Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.     

Measurement of messenger RNA for luteinizing hormone beta-subunit and alpha-subunit during gestation and the postpartum period in ewes

Pituitary content of luteinizing hormone (LH) and mRNAs for LH beta-subunit (LH beta), alpha-subunit, prolactin, and growth hormone were measured in ewes on Days 50 and 140 of gestation and on Days 2, 13, 22, and 35 postpartum. Content of LH in dissociated anterior pituitary cells declined (P less than 0.05) between Days 50 and 140 of gestation and remained low at 2 days postpartum. By 22 days postpartum, pituitary concentrations of LH were comparable to concentrations in normally cycling ewes. During gestation concentrations of mRNA for LH beta and alpha-subunit paralleled changes in cellular content of LH, reaching minimal levels on Day 140. By Day 2 postpartum, pituitary concentrations of mRNAs for LH beta and alpha-subunit began to increase; they reached maximum levels by Day 13 postpartum. There appeared to be a gradual linear increase in mRNA for prolactin through gestation and the postpartum period. No changes in mRNA for growth hormone were noted during the prepartum or postpartum periods. These data suggest that the decline in pituitary concentrations of LH during gestation is due to a decrease in cellular mRNA for LH beta and alpha-subunit. The increase in mRNA for LH beta and alpha-subunit appears to precede an increase in cellular content of LH in the postpartum ewe by several days.     

Differential patterns in luteal prolactin and LH receptors during pregnancy in sows and ewes

In the sow, a dramatic increase of LH specific binding was observed during the second half of pregnancy. This was due to an increase in receptor number (41 fmol and 95 fmol/mg protein at Days 50 and 105 respectively). The apparent association constant was unchanged. The pattern of prolactin receptor content showed two peaks at Day 60 and Day 105. Prolactin receptors increased earlier during pregnancy than did LH receptors, suggesting a possible role of prolactin in the induction of LH receptors. In the ewe, the receptor content of LH and prolactin did not change very much during pregnancy. The corpus luteum showed normal luteal function until parturition although it was not necessary for maintenance of pregnancy in the ewes.     

Evidence that the corpus luteum of pregnancy contributes to the control of tonic secretion of LH in the ewe

Concentrations of LH and FSH were measured in blood samples collected from the jugular vein at 20-min intervals for 7 h (09:00-16:00 h) on Days 60, 80, 100 and 120 of pregnancy in 5 intact ewes and 5 from which the CL had been excised on Day 70. In the 5 intact ewes, plasma LH concentrations remained low and unchanged between Days 60 and 120. During this period, pulsatile release of LH occurred irregularly and infrequently. Removal of the CL resulted in an increase in the basal values of LH and in the frequency and amplitude of LH pulses. Concentrations of FSH were relatively constant in all stages of pregnancy examined and were similar in both groups of ewes. These results show that (1) LH concentrations are low during the second half of pregnancy; and (2) LH, but not FSH, increases after CL excision, presumably by removing some luteal factor inhibitor of LH secretion.     

Influence of progesterone concentrations on secretory functions of trophoblast and pituitary during the first trimester of pregnancy in dairy cattle

The essential role played by progesterone in the maintenance of pregnancy is unequivocal; however, the effects of progesterone on the secretory patterns of placental and pituitary molecules during the gestation period are not well defined. The objective of this study was to describe pregnancy-associated glycoprotein (PAG) concentrations (measured by RIA-497 and RIA-Pool) in pregnant females with progesterone concentrations lower (low-P4 group, n=20) or higher (high-P4 group, n=17) than the mean of 8.74 ng/mL on Day 21 (AI=Day 0). Luteinizing hormone (LH) and prolactin concentrations were also measured in both groups. Throughout the study period, blood samples were collected on Days 0, 21, 45, 60, and 80 from 37 females that were confirmed to be pregnant. PAG concentrations measured by both RIA-497 and RIA-Pool tended to be higher in high-P4 group than in low-P4 group from Day 30 until Day 80. On Day 80, plasma PAG concentrations that were measured using RIA-497 were observed to be higher (P<0.05) in the high-P4 group than in the low-P4 group (10.2+/-8.7 ng/mL versus 6.9+/-3.8 ng/mL). Concentrations of LH on Day 60 and prolactin on Day 80 were observed to be significantly lower (P<0.05) in the high-P4 group. There was a tendency for the concentrations of LH (Days 45 and 80) and prolactin (Days 30, 45, and 60) to be lower in cows in the high-P4 group than in the low-P4 group. Our results suggest the existence of a relationship among the concentration levels of progesterone, PAG, LH, and prolactin during early pregnancy.     

Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA.

Using MA-10 Leydig tumor cells as a model system we have examined the possibility that the lutropin/choriogonadotropin (LH/CG)-induced down-regulation of the LH/CG receptor is accompanied by changes in LH/CG receptor mRNA. We show that LH or CG are indeed capable of reducing the levels of LH/CG receptor mRNA, but that the time course and magnitude of the reduction in receptor mRNA are such that this phenomenon cannot account entirely for the down-regulation of the receptor. In fact, we estimate that LH/CG can reduce the number of LH/CG receptors by at least 80% with little or no change in the levels of LH/CG receptor mRNA. These data are consistent with our previous hypothesis that the LH/CG-induced down-regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor that occurs as a result of the receptor-mediated endocytosis of LH/CG. Our studies also show that the LH/CG-induced down-regulation of the LH/CG receptor mRNA is mediated by cAMP. Thus, addition of 8-bromo-cAMP to MA-10 cells leads to a similar reduction in the levels of LH/CG receptor and receptor mRNA; while deglycosylated human CG, a hormone derivative that binds to the LH/CG receptor but has a reduced ability to stimulate cAMP synthesis, does not reduce the levels of LH/CG receptor mRNA. Last, human CG or 8-bromo-cAMP are unable to reduce LH/Cg receptor mRNA in a mutant MA-10 cell line that express a cAMP-resistant phenotype.     

