Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes

Prolactin (PRL) is one of the pituitary hormones that participate in controlling mammalian folliculo-and oogenesis. In the present study, the combined action of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of the surrounding cumulus cells was investigated in vitro. It has been shown that PRL completely suppresses the inhibitory effect of dbcAMP on meiosis reinitiation and completion of the oocyte nuclear maturation. Moreover, PRL partly inhibited the dbcAMP-induced cumulus expansion, although it produced an opposite effect in the control medium. In the presence of PRL, the inhibitory effect of dbcAMP on the proliferative activity of cumulus cells, as well as on the PRL-induced suppression of destructive processes in these cells, was revealed. In cumulus cells, the mRNA expression of the long PRL receptor isoform was established by the RT-PCR method. The obtained data indicate an interaction of signal cascades induced by PRL and cAMP in the bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes seeming to differ from that in cumulus cells.     

Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes

Prolactin (PRL) is one of the pituitary hormones participating in the control of mammalian folliculo- and oogenesis. In the present study, the joint effect of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. It has been shown that PRL totally suppresses the braking impact of dbcAMP on meiosis reinitiation and the completion of oocyte nuclear maturation. Furthermore, PRL partly inhibited cumulus expansion induced by dbcAMP, although it exerted the opposite effect in the control medium. In the presence of PRL, the inhibitory impact of dbcAMP on the proliferative activity of cumulus cells and on the PRL-elicited braking of destructive processes in the cells has been found. In cumulus cells, mRNA expression of PRL receptor long isoform was revealed by the RT-PCR method. The data obtained suggest an interaction of signal cascades induced by PRL and cAMP in bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes being apparently different from that in cumulus cells.     

Postnatal ovary is necessary for the maturation of estradiol-induced luteinizing hormone and prolactin surges in female rats

The role of postnatal ovary in the maturation of estradiol (E2)-induced luteinizing hormone (LH) and prolactin (PRL) surges was examined in female rats of Wistar-Imamichi strain. Animals were bilaterally ovariectomized at 24 h after birth, 1 week (w), 2 w, 3 w, 4 w or 6 w of age. At about 10 w of age, every group was primed with estradiol benzoate (E2B) for two days, and on the third day was decapitated at either 0900 h or 1900 h. Anterior pituitary (AP) LH and PRL content was determined in every group of no E2B treatment. Surge-like secretions of LH and PRL were observed at 1900 h, only in rats ovariectomized on or after 4 w of age. AP LH and PRL content was the higher, as ovariectomy was delayed. These results indicate that postnatal ovary is necessary for the maturation of E2-induced LH and PRL surges. Such an effect of ovary is mediated at least by its stimulation of AP LH and PRL content.     

Effect of prolactin on ovarian steroidogenesis

This review summarizes evidence that prolactin (PRL) is involved directly in the regulation of ovarian steroidogenesis. The scope of this paper will be limited to two areas. The first area involves the regulation of the corpus luteum function and the second the effect of PRL on steroidogenesis during the follicular phase of the oestrous cycle. Cyclic changes of plasma PRL levels and the ovarian receptors which bind PRL have been observed during the oestrous cycle of domestic animals. The luteotropic effect of PRL and its failure is also presented. PRL may exert a physiological effect on follicle steroidogenesis but its effect depends on the level of follicle maturation. The effect of PRL treatment on estrogen production by follicular cells in vivo and in vitro was studied. The results of many papers indicate suppression of estrogen secretion by the direct action of PRL at the ovarian level. However, there is abundant evidence, that PRL is involved directly in regulation of ovarian steroidogenesis, the precise mechanism remains to be discovered.     

Realization pathways of prolactin modulating effect on the cAMP-dependent mechanism of meiosis regulation in bovine oocytes

Cyclic AMP is one of the key regulators of mammalian meiosis. In the present work, realization pathways of the previously revealed modulating effect of prolactin (PRL) on the cAMP-dependent mechanism of meiosis regulation in bovine oocytes were studied. A comparative investigation of individual and combined effects of PRL (50 ng/ml) and an activator of adenylate cyclase forskolin (FK, 20 μM) on the meiotic reinitiation and completion of nuclear maturation in cumulus-surrounded and cumulus-free oocytes was performed. It has been shown that the pattern of the effects of PRL on the meiotic resumption in oocytes devoid of cumulus cells depends on the presence of FK in the culture medium. Furthermore, the realization of this effect is not associated with the activation of cytoplasmic isoforms of protein kinase C. It has also been found that PRL inhibits the retarding action of FK on the completion of oocyte nuclear maturation both in the presence and absence of cumulus cells. These findings suggest that PRL may modulate the functioning of the cAMP-dependent mechanism of meiosis regulation by the direct action on bovine oocytes, with realization of this action being independent of the metabolic coupling of oocytes with cumulus cells.     

