Inhibition of human chorionic gonadotropin-induced ovulation and steroidogenesis by short-term hyperprolactinemia in female rabbits

To clarify the possible direct effects of hyperprolactinemia on the ovulatory process, we experimentally established hyperprolactinemia in female rabbits with 4 daily injections of sulpiride (SLP) at different doses and induced ovulation with human chorionic gonadotropin (hCG). Plasma levels of prolactin (PRL) were increased significantly before hCG injection in each SLP-treated group compared with the corresponding values for the controls. The ovulation rates at 14 h after hCG were significantly reduced in the 16 and 24 mg/kg/day SLP-treated groups. An inverse correlation (r = -0.74, P less than 0.001) was found between the ovulation rate and the increasing in plasma PRL measured just prior to hCG injection. The increase in peripheral as well as ovarian venous progesterone and 20 alpha-hydroxypregn-4-en-3-one(20 alpha-OHP) at 4 and 14 h after hCG injection in inhibited ovulation groups was much less than in the control group. However, the estradiol, androstenedione and testosterone concentrations were comparable with the control values. These results indicate that hypersecretion of PRL induced by SLP has a direct effect on ovary by inhibiting follicular rupture induced by hCG and this inhibitory effect was partly due to the suppression of progesterone secretion during the course of ovulation. This may be one of the causes leading to hypogonadism during hyperprolactinemia.     

Effects of prolactin on ovarian plasmin generation in the process of ovulation.

The present study was undertaken to assess the effects of prolactin (PRL) on gonadotropin-induced plasmin generation in the in vitro-perfused rabbit ovary. The ovarian plasmin activity was determined by measuring plasmin bound to its major inhibitor, alpha 2-plasmin inhibitor (alpha 2 PI-Plm). In the first experiment, exposure to hCG enhanced ovarian alpha 2 PI-Plm generation from 1.2 +/- 0.3 ng/min/ovary in unstimulated ovaries to 2.9 +/- 0.3 ng within 2 h. The concentration of alpha 2 PI-Plm reached a maximum at 4 h and then declined. A second peak occurred 8 h after hCG administration; however, the ovarian alpha 2 PI-Plm generation without hCG was very low throughout the entire perfusion period. In the subsequent experiment, the addition of PRL(10-10(3) ng/ml) to the perfusate inhibited hCG-induced ovulation in a dose-dependent manner. Exposure to PRL at 10(3) ng/ml significantly (p less than 0.05) inhibited hCG-induced alpha 2 PI-Plm generation in ovaries throughout the entire perfusion period. Furthermore, PRL inhibited hCG-stimulated alpha 2 PI-Plm generation at 4 h after hCG administration in the perfused rabbit ovaries in a dose-dependent manner. In conclusion, PRL directly inhibits hCG-induced ovulation in rabbit ovary, at least in part, by a mechanism depending upon inhibition of the plasmin-generating system in the preovulatory follicles.     

Hormonal regulation of testicular prolactin receptors and testosterone synthesis in golden hamsters

A study was conducted with hypophysectomized hamsters to determine effects of administration of prolactin (PRL), luteinizing hormone (LH), and follicle-stimulating hormone (FSH)-alone or in combination-on testicular PRL receptors and in vitro testosterone production. Hormonal injections commenced the second day after hypophysectomy, and hamsters were killed on Day 5, approximately 13 h after the last hormonal injection. PRL receptor numbers were reduced by hypophysectomy, and PRL administration alone lessened the extent of this decrease. By themselves, neither LH nor FSH affected PRL receptors, but a combination of PRL + FSH + LH produced the greatest effect on these receptors. Receptor affinity was only modestly affected by any treatments. In vitro testosterone synthesis was measured after addition of 0, 2, 10, and 50 mIU of human chorionic gonadotropin (hCG) to incubations of testicular tissue. Neither PRL nor FSH by themselves in vivo affected basal or hCG-stimulated testosterone production. However, PRL + FSH increased (p less than 0.05) the magnitude of the in vitro testosterone response to hCG, as well as the sensitivity of that response (slope of the dose-response curve). LH alone increased both basal and hCG-stimulated testosterone production. PRL + LH provided no additional increase in the magnitude of the testosterone response, but increased (p less than 0.05) the sensitivity. PRL + FSH + LH in vivo provided for the greatest sensitivity of the testosterone response to hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

