Prolactin maintains relaxin and progesterone secretion by aging corpora lutea after hypophysial stalk transection or hypophysectomy in the pig

Porcine corpora lutea persist beyond 150 days in hysterectomized animals compared with about 114 days during normal pregnancy. To explore the mechanism(s) regulating the peak release of relaxin and secretion of progesterone by aging corpora lutea and to examine the direct effect of purified porcine (p) PRL on such corpora lutea, hypophysial stalk transection (HST), hypophysectomy (HYPOX) with or without PRL replacement, and sham operation control (SOC) were conducted on day 110 (estrus = day 0) on purebred Yorkshire gilts that were hysterectomized on days 6-8. The pPRL (0.5 mg every 6 h daily) or PBS (0.5 ml every 6 h daily) was given iv from days 110-120. HYPOX + pPRL, HYPOX + PBS, HST + PBS, and SOC + PBS formed four experimental groups. Peak relaxin concentrations in peripheral plasma (mean values ranged from 22-24 ng/ml) occurred on about day 113 for all groups [113.4 +/- 0.3 days (+/- SE)] regardless of the different surgical interventions. After peak release, relaxin decreased steadily in the HYPOX + PBS group, falling to less than 1.0 ng/ml by 6 days later, whereas relaxin in other groups remained elevated (approximately 7 ng/ml). In the HYPOX plus PBS group, progesterone decreased abruptly, remaining below 1 ng/ml from 1 week onward, lower (P less than 0.01) than that in controls (approximately 19 ng/ml); in the HYPOX + pPRL group, progesterone levels (approximately 17 ng/ml) remained similar (P greater than 0.05) to those in controls (approximately 19 ng/ml) and the HST + PBS group (approximately 15 ng/ml). These results clearly reveal that the pituitary gland plays no direct role in regulating the timed peak release of relaxin from aging corpora lutea in hysterectomized gilts and that the peak release of relaxin on about day 113 is preprogrammed and inherent within such aging luteal cells. This study provides strong evidence that purified pPRL maintains both relaxin and progesterone secretion as well as the morphology of aging corpora lutea for at least 10 days after hypophysectomy in hysterectomized gilts.     

The effect of relaxin infusion on prolactin and growth hormone secretion in monkeys

To test the hypothesis that relaxin may affect pituitary hormone secretion, synthetic human relaxin was infused into cycling and pregnant rhesus monkeys. Doses ranging from 0.154-1540 ng/kg.min were calculated to achieve circulating relaxin concentrations of 1 pM to 10 nM. Low (0.154 and 1.54 ng/kg.min), intermediate (15.4 and 154 ng/kg.min), and high (1540 ng/kg.min) doses of relaxin were infused for 15 min each hour into ovulating monkeys at the midluteal phase of the menstrual cycle in two separate experiments. Serum GH and PRL were measured by RIA, and serum relaxin was determined by enzyme-linked immunosorbent assay. Relaxin was undetectable in peripheral plasma during the control saline infusion and during infusion of the lowest dose of relaxin. Serum relaxin levels reached 0.011, 0.119, 0.965, and 13.0 nM with 1.54, 15.4, 154, and 1540 ng/kg.min, respectively. Serum GH was significantly elevated over basal levels upon infusion of relaxin from 1.54-1540 ng/kg.min; however, a plateau was observed with the intermediate doses, and a decrease in the magnitude of the response was observed at the highest dose. Serum PRL increased over basal levels with 15.4 and 154 ng/kg.min, but there was no difference in the magnitude of the increase between these doses. PRL levels during infusion of the highest dose of relaxin were similar to control levels. These data suggest that relaxin can stimulate secretion of GH and PRL in cycling monkeys within a defined dose range, but that a decrease in pituitary responsiveness occurs at higher doses. One high dose of relaxin (2600 ng/kg.min) was infused for 1 h into the maternal and then the fetal circulations of chronically catheterized and tethered pregnant monkeys between 120-140 days gestation. Upon infusion of relaxin into the maternal circulation, there was a significant elevation of PRL in the mother but not the fetus. Upon infusion of relaxin into the fetus, there was no consistent change in PRL secretion in either the mother or the fetus. In conclusion, relaxin may have a heretofore undescribed role in pituitary physiology during the menstrual cycle and in pregnancy.     