Prolactin modulation of theophylline inhibitory action on maturation of bovine oocyte-cumulus complexes in vitro

The comparative investigation of the individual and joint impact of prolactin (PRL, 50 ng/ml) and theophylline (TP), a nonselective inhibitor of phosphodiesterases, on nuclear maturation of bovine oocytes and the expansion of cumulus cells enclosing the oocytes was carried out using a model of in vitro culturing. It has been shown that TP (5 mM) exerts a short-term inhibitory action on oocyte meiosis reinitiation and blocks it at diakinesis and metaphase I stages as well as inhibits the cumulus expansion. The addition of PRL to the medium containing TP caused the decrease in the rate of oocytes at diplotene stage after 6 h of culturing and the increase in the rate of oocytes attained the closing stages of maturation after 24 h of culturing. Furthermore, PRL suppressed partly the inhibitory impact of TP on the expansion of cumulus cells. The data obtained suggest the signal cascade induced by PRL in bovine oocyte-cumulus complexes to be compled with cAMP-dependent intracellular pathway.     

Prolactin and growth hormone in the regulation of the immune system

Evidence implicating prolactin (PRL) and growth hormone (GH) in the regulation of the immune system has been reviewed. Hypophysectomized animals have deficiencies in both cell-mediated and humoral immunological functions and either PRL or GH corrects these deficiencies. Animals administered bromocryptine, a drug that specifically blocks PRL release, have impaired immune responses similar to hypophysectomized animals, and again both PRL and GH correct these deficiencies. Genetically dwarf animals, which lack both PRL and GH, are also immunocompromised, and once again PRL and GH can correct the deficiencies. In dwarf animals, however, fewer studies have examined PRL actions. In growth-deficient children, immune function is not dramatically altered and basal secretion of GH has been reported. Very few clinical studies have examined whether PRL secretion is also deficient, and this may explain why a clear loss in immune function is not evident in growth-deficient children. In a number of species, including man, both PRL and GH stimulate thymic function and increase the secretion of thymulin, a thymic hormone. No studies, however, have reported on the effects of PRL and GH on other thymic hormones. A number of studies have reported in vitro effects of PRL and GH on cells involved with immunity, and the presence of high-affinity PRL and GH receptors have been observed on a number of these cells. The action of GH on the proliferative response of cells involved with immunity in vitro appears to be mediated by the production of insulin-like growth factor I. The effect of PRL on insulin-like growth factor I production by these cells has not been examined. One of the most consistent findings from in vitro studies is that prolactin antisera blocked a number of immune reactions. This led to the discovery that cells involved with immunity appear capable of producing PRL and GH, but the physiological significance of these observations have not been explored. There is a great need to identify the cell types responding to PRL and GH and this should be a goal of future investigations. There is also a need for investigators to be aware that both PRL and GH are involved in the regulation of the immune system and to design experiments to elucidate where each functions in the maturation cascade of cells involved with immunity. From the evidence available, it is apparent that PRL and GH have an important function in the immune system and future investigations should be directed toward elucidating their site(s) of action.     

Effect of hypo-osmotic environmental changes on the expression of gonadotropin-releasing hormone,its receptor,and gonadotropin hormone subunit mRNA in adult chum salmon (Oncorhynchus keta)

Migrating fish such as salmonids are affected by external environmental factors and salinity changes are particularly important, influencing spawning migration. The aim of this study was to test whether changes in salinity would affect the expression of the hypothalamic-pituitary-gonadal (HPG) axis hormones (gonadotropin-releasing hormones (GnRHs) [salmon GnRH and chicken GnRH-II], GnRH receptors [GnRHR1 and GnRHR5], and mRNA of the gonadotropin hormone [GTH] subunits [GTHα, follicle stimulating hormone β, and luteinizing hormone β]) in chum salmon (Oncorhynchus keta). Fish were progressively transferred from seawater (SW) through 50% SW to freshwater (FW), and the relationship between the osmoregulatory hormone prolactin (PRL) and sexual maturation was determined. The expression and activity of HPG hormones and their receptors, and levels of estradiol-17β and PRL increased after fish were transferred to FW, demonstrating that changes in salinity stimulate the HPG axis and PRL production in migrating chum salmon. These findings reveal details about the role of the endocrine system in maintaining homeostasis and stimulating sexual maturation and reproduction in response to salinity changes in this species.     