催乳素对培养的绵羊睾丸间质细胞睾酮分泌的影响

在绵羊睾丸间质细胞体外无血清长期培养的条件下,研究了催乳素对睾丸间质细胞睾酮分泌的调节作用。实验结果表明,催乳素可增强细胞对人绒毛膜促性腺激素(hCG)刺激的反应。催乳素的这种作用呈双相调节。睾酮分泌量显著高于hCG和催乳素单独作用时的总和。在hCG存在下,不同的底物转化为睾酮的量不同。其中雄烯二酮和孕酮转化为睾酮的方式存在着双相性。脱氢表雄酮转为睾酮的量少,不存在双相性,而与其剂量成正比。催乳素在hCG存在下可调节底物转化为睾酮。低剂量的催乳素(1ng/ml)可使一定剂量的孕酮(10～30ng/ml)转化为睾酮的量明显增加,而高剂量的催乳素(>10ng/ml)却明显地抑制孕酮转化为睾酮。催乳素可明显地抑制雄烯二酮转化为睾酮,与剂量无关。可见催乳素对于孕酮和雄烯二酮这两个关键底物转化为睾酮的调节是不同的。催乳素增强hCG刺激睾酮分泌的作用可能部分是通过其促进孕酮转化为睾酮来实现的。     

Effects of prolactin on the morphology of cultured rat granulosa cells

Morphological changes were correlated with biochemical data induced by prolactin (PRL) in cultured rat granulosa cells from large preovulatory follicles. Biochemical results indicated that PRL exerted a significant dose-dependent inhibition in gonadotrophin-induced secretion of progesterone and 17 beta-oestradiol. PRL alone failed to affect basal steroidogenic secretion. In parallel morphological experiments, using phase-contrast microscopy, untreated and 100 ng/ml PRL-treated cells appeared as a monolayer of flattened, fibroblast-like cells. Upon exposure to 0.4 IU/ml human chorionic gonadotrophin (hCG), aggregates of rounded, epithelioid-shaped cells were formed. The addition of PRL to hCG in the same doses minimized the changes induced by hCG. Similarly, electron microscopy of untreated and PRL-treated cultures revealed flat cells devoid of microvilli, with evenly dispersed microfilaments. The addition of hCG caused rounding of the cells and was accompanied by the appearance of microvilli and by pronounced steroid-producing organelles. Bundles of microfilaments were noted at the cell periphery. PRL added to hCG caused a reduction of the hCG effects, and the cell morphology was intermediate to that seen in untreated and hCG-treated cultures. The finding that PRL can prevent or minimize morphological changes caused by hCG in rat cultured granulosa cells correlates with the biochemical changes induced by PRL, and supports the concept that PRL is a modulator of gonadotrophic action in the ovary.     

Natural and artificially induced ovulatory models related to lactation in the rat: role of prolactin

The presence and the importance of a preovulatory prolactin (PRL) peak was determined in four, natural or artificially induced, ovulatory models related to lactation in the rat. Gonadotrophin peaks were determined in the afternoon preceding ovulation in four models: postpartum ovulation (PPO), ovulation after the lactational period (AL) (natural models), ovulation after litter removal at midlactation (ML), and ovulation in lactating rats (LR) (artificially induced models). In PPO, AL, and ML rats a preovulatory PRL surge was detected, showing that its presence is a common characteristic of ovulation in the rat. Bromocriptine inhibition of PRL levels in PPO and AL rats did not modify the percentage of rats which ovulated. In contrast, this treatment was able to significantly increase ovulation percentage in ML rats. Moreover, in LR rats strong dopaminergic inhibition of PRL levels, induced by pergolide, was necessary for ovulation to take place, but if pergolide-treated rats were injected with ovine PRL ovulation was completely inhibited. These data suggest that while a PRL surge seems to be always present in natural ovulatory models, it is not essential for ovulation to take place. On the other hand, in artificially induced ovulatory models, suppression of prolactinemia is able to induce ovulation or to increase the percentage of rats which ovulated. This effect of PRL on ovulation may be direct or indirect.     

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.     