An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys

A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.     

Divergent effects of antiprogesterone, RU 486, on progesterone, relaxin, and prolactin secretion in pregnant and hysterectomized pigs with aging corpora lutea

Porcine corpora lutea produce progesterone and relaxin during pregnancy and after hysterectomy. Peak amounts of relaxin are released into peripheral blood in both pregnant and hysterectomized animals on about day 113 (estrus = day 0 and term = 114), and this release coincides with an abrupt decrease in the progesterone concentration. RU 486, a progesterone receptor antagonist, was used to investigate the effects of interruption of progesterone binding to its receptor on luteal function and gonadotropin secretion of pigs with aging corpora lutea. RU 486 was administered orally to hysterectomized gilts (surgery on day 8) once a day (0800 h) on days 111-115 at two dosages (group 1, 2 mg/kg BW; group 2, 4 mg/kg BW). During 5 days of RU 486 treatment, plasma progesterone concentrations in both treated groups were markedly elevated (32 and 37 ng/ml for groups 1 and 2) compared with 22 ng/ml in the controls (group 3; P less than 0.01). PRL concentrations increased in both groups (9 and 13 ng/ml) and differed significantly from those of the controls (3 ng/ml) (P less than 0.04). RU 486 treatment delayed the time of relaxin peak to days 116.1 and 117.0 in groups 1 and 2 compared with day 114.1 in the controls (P less than 0.01). Pregnant gilts received RU 486 orally once a day (0800 h) at 4 mg/kg BW beginning on day 111 until parturition occurred. Parturition was induced on day 112.7 after only two RU 486 treatments compared with day 114.7 in the control group (P less than 0.01). Progesterone decreased abruptly from a pretreatment mean of 11 to less than 0.6 ng/ml during the 2 days that RU 486 was given compared with a shift from 12 to 6 ng/ml during the same period in the controls (P less than 0.01). The time of the relaxin peak was advanced to day 112.1 in RU 486-treated gilts compared with day 113.9 in the controls (P less than 0.01). Results from this study provide strong evidence that the antagonistic effect of RU 486 on progesterone receptor results in an abrupt increase in PRL and progesterone secretion in hysterectomized gilts with aging corpora lutea. In marked contrast with hysterectomized animals, the acute luteolytic effects of RU 486 depend on the presence of the uterus and/or conceptuses in the pig. Disruption of the regulatory loop of progesterone secretion by RU 486 alters the ability of corpora lutea to produce and release peak quantities of relaxin.     

Relaxin production and release after hysterectomy in the pig

Relaxin secretion by luteal tissue and into peripheral blood was examined in relation to changes in the fine structure of aging porcine corpora lutea. Sequential bleedings every sixth day reveal a similar increase in serum relaxin concentrations from days 6-114 in hysterectomized and pregnant gifts. The span of the estrous cycle is about 21 days, and the length of pregnancy is about 115 days in pigs. Estrone (E1) and 17 beta-estradiol (17 beta-E2) serum levels increase with placental development; after hysterectomy, they are low from days 12-168. In luteal tissue, peak quantities of relaxin and electron-dense granules occur on day 100 in hysterectomized and pregnant gilts. By day 112, luteal tissue relaxin levels and granule populations decrease by about half, while relaxin secretion into peripheral blood approaches peak concentrations about day 114 in hysterectomized as well as pregnant gilts. Between days 114 and 120, serum relaxin decreases by half even though the corpora lutea persist to day 150 in hysterectomized animals. Thereafter, cytoplasmic granules and blood and luteal tissue levels of relaxin decrease gradually to day 150 in hysterectomized gilts; in contrast, they disappear abruptly after parturition. In hysterectomized gilts, relaxin release coincides with diminished populations of granules in corpora lutea, and the initiation of these events seems to be precisely timed. E1 and 17 beta-E2 secretion is unrelated to relaxin secretion in pregnant and hysterectomized gilts. High peripheral blood levels of E1 and 17 beta-E2, primarily of placental origin, or even the presence of the uterus are not required in the release of relaxin at day 114. By using steroidogenic and protein synthetic capabilities of luteal cells as criteria essential to the evolution of reproduction in this species, the results presented here suggest that 114 days rather than 21 days characterize the reproductive cycle, even in the absence of the uterus. These results indicate that the production and, particularly, the release of relaxin into peripheral blood on about day 114 in hysterectomized gilts as well as pregnant animals may be genetically controlled.     