Role of luteinizing hormone-releasing factor (LH-RF) and LH on maintenance of early gestation in rats.

The role of luteinizing hormone (LH) and LH-releasing hormone (LH-RH) in the maintenance of early pregnancy in rats was studied. Serum levels of progesterone (P) and LH were measured daily in untreated pregnant rats from Day 4 through parturition. Serum levels of P and LH were determined on Days 11 and 15 of pregnancy in animals treated with antisera to LH (LH-A/S) and to LH-RH (LH-RH-A/S) on Days 8-10. Serum levels of P peaked on Days 7 and 16 in untreated animals, after which they declined sharply just before delivery. Serum LH fluctuated between 30-160 ng/ml during pregnancy but did not exhibit any distinctive peaks. Treatment with .2 ml LH-A/S on Days 8-10 reduced serum P to virtually undetectable levels on Day 11, and only a slight recovery was evident on Day 15. Lower doses of LH-A/S had no effect. Administration of 1.3 ml LH-RH-A/S had no effect on serum levels of P or LH, and did not impede fetal development. The results indicate that LH is essential to the luteotropic complex of early pregnancy in the rat, and also that LH-RH-A/S can maintain to some extent basal levels of P and LH during early pregnancy.     

Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy

The present study was undertaken to determine the expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in the porcine corpus luteum (CL) during the estrous cycle and early pregnancy. Immunohistochemical studies localized proteins of VEGF ligand-receptor system in the cytoplasm of luteal cells and in some blood vessels. Western blot analysis revealed significantly higher levels of VEGF protein during early and mid-luteal phase (vs. late luteal phase; P<0.001 and P<0.01, respectively). Quantification of VEGF mRNA in the CL showed increased mRNA levels during entire luteal phase (vs. Days 16-17; P<0.05). Expression of Flt-1 protein remained high during luteal phase (P<0.001), but the mRNA levels tended to increase from the early to the late luteal phase. Elevated protein expression of Flk-1/KDR was found in the mid-luteal phase (vs. Days 16-17; P<0.05). However, induction of Flk-1/KDR mRNA expression occurred earlier, in early luteal phase. The lowest VEGF, Flt-1 and Flk-1/KDR mRNA and protein levels were observed in regressed CL (P<0.001). During pregnancy, VEGF, Flt-1 and Flk-1/KDR mRNA and protein expression was comparable to the mid-luteal phase. In conclusion, the present study has demonstrated dynamic expression of VEGF and its receptors in the porcine CL during the estrous cycle and early pregnancy. These data suggest that the VEGF ligand-receptor system may play an important role in the development and maintenance of the CL in pigs.     

Urine collection in the common marmoset (Callithrix jacchus) and its applicability to endocrinological studies

We investigated a new method of urine collection in the common marmoset. We entered the cage as soon as the light cycle started in the breeding room and collected urine from the animal directly without any restraint. We were able to take separate samples from completely different individuals housed together for behavioral studies in the same cage. Urine and blood samples were taken from individuals from late pregnancy through postpartum nursing period. Cortisol and prolactin concentrations measured in urine were compared to those measured in blood to evaluate this collection method. LH/CG level in the urine samples was also measured. Urine data in females indicated a tendency toward high cortisol values during late pregnancy, a sharp drop before parturition, and increase after delivery. In females cortisol levels measured in blood closely resembled concentrations measured in urine. Urine cortisol in males clearly indicated an increase postpartum, but the increase was not indicated in plasma. Plasma and urine prolactin concentrations in females made a similar increase during lactation. Male's plasma prolactin clearly indicated an increase directly proportional to strong behavioral contact with the infant. We also confirmed hormonal changes during pregnancy, and postpartum ovulation and subsequent pregnancies, from urine LH/CG data. We found this method extremely useful because of the high correlation between cortisol, prolactin and LH/CG data from blood and urine. Additionally, we collected urine samples with little stress to the animal from fear, irritation, pain, etc.     

Luteal LH receptors during the oestrous cycle and early pregnancy in the pig

The concentration, affinity constant and occupancy of the LH receptor have been measured in corpora lutea, from 29 pigs at Days 6--16 of the oestrous cycle, and from 25 pigs at Days 12--30 of pregnancy, by using 125I-labelled porcine LH tracer. Investigation of the specificity of the receptor showed that cross-reactions of other pituitary hormones were accounted for by their contamination with LH. Luteal concentration of unoccupied receptors doubled between Days 6 and 10 of the cycle, and decreased between Days 12 and 14; it increased 3-fold between Days 20 and 30 pregnancy, but was lower on Day 12 of pregnancy than at a comparable stage of the cycle. Concentrations of receptors occupied by LH increased early in the oestrous cycle, in parallel with the total receptor concentration; in pregnancy the percentage occupancy dropped dramatically as total receptor concentration increased between Days 20 and 30. Receptor affinity constants increased towards the end of the cycle and decreased between Days 20 and 30 of pregnancy. It is suggested that (1) the lower concentration of receptors in early pregnancy may reflect down regulation by circulating LH, concentrations of which are higher in early pregnancy than during the cycle; (2) the increase in receptor concentration between Days 20 and 30 or pregnancy may be due to a rise in circulating oestrogen levels; and (3) the decrease in occupancy at this time may be caused either by a decrease in affinity constant or by placental production of a chorionic gonadogrophin-like compound.     

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.