Identical changes in Bax expression, but not Fas ligand expression, occur in structural luteolysis in gonadotropin releasing hormone agonist- and prolactin-treated superovulated rats

Structural luteolysis induced by gonadotropin releasing hormone agonist (GnRHa) or prolactin (PRL) is defined as histological involution of the corpus luteum. We reported that one of the mechanisms of structural luteolysis induced by PRL was tissue remodeling by matrix metalloproteinase (MMP) and also apoptosis in superovulated rats. We also reported that GnRHa induced structural luteolysis with elevation of MMP. In this study, we investigated whether GnRHa caused apoptosis in mature corpus luteum of superovulated rats and also examined the expression of apoptosis-related molecules (Fas, Fas ligand (FasL), Bcl-2, Bax). We gave 4-day GnRHa treatment 5 days after hCG injection to immature female rats treated with pregnant mare surum gonadotrophin (PMSG) and hCG to induce structural involution of mature corpus luteum. PMSG-hCG-treated rats without GnRHa treatment, rats treated with bromocryptine (Brom) to induce functional luteolysis and rats treated with Brom followed by PRL (Brom+PRL) to mimic the PRL surge to induce structural luteolysis as we previously reported were used for comparison. GnRHa treatment caused structural luteolysis characterized by structural involution, a decrease in the serum progestin level, and apoptotic bodies as well as structural luteolysis induced by Brom+PRL. FasL expression in corpora lutea was elevated after Brom treatment, but there was no elevation of FasL after GnRHa treatment started. FasL expression decreased and Bax expression increased in structural luteolysis induced by GnRHa as well as Brom+PRL treatment, although Fas and Bcl-2 expression did not change throughout the luteal phase. In summary, both GnRHa and Brom+PRL caused structural luteolysis, one of whose mechanisms was apoptosis with an increase in Bax expression, but not with an identical change in FasL expression. It is speculated that the significance in alteration of FasL may involve some mechanism other than apoptosis.     

Dose response of luteinized porcine granulosa cells in vitro to prolactin: dependency on pre-exposure to human chorionic gonadotrophin

This study examined the hypothesis that human chorionic gonadotrophin (hCG) increases prolactin (PRL) stimulation of the utilization of lipoprotein-borne cholesterol by pig luteinized granulosa cells in culture. These cells, which luteinize in culture, were harvested from 6-mm or greater diameter follicles and cultured in the presence of 1% fetal calf serum and 1 microgram/mL insulin for 48 h. On the third day, the media were replaced with fresh serum-free media, with the same dose of insulin, and on the following day (day 4) the media were replaced with serum- and insulin-free media. At this time (day 4) hCG was added to some cultures. On day 5, cells from the group with hCG and cells from the group without hCG were treated with graded doses of ovine PRL (0.1-3.0 micrograms/mL). To a second set of cells, likewise treated, 100 micrograms of porcine low density lipoprotein (LDL) was added. Two days later (day 7) media were sampled and replaced with media alone or media containing hormones and (or) LDL. On day 9 cultures were terminated. In the cells pre-exposed to hCG, PRL (1 microgram/mL) in the presence of LDL increased progesterone production 1.7-fold (p less than 0.01) on day 7 and 2.2-fold (p less than 0.01) on day 9. In the granulosa cells in culture pre-exposed to hCG, the effect of PRL on LDL utilization was dose dependent and saturable at 1 microgram/mL on days 7 and 9. We conclude that brief pretreatment of luteinized pig granulosa cells with hCG results in a dose-dependent PRL-induced utilization of LDL for progesterone synthesis.     

Effects of cycloheximide during the periovulatory period on ovarian follicular FSH, hCG, and prolactin receptors and on follicular maturation in the hamster

Hamsters were injected sc at 1400 hr on proestrus with either 4 mg cycloheximide (which blocks ovulation but only transiently affects ovarian protein synthesis) or saline and killed at 2-hr intervals until 0400 hr on estrus. After cycloheximide, the first surge of FSH (at 1600 hr) was half the normal value and the second surge of FSH (beginning at 2200 hr) was eliminated. Control follicles at 1400 hr had approximately the same number of FSH and hCG receptors with about one-third as many PRL receptors. Down regulation of FSH and hCG receptors for control follicles occurred by 2400 hr while PRL receptors dropped abruptly 4 hr earlier. Compared to the 1400-hr control values, the maximal loss of FSH, LH, and PRL receptors was 40, 45, and 85%, respectively. Although cycloheximide tended to slightly delay the loss of FSH receptors at 2000-2200 hr it did not prevent the ultimate fall in FSH and hCG receptors; the loss of PRL receptors was accelerated by 4 hr. Cycloheximide prevented or delayed follicular growth, resumption of meiosis, and cumulus expansion. The altered proestrous profile of steroids after cycloheximide (prolonged follicular estradiol and reduced progesterone) is therefore not associated with drastic alterations in the number of FSH and hCG binding sites. On the other hand, PRL receptors represent fast turnover protein(s).     