Precise timing for peak relaxin and decreased progesterone secretion after hysterectomy in the pig

Relaxin and progesterone secretion by aging corpora lutea (days 90-120) was examined in pregnant and lactating gilts compared with that in hysterectomized animals. The length of pregnancy is about 115 days in pigs. Unmated gilts were hysterectomized on day 6 (estrus = day 0). From days 90-101, relaxin concentrations in peripheral plasma remained consistently low in pregnant gilts (range, 0.7-1.5 ng/ml) and less (P less than 0.05) than those in hysterectomized animals (range, 0.9-3.5 ng/ml). Relaxin increased abruptly (P less than 0.01) to a peak of 66 ng/ml in pregnant gilts and 37 ng/ml in hysterectomized animals. Relaxin peaked in pregnant animals at 113 +/- 0.7 days (+/- SE) and in hysterectomized gilts at 113 +/- 0.7 days; gestation length averaged 114 +/- 0.8 days. In pregnant gilts, relaxin decreased from a peak of 66 to 11 ng/ml within 1 day and remained low (less than 1.0 ng/ml) in these lactating dams until day 120. In hysterectomized gilts, peak relaxin also decreased abruptly from 37 to 4.2 ng/ml, but remained consistently greater (P less than 0.05) than that in lactating dams. Although there were abrupt shifts in relaxin concentrations within 20 min, there was no evidence for consistent episodic relaxin release between days 112-116. Plasma progesterone concentrations were consistently greater (P less than 0.05) in hysterectomized than in pregnant gilts from days 102-110. Progesterone decreased abruptly in prepartum gilts (days 111-114) from 16 to 1.2 ng/ml and remained low during lactation (0.5 ng/ml). In hysterectomized animals, it decreased abruptly on days 110-113, ranging from 20-12 ng/ml, and remained at this lower level until day 120. These results clearly indicate that a precisely timed peak release of relaxin and coincident decrease in progesterone secretion occur in unmated hysterectomized gilts at the same time as those found a few hours preceding parturition during normal pregnancy. These abrupt shifts in relaxin and progesterone secretion on days 112-113 in both hysterectomized and pregnant gilts may be regulated autonomously from within the ovary or from the central nervous system and pituitary gland.     

Relaxin and progesterone secretion as affected by luteinizing hormone and prolactin after hysterectomy in the pig

Plasma levels of relaxin and progesterone in hysterectomized and pregnant gilts were determined from days 100-120 to evaluate the effects of purified porcine (p) LH and pPRL on the secretory activity of the aging corpora lutea. Gilts were bred on the second observed estrus or were hysterectomized between 6-8 days after estrus (estrus = day 0) and were assigned randomly to one of three treatment groups; saline-treated control, im injections of pLH, and iv injections of pPRL from days 110-120. In control, pLH-treated, and pPRL-treated animals, average gestation lengths were 114 +/- 0.8, 116 +/- 1.9, and 115 +/- 0.5 days (+/- SE), respectively. The relaxin level in mated gilts on day 100 was less than 2 ng/ml; it began to increase after day 110 and peaked in control animals on day 113 (66 ng/ml), whereas in pLH- and pPRL-treated animals, prepartum peak values were greater (P less than 0.01) and occurred on days 113 (104 ng/ml) and 114 (117 ng/ml), respectively. Relaxin dropped to basal levels (less than 1 ng/ml) by day 115 in controls and by day 116 in both pLH- and pPRL-treated gilts. Although pLH and pPRL treatments markedly accentuated peak relaxin secretion, they did not significantly accelerate or delay parturition or delay the abrupt demise of the corpora lutea immediately postpartum. In hysterectomized gilts, relaxin began to increase after day 110, peaked in control animals on day 113 (27 ng/ml), and decreased abruptly thereafter to less than 4 ng/ml. In contrast, pLH caused an immediate release of relaxin on day 111 (23 ng/ml) and sustained elevated levels (P less than 0.01) of relaxin until day 118, but the original corpora lutea regressed. Relaxin in pPRL-treated animals increased steadily after day 110, reaching peak values by day 115 (29 ng/ml), and remained consistently elevated (P less than 0.01) until day 120. Progesterone secretion was maintained in the pPRL-treated hysterectomized gilts from days 110-120 by the original corpora lutea and with no luteinization of follicles or formation of new corpora lutea. It is evident from this study that administration of pPRL starting on day 110 enhanced and prolonged the preprogramed release of relaxin and maintained progesterone secretion by aging corpora lutea in hysterectomized animals until day 120.     

