyrazolo-[3,4d]pyrimidine (4-APP), aminoglutethimide and/or estradiol to day 12 hypophysectomized-hysterectomized pregnant rats. Estradiol treatment markedly increased mRNA levels of HMG-CoA reductase in the corpus luteum. This stimulatory effect of estradiol was specific for the reductase since the mRNA for luteal P-450scc did not increase following estradiol treatment. To determine whether estradiol's action on HMG-CoA reductase mRNA was mediated by changes in cellular free cholesterol, the separate and combined action of estradiol and either 4-APP or aminoglutethimide on both sterol content and HMG-CoA reductase expression was examined. The estradiol induced rise in HMG-CoA reductase was not accompanied by a decrease in luteal cholesterol. 4-APP treatment depleted cholesterol in the corpus luteum and induced a greater increase in HMG-CoA reductase mRNA than estradiol. Combined treatment with estradiol and 4-APP resulted in a synergistic increase in HMG-CoA reductase mRNA despite the fact that estradiol did not further reduce the 4-APP induced decrease in luteal sterol content. This indicates that luteal cells possess an estradiol-mediated mechanism for up-regulation of HMG-CoA reductase gene expression which is distinct from the cholesterol negative feedback regulation. However, estradiol stimulation of HMG-CoA reductase was totally inhibited when sterol content was elevated by aminoglutethimide, suggesting that estradiol stimulation of HMG-CoA reductase expression is not sufficient to overcome the negative regulatory effect of cholesterol. In contrast to HMG-CoA reductase mRNA which appears to be controlled by sterol and estradiol, P-450scc mRNA levels remained constant in corpora lutea despite wide changes in sterol and steroid content induced by either 4-APP, aminoglutethimide or estradiol treatment. However, interestingly, aminoglutethimide increased substantially the content of P-450scc protein. This increase in P-450scc enzyme was not due to a greater amount of translatable message since similar levels of immunoprecipitable 35S-P-450scc were translated in vitro from luteal mRNA of rats treated with or without aminoglutethimide. In summary, results of this investigation have revealed that the expression of HMG-CoA reductase and cytochrome P-450scc are controlled by totally different mechanisms in the rat corpus luteum.(ABSTRACT TRUNCATED AT 400 WORDS)