Effect of prolactin and cyclic AMP on 125I-labelled low density lipoprotein uptake and metabolism by luteinized porcine granulosa cells in culture

The present study examines the effect of prolactin (PRL) and N6-2(1)-O-dibutyryladenosine 3'5'-cyclic monophosphate (cAMP) on low density lipoprotein (LDL) uptake and metabolism by luteinized porcine granulosa cells in culture. Granulosa cells from preovulatory follicles were plated with 1% serum and 1 microgram/mL of insulin for the first 48 h. Following plating (day 3) the cells were cultured in serum-free media with the same dose of insulin. The next day the medium was replaced with serum- and insulin-free medium, and to some cultures 1.23 IU/mL of human chorionic gonadotrophin (hCG) was added. On day 5 the medium was again replaced and graded amounts of PRL (0, 0.03, 0.3, and 3 micrograms/mL) were added. Following 48 h of incubation with PRL, 20 micrograms/mL of 125I-labelled LDL was added to cultures. Surface-bound, internalized, and degraded LDLs were quantitated at 12 h following addition of LDL. To examine the effect of cAMP on LDL metabolism, the cells were exposed for 24 h to cAMP (3mM) on day 6 of culture. PRL had a stimulatory effect on LDL degradation by luteinized granulosa cells. Pre-exposure of cells to hCG augmented the stimulatory effect of PRL. Addition of cAMP also enhanced LDL degradation by luteinized granulosa cells. Both PRL and cAMP increased surface binding of LDL in cells pre-exposed to hCG, but there was no effect on internalization. The increase in cell surface binding of LDL with PRL and cAMP was less than their effect on LDL degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactational [correction of Lacational] anovulation in rats and its dependency on progesterone

The relationship between prolactin (PRL) secretion and anovulation in lactating rats was studied. Normal lactating rats and lactating rats treated with antiserum against luteinizing hormone-releasing hormone at the time of postpartal ovulation were used. Normal lactating rats were treated with either a dopamine agonist (CB-154, 150 micrograms/rat) on Day 10 or 13, or pups removal on Day 7 or 10, and thereafter luteolysis and inhibition on PRL secretion were assessed. With the CB-154 treatment, the incidence of luteolysis increased as the lactational period advanced (42% vs 72%), whereas it decreased (73% vs 14%) with the pups removal. Thus, dopamine effectively inhibited PRL secretion during the later lactational stage, but could not do so during the earlier stage when there were mechanisms other than dopamine stimulating PRL secretion. Following luteal regression induced by CB-154, ovulation did not occur if the rats were treated with CB-154 on Day 10, whereas 50% of the rats ovulated within 4 days if treated on Day 13. Furthermore, in the lactating rats treated with anti-luteinizing hormone-releasing hormone serum during late pregnancy, ovulation was not observed until Day 10 of lactation. Since the serum progesterone levels were low in these rats due to the absence of ovulation and lactational corpora lutea, the blockade of ovulation was not due to elevated circulating progesterone during the early lactational period. The mechanism of ovulation blockade during lactation thus seems to shift from being progesterone independent to progesterone dependent at a similar period when the neuroendocrine control of PRL secretion shifts from dopamine independent to dependent.     

Effects of deletion of the prolactin receptor on ovarian gene expression

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

Effects of neonatal treatment with diethylstilbestrol and 17 alpha-hydroxyprogesterone caproate on in vitro pituitary prolactin secretion

The effects of neonatal hormone treatment with diethylstilbestrol (DES) and 17 alpha-hydroxyprogesterone caproate (HPC) on days 1-5 of life on serum prolactin (PRL) levels and 3H-PRL synthesis and release were studied in C3H/MTV+ mice at 2, 4, 6, 8 and 10 weeks of age. Neonatal treatment of mice with 2.5 micrograms/day DES was the only treatment that affected the developmental pattern of serum PRL levels. Serum PRL levels were significantly decreased at 6 wks of age with this dose of DES. Neonatal treatment with 2.5 micrograms/day DES and 150 micrograms/day HPC affected the developmental pattern of H-PRL synthesis by the pituitary. At 10 wks of age 3H-PRL synthesis was significantly decreased by these doses of DES and HPC. The percent of 3H-PRL released did not differ between neonatally hormone treated and control animals, suggesting that neonatal treatment affected mechanisms that regulate PRL synthesis but not those that regulate release.     