Progesterone secretion as affected by 17 beta-estradiol after hysterectomy in the pig

Serum levels of progesterone in hysterectomized gilts were determined to evaluate the effects of exogenous 17 beta-estradiol (17 beta-E2) on the secretory activity of aging corpora lutea. Gilts were hysterectomized on day 6 of the estrous cycle and given im injections of 17 beta-E2 to mimic blood levels of endogenous estrogen during normal pregnancy. Serum progesterone was determined every third or sixth day, and estrone and 17 beta-E2 were measured at 12-day intervals from days 42-192. Progesterone decreased (P less than 0.01) after day 104 in hysterectomized gilts given sesame oil. Exogenous estrogen consistently decreased (P less than 0.02) progesterone secretion during an extended period (days 45-108) in aging corpora lutea of hysterectomized gilts. Abrupt decreases in estrone, 17 beta-E2, and progesterone levels occurred after termination of estrogen injections on day 114. The decrease in the secretion of these steroids in hysterectomized gilts was similar to that observed in previous studies at parturition and during the early postpartum period. Estrogen treatment beyond day 114 inhibits follicular growth and suppresses ovulation, but behavioral estrus was induced during the terminal stages of luteal activity. In the absence of the uterus, aging corpora lutea respond to exogenous estrogen by decreased progesterone secretion, which is similar to that observed during normal pregnancy in the pig.     

Neuroendocrine regulation of anterior pituitary function in patients during and after molar pregnancy

Only partial studies evaluating the endocrine profile in molar pregnancy have been performed. In order to characterize the neuro-endocrine pattern during and after molar pregnancy, we studied the basal hormonal levels of hCG, human placental lactogen (hPL), FSH, GH, TSH, free thyroxine index (FTI), oestradiol-17 beta (E2), and progesterone (PG), as well as the anterior pituitary response to TRH, GnRH, and hypoglycaemia induced by insulin in 7 patients during molar pregnancy and one week after molar abortion. hCG showed significantly higher serum levels during rather than after molar pregnancy and hPL was detectable in only 4 patients during, but in none after molar pregnancy. FSH values were in the follicular phase range before and after molar abortion (12.7 +/- 0.8 and 12.7 +/- 3.5 IU/l). PRL had elevated basal levels before and after molar abortion; 103.0 +/- 16.5 and 43 +/- 10.6 micrograms/l, respectively (P less than 0.05). GH levels were distinctly elevated in 3 patients during molar pregnancy; after molar abortion, the basal GH values were normal in all patients less than 10 micrograms/l. Basal cortisol and TSH levels were in the normal range before and after molar abortion. The FTI was above the normal range in 3 patients during molar pregnancy, whereas after molar abortion the values were normalized. E2 levels were elevated before and after molar abortion, 1881 +/- 477 and 96.5 +/- 39.2 ng/l, respectively (P less than 0.01). PG levels before and after molar abortion were 30.9 +/- 5.4 and 10 +/- 6.7 micrograms/l, respectively (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

The importance of a luteolytic effect of the pituitary in understanding the placental control of the rat corpus luteum

In rats bearing a full complement of conceptuses (FC-bearing rats) progesterone secretion increases about 1.5 times and the corpora lutea double in weight between days 12 and 16. Reducing the number of conceptuses to one (by aspirating all but the single conceptus) at any time between days 7 and 12 of pregnancy (day 1 = insemination) caused progesterone secretion and corpus luteum weight to remain at or below the day 12 level until term. When rats bearing one conceptus were hypophysectomized on day 12, however, serum progesterone increased and fell after day 12 in a pattern almost identical to that in FC-bearing rats, except that the peak and trough occurred about 2 days later; hypophysectomy on day 15 or 18 also increased serum progesterone to values approaching those in intact FC-bearing rats. In a series of rats bearing experimentally determined conceptus numbers between 0 and 5 or naturally occurring ones of 6-16, the serum level of placental lactogen on day 12 (the day of the first peak of secretion of this hormone) was directly proportional to conceptus number over the entire range. The pattern of rat placental lactogen secretion between days 7 and 23 in FC-bearing rats included two peaks, on day 12 and on days 18-21, but in one-conceptus-bearing rats, the level was only slightly higher than the PRL level, and it remained unchanged, even by hypophysectomy, until term. Serum PRL levels bore no relation to conceptus number, were very low, and were unaffected by hypophysectomy except in the FC-bearing rats, in which lypophysectomy prevented the rise that normally occurs after day 21. These results, especially in relation to others previously reported, show that by day 12 of pregnancy, the rat pituitary, instead of merely becoming luteotropically inactive, produces a substance that inhibits further development of the corpora lutea. The results also imply that the placentas maintain luteal growth and progesterone secretion as much by suppressing this luteolytic activity as by stimulating the corpus luteum directly.     

The impact of unabated stimulation by human chorionic gonadotropin on the steroid hormone environment of pregnant rats and the spontaneous expression of ovarian cysts in female progeny

Unabated stimulation by low doses of human chorionic gonadotropin (hCG) induces ovarian cysts in pregnant rats. In order to determine the impact of these in vivo treatments on the hormonal milieu of pregnancy, and the potential impact of an aberrant cystic-ovary state during pregnancy on the resulting female offspring, pregnant rats were treated with either 0 (control), 1, or 3 IU hCG twice daily for at least 9 days, beginning on day 13 of pregnancy. Serum was harvested from control and hCG treated animals on days 15, 17, 19, and 22 of pregnancy. Control pregnant rats and animals treated with 1 IU hCG shared similar serum profiles for progesterone (P4), androstenedione (A4), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), androsterone (A5), and estrone (E1) between days 15 and 22 of pregnancy. Testosterone serum concentrations were similar for control and 1 IU hGG-treated pregnant rats between days 15 and 19 of pregnancy; whereas, on day 22, 1 IU hGG-treated pregnant rats displayed lower serum testosterone than control pregnant rats (P < or = 0.05). In contrast, serum estradiol (E2) concentrations for 1 IU hCG- treated pregnant rats were greater than E2 values observed for control rats on days 15-19 of pregnancy (P < or = 0.05). Serum testosterone and 3alpha-diol values for 3 IU hCG-treated pregnant rats differed from those of control pregnant rats only on day 19 when these values were transiently greater for these hCG-treated animals compared with serum values for control pregnant rats (P < or = 0.05). Serum A4 values for 3 IU hCG-treated pregnant rats were elevated compared to values for control pregnant rats only on days 15 and 17 (P < or = 0.05). In contrast, serum E1, A5, and E2 were elevated on days 19-22, 17-22, and 15-22, respectively, in 3 IU hCG-treated pregnant rats compared to control pregnant rats (P < or = 0.05). No pups from control pregnant rats displayed ovarian cysts during the time they were observed postnatally. In contrast, 6 of 25 pups from 3 IU hCG-treated pregnant rats displayed cystic ovaries, without corpora lutea, on day 55 of age. Serum steroid concentrations for these cyst-bearing progeny were similar to those of female progeny from control pregnant rats, whereas female progeny without ovarian cysts from 3 IU hCG-treated pregnant rats displayed differences in serum steroid values from those of progeny from control pregnant rats (P < or = 0.05). The data support the concept that an aberrant, yet physiologic hormonal environment associated with the induction of ovarian cysts during pregnancy in rats, can lead to the spontaneous establishment of an ovarian cystic state in at least a subset of the female progeny. Further, the date suggest that tonically increased ovarian estrogen production during pregnancy, reflected by tonically elevated peripheral serum estrogen concentrations, may play a pivotal role in the etiology of an ovarian cystic state in this subset of daughters from hCG-induced, cyst-bearing pregnant rats.     

Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells

Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).     

Influence of light-dark cycle on antepartum serum relaxin and progesterone immunoactivity levels and on birth in the rat

Relaxin and progesterone are produced by the corpora lutea in the pregnant rat. Relaxin immunoactivity levels are elevated in peripheral sera during the last 12 days of pregnancy. In rats maintained under a conventional photoperiod of 14 h of light (0600-2000 h) and 10 h of darkness (2000-0600 h), there is an antepartum elevation in serum relaxin to maximal levels coincident with the rapid decline in serum progesterone to basal levels during the light phase of the photoperiod 1 day before birth. Therefore, we postulated that this maximal elevation in serum relaxin levels may be temporally associated with functional luteolysis and linked to the photoperiod. In the present study the photoperiod was advanced near midpregnancy in order to examine further the relationship of the antepartum elevation in serum relaxin levels with both functional luteolysis and the photoperiod. Three groups of rats were maintained under a conventional photoperiod of 14 h of light (0500-1900 h) and 10 h of darkness until days 7 and 8 of pregnancy when the photoperiod was advanced 8 h in group 2 (G2) and advanced 18 h in G3 relative to the conventional photoperiod that was maintained in G1. Serum relaxin and progesterone levels were determined in blood samples obtained at 4-h intervals from 2000 h on day 19 of pregnancy until birth. The times of occurrence of birth, maximal relaxin levels, and decline of progesterone to basal levels in G2 and G3 were generaly advanced 50-60% of the advancement of the photoperiod. There was a close temporal association between the attainment of maximal relaxin levels and basal progesterone levels; they both occurred during the light phase of the photoperiod, approximately 24 h before birth in all three groups. We conclude that the antepartum elevation of serum relaxin to maximal levels may be associated with functional luteolysis and that its time of occurrence is influenced by the photoperiod. This study also provides evidence that the antepartum elevation of relaxin levels consists of two phases which occur at a 24-h interval. It is proposed that these two phases in the elevation of relaxin levels may be indicative of an increasingly effective endogenous circadian luteolytic process whose time of occurrence is influenced by the light-dark schedule.     

Influence of starvation on hormonal control of hypophyseal secretion in rats

The reduction of hypophyseal hormone secretion during starvation is not completely understood. A previous study showed that the concomitant reduction of plasma TSH and T3 may be related to an increased sensitivity of the thyrotrope cell to T3. This suggests that regulation of hypophyseal secretion by peripheral hormones may be altered in starved rats. As GH and PRL secretion are under the control of thyroid and steroid hormones, the aim of the present study was to investigate the modification of feed-back control by T3 or E2 on hypophyseal secretion during starvation. For this purpose, pituitary GH, PRL and TSH contents and their plasma responses to TRH injection were measured in euthyroid, thyroidectomized (Tx), T3-supplemented Tx and E2-treated male Wistar rats before and after a 3-day starvation. TRH (0.25 micrograms/100 g) was injected iv through a chronically-implanted catheter. Our results show that GH content and GH plasma response to TRH are dramatically increased in T3-treated Tx starved rats, suggesting that starvation also increases the effectiveness of T3 influence on somatotrope cell secretion. By contrast, effects of T3 on PRL secretion remain unchanged during starvation. Furthermore, starvation in E2-treated rats is associated with a marked rise in the PRL and GH responsiveness to TRH without any significant change of hormonal pituitary content. This suggests that, in starved rats, E2 increases the effects of TRH on lactotrope and somatotrope secretion. No significant effect on TSH secretion could be demonstrated. Thus, starvation seems to act differentially on the feed-back mechanisms controlling the hormonal secretion of the three adenohypophyseal target cells to TRH.     