Circulating hormone concentrations in hypothyroid rats with induced polycystic ovaries

The induction of polycystic ovaries in hypothyroid rats by human chorionic gonadotropin (hCG) has been studied for many years. A complete understanding of this phenomenon requires information regarding the circulating levels of the hormones of the hypophyseal-gonadal axis. In this study, serum prolactin (PRL), luteinizing hormone (LH), estradiol, testosterone, and progesterone were measured by radioimmunoassay at intervals during the 40-day period in which large ovarian cysts were induced in hypothyroid rats by daily injections of hCG. After 20 injections, ovaries increased in weight 10-fold, and well-developed ovarian cysts were present, accompanied by lutein tissue; cyst development continued for the subsequent 20 days of hCG. Both PRL and LH rose during the first 5 days of treatment and were maintained at high levels from day 20 on. The pattern of change of gonadal steroids showed greater increases with hCG in hypothyroid than in euthyroid rats. Levels of estradiol in hypothyroid, hCG-injected rats increased in parallel to ovarian hypertrophy, whereas progesterone was high in initial stages and then declined. Testosterone increased in both euthyroid and hypothyroid animals, with no clear pattern coincident with cyst formation. The data suggest that the formation of polycystic ovaries in the hypothyroid rat is associated with high levels of PRL and LH followed by elevations of estradiol, which may serve to maintain continuous PRL, as well as LH, stimulation of the ovary.     

In vivo and in vitro effects of prolactin and growth hormone on lipid metabolism in a teleost, Anabas testudineus (Bloch)

Prolactin (PRL) has an important role in the regulation of water and electrolyte homeostasis in teleosts. The present study was designed to evaluate the role of PRL and GH on malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) in Anabas testudineus. Ovine prolactin significantly inhibited ME, G6PDH and ICDH activities when administered in vivo compared to vehicle treated controls. In vivo administration of PRL reversed the action of bromocryptine on enzyme activities. Ovine growth hormone in vivo also modified the effect of bromocryptine but not to the level of prolactin. Combined action of PRL+GH in vivo was most effective in keeping the enzyme activities at normal level after bromocryptine treatment. Prolactin in vitro also reversed the action of bromocryptine on enzyme activities, while GH in vitro failed to do so. Hence, prolactin seems to have an inhibitory effect on lipid metabolism in this teleost. Combined action of PRL+GH is more prominent in in vivo conditions at low PRL levels. Dopaminergic pathways may be involved in the control of prolactin and to some extent on growth hormone secretion.     

FSH-induced ovulation in intact and hypophysectomized mice

A single, ovulatory dose of 25 micrograms of a highly purified preparation of ovine FSH caused ovulation in 89% of hypophysectomized and 91% of intact female mice primed 48 h earlier with PMSG; the number of oocytes recovered (29.4 +/- 4.7 and 22 +/- 2.7/mouse ovulating, respectively) compared favourably with the 20.0 +/- 2.9 oocytes per ovulating female recovered from animals that received PMSG + hCG. After oFSH injection, 82% of oocytes released were fertilized and developed to blastocysts. That the trace contamination (less than 0.2%) of the oFSH with oLH was not responsible for the ovulation was shown by the markedly reduced number of oocytes collected from ovulating females that were injected with equivalent low levels of hCG (0.001 micrograms) or oLH (1 microgram) (9.0 +/- 3.3 and 8.0 +/- 3.1, respectively). These results demonstrate that oFSH is as effective as LH in inducing ovulation of competent oocytes in the mouse.     

Suppression of the proestrus prolactin surge in the rat estrous cycle by urethane anesthesia

Urethane-anesthetized male rats have been used for the analysis of prolactin (PRL)-releasing substances on PRL secretion. However, there are only a few reports investigating the effect of urethane anesthesia on PRL secretion in female rats. In this study, we intended to examine the effects of urethane anesthesia on PRL secretion during proestrus in the rat. Proestrus PRL surge was completely blocked when urethane was administered to rats prior to the critical period of proestrus both at doses of 1.0 g/kg and 1.5 g/kg. Additionally, urethane, at a dose of 1.5 g/kg, was also effective in blocking spontaneous ovulation. An experiment examining pituitary PRL concentration at 1800 h confirmed that urethane (1.0 g/kg) anesthesia prevents the PRL surge from the pituitary. Similarly, urethane anesthesia blocked the LH surge from the pituitary, but LH levels in the urethane-treated group were higher than those in the pentobarbital-treated group.