Differential effects of progestin and relaxin on the synthesis and secretion of immunoreactive prolactin in long term culture of human endometrial stromal cells

PRL secretion from human endometrium is a continuous process extending from the luteal phase of the menstrual cycle throughout the entire gestational stage. We have developed a long term primary cell culture system to elucidate the hormonal requirements for this sustained production of PRL. The effects of medroxyprogesterone acetate (MPA), progesterone, and relaxin (RLX) on the production of immunoreactive PRL were investigated. MPA stimulated cell growth and PRL production rate during days 5-20 of culture. Progesterone was 20-40% less effective in stimulating PRL than MPA. Stimulation of PRL was continued 1-2 weeks after MPA withdrawal. Relaxin did not promote cell growth. However, it induced the PRL production which fluctuated during the long term culture. The maximal response to RLX was 2- to 3-fold higher or similar to that of MPA. Only five of nine endometrial specimens examined responded to RLX alone. The effect of MPA plus RLX was significantly greater than that of MPA or RLX alone. The highest production rate was shown in cells treated with MPA and then RLX in sequence. After a month of culture, the production rates (micrograms of PRL per 0.1 mg cell DNA/day) under various culture conditions (A, control; B, MPA; C, MPA for 10-15 days and no hormone afterward; D, both MPA and RLX; and E, MPA and RLX in sequence) were: A, about 0-0.01 (n = 12); B, 2.5 +/- 0.9 (n = 8); C, 4.8 +/- 2.5 (n = 8); D, 5.7 +/- 3.0 (n = 5); and E, 11 +/- 3.7 (n = 7); mean +/- SD; n, number of specimens). Endometrial stromal cells were incubated with [35S]methionine, and [35S]immunoreactive PRL and other secretory proteins were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to characterize the size and isoforms of immunoreactive PRL. PRL was one of the five major secretory proteins (23-25K, 32K, 42K, 78K, and 150K daltons, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition) induced by MPA and RLX in endometrial stromal cells. More than 90% of immunoreactive PRL was secreted into the medium. The apparent mol wt of immunoreactive PRL were 21K, 23K (the predominant size), and 25K daltons. Results obtained from the incorporation of [14C]glucosamine into immunoreactive PRL indicated that both 23K and 25K PRL contained glycosylated PRL. A 45K-dalton glycosylated immunoreactive PRL was also present in the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of mammary gland development in estrogen receptor-alpha knockout mice

Mammary glands from the estrogen receptor-a knockout (alphaERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERalpha signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the alphaERKO mammary phenotype. We report that circulating PRL is reduced in the female alphaERKO mouse. Implantation of an age-matched, heterozygous ERalpha pituitary isograft under the renal capsule of 25-day-old or 12-week-old alphaERKO mice increased circulating PRL and progesterone levels, and induced mammary gland development. Grafted alphaERKO mice also possessed hypertrophied corpora lutea demonstrating that PRL is luteotropic in the alphaERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in alphaERKO mice despite elevated PRL levels. Hormone replacement using pellet implants demonstrated that pharmacological doses of estradiol induced limited mammary ductal elongation, and estradiol in combination with progesterone stimulated lobuloalveolar development. PRL alone or in combination with progesterone or estradiol did not induce alphaERKO mammary growth. Estradiol and progesterone are required for the structural development of the alphaERKO mammary gland, and PRL contributes to this development by inducing ovarian progesterone levels. Therefore, the manifestation of the alphaERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen signaling at the hypothalamic/pituitary axis.     

THE SENSITIVITY OF GROWTH HORMONE AND PROLACTIN SECRETION TO THE SOMATOSTATIN ANALOGUE SMS 201–995 IN PATIENTS WITH PROLACTINOMAS AND ACROMEGALY

The somatostatin analogue SMS 201-995 has recently been shown to be effective in suppressing GH secretion in most acromegalic patients. In the present study it was investigated whether PRL release in prolactinoma and acromegalic patients might also be sensitive to SMS 201-995 and whether co-secretion of PRL in acromegaly plays a role in determining the sensitivity of GH secretion to SMS 201-995. The s.c. administration of 50 micrograms SMS 201-995 did not affect high plasma PRL levels in four microprolactinoma patients. Therapy of one of these patients for 3 d with 50 micrograms three times a day also did not affect PRL levels. The single administration of 50 micrograms SMS 201-995 in 22 acromegalic patients lowered plasma GH levels for 2-6 h to less than 5 micrograms/l in 14 patients and to less than 50% of control values in 16 patients. In 18 of these 22 patients the immunohistochemical picture of the pituitary tumour was known. Eleven patients had pure GH-containing tumours and in seven patients there were mixed GH/PRL-containing tumours. In two of these latter patients there was evidence for GH and PRL being secreted by the same tumour cells. The sensitivity of GH secretion to SMS 201-995 did not differ between the patients with pure GH or mixed GH/PRL-containing adenomas. Plasma PRL levels were not affected by SMS 201-995 in the patients with pure GH-secreting tumours, but were significantly suppressed in four of the seven patients with mixed GH/PRL containing tumours. Chronic treatment for 16 weeks of one patient with a mixed GH/PRL-containing tumour with SMS 201-995 (100 micrograms three times a day) resulted in normalization of both the increased GH and PRL levels. It is concluded that SMS 201-995 does not affect tumorous PRL secretion in patients with pure prolactinomas. In acromegalic patients with mixed GH/PRL-containing tumours PRL secretion in some patients is sensitive to SMS 201-995, making these patients good candidates for chronic treatment with the analogue. The simultaneous presence of PRL in the GH-secreting pituitary tumour or the presence of hyperprolactinaemia in acromegalics does not play a role in the sensitivity of GH secretion to the somatostatin analogue.     

Role of the ovary in the synthesis of placental protein-14.

Women with absent ovarian function provide an opportunity to investigate the ovarian contribution to secretion of placental proteins (PP), such as PP14, in early pregnancy. We present data on serum PP14 levels in 12 women (median age, 30.5 yr; range, 26-37 yr) with premature ovarian failure (POF) who conceived after ovum donation and embryo transfer with exogenous sex steroid support using transdermal (n = 5) or oral (n = 7) estradiol and vaginal (n = 8) or im (n = 4) progesterone. The women were closely monitored throughout early pregnancy, with measurement of serum levels of estradiol (E2), progesterone (P4), and PP14. Levels of E2 and P4 were entirely normal. Levels of PP14 were significantly subnormal (P = 0.008) in all 12 agonadal women compared with levels of PP14 in a control group of women with normal ovarian function between 6-12 weeks gestation. Basal and peak levels for subjects with absent ovarian function were 40 and 124 micrograms/L, respectively. For each week between 6-12 weeks of pregnancy, the mean serum levels of PP14 for women with normal ovarian function were between 706-940 micrograms/L. These observations support the concept that PP14 arises from the ovary in early pregnancy or that factors under the control of the maternal ovary are involved in its production by the endometrium.     

Relative sensitivity of the corpus luteum of different days of pregnancy to LH-deprivation in the rat and hamster.

Deprivation of endogenous LH by LH antiserum (LH A/S) in 6-day pregnant rats did not affect the luteal or serum progesterone within 24 h. LH A/S treatment on day 7 or 8 of pregnancy, however, caused a 70 and 92% reduction in luteal progesterone, respectively, within 24 h. Serum levels of progesterone showed a similar reduction. In the case of pregnant hamster, unlike the rat, there was a significant decrease in progesterone in the serum, luteal and non-luteal compartments whether the A/S was administered on day 4, 5 or 6. There was more than a 10-fold increase in the luteal cholesterol esters within 24 h whether the A/S was given on day 6, 7 or 8 of pregnancy in the rat. Rat corpora lutea of days 6 and 8 of pregnancy reacted in a like manner to LH-deprivation, showing an increased utilization of [U-14C]glucose to form 14CO2 in vitro. In the rat, LH (25 mug NIH-S19) administration in vivo either on day 6 or day 8 of pregnancy, caused within 2 h an increase in serum and non-luteal progesterone, but luteal progesterone was unchanged. On the other hand, LH administration to hamsters on day 8 of pregnancy caused an increase in progesterone levels in serum, luteal and non-luteal tissue. Incubation of corpora lutea isolated from untreated 6- and 8-day pregnant rats with LH brought about an increase in progesterone secretion into the medium in both cases. The results show that, even though LH-deprivation does not apparently affect progesterone concentration in the corpus luteum of 6-day pregnant rats, it does affect other metabolic parameters such as glucose utilization and cholesterol turnover, suggesting that the corpus luteum of early pregnancy exhibits a continuous dependency on LH for the maintainence of metabolic functions.