Impact of prolactin on the growth rate pattern and biochemical components of accessory sex organs of male albino rats during puberal transition

Prolactin (PRL) was administered subcutaneously to different age-groups of male albino rats across the puberal transition. The tissue somatic indices (TSI) and organic components of accessory sex organs were increased in the presence of PRL at all the age-groups. The participation of PRL in the acceleration of maturation events of accessory sex organs was age-dependent.     

Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro

At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals. We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ([Ca2+]is) in the oocytes. Cumulus-oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3). Levels of [Ca2+]is in oocytes were determined by using the fluorophore chlortetracycline. The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3). Compared with the control, lower levels of [Ca2+]is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs. 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs. 1.52+/-0.04 AU in System 3, P<0.001). These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in [Ca2+ ]is levels at early stages of oocyte maturation.     

Participation of granulosa cells in mediation of prolactin and somatotropin action on bovine oocyte-cumulus complexes in vitro

Maturation of bovine oocyte-cumulus complexes (OCC) in media derived following granulosa cell culturing with prolactin (PRL, 50 ng/ml) and somatotropin (ST, 10 ng/ml) was studied. A medium conditioned by granulosa cells in the presence of PRL or ST exerted a stimulating effect on the proliferative activity of cumulus cells. ST introduction into the granulosa cell culture also caused a decrease in the rate of cumulus cells with degenerated chromatin at a subsequent OCC culturing. At the same time, the expansion of cumulus did not depend on hormone availability in the culture medium for granulosa cells. When OCC matured in conditioned media, a short-term inhibition of oocyte meiosis reinitiation (after 6 h of culturing) was revealed in both the experimental groups, as compared to the control. Furthermore, the addition of ST and PRL to granulosa cell culture resulted in a subsequent decline in the rate of oocytes with signs of chromosome degeneration, observed as early as by 6 h of incubation and to be retained throughout the whole period of OCC culturing. In this case the earlier resumption of meiosis was associated with a higher rate of degeneration of the nuclear material in oocytes. The results of the present study suggest that granulosa cells may mediate, at least in part, PRL and ST impacts on in vitro maturation of bovine OCC, with no contact between OCC and granulosa cells being required for hormonal signaling.     

Postnatal development of growth hormone and prolactin cells in male and female rat pituitary. An immunocytochemical light and electron microscopic study

Localization and ultrastructural maturation of prolactin (PRL) and growth hormone (GH) cells were studied in pituitaries from neonatal, immature (4-6 weeks old), and adult rats (2-3 months old) by light and electron microscopic immunocytochemistry. The distribution pattern of these cells did not change with age. Both cell types were concentrated laterodorsally, with PRL cells adjacent to the intermediate lobe and GH cells nearer the center of the pars distalis. Labeling density of the immunogold reaction was highest for both hormones in immature rats. In neonatal and immature rats, one PRL cell type with granules 200 nm in diameter was present. In adult rats, two types of PRL cells were present: one containing polymorphous granules measuring about 500 nm (prevalent in female rats), the other with spherical granules about 200 nm (prevalent in male rats). No changes were detected in GH cells during maturation.     

Effect of taurine on growth hormone and prolactin secretion in rats: possible interaction with opioid peptidergic system

The effect of taurine on growth hormone (GH) and prolactin (PRL) secretion was investigated in the urethane-alpha-chloralose anesthetized rats, considering the interaction with endogenous opioid peptidergic system. Intraventricular injection of taurine (0.25 and 1.0 mumol) stimulated GH and PRL secretion in a dose-dependent manner. However, 4.0 mumol taurine failed to show these effect. The intravenous infusion of naloxone (4 mg/kg b.w.) completely inhibited both the GH and PRL secretion induced by taurine (1.0 mumol). The combined treatment of taurine (1.0 mumol) and FK33-824 (Met-enkephalin derivative, 100 micrograms/kg b.w., i.v.) significantly increased GH and PRL responses induced by taurine or FK33-824 alone. These results indicate that taurine is an effective stimulator of GH and PRL secretion in rats, and that the mechanism of this action involves the opioid peptidergic system in the hypothalamus.     

A permissive role for extracellular Ca2+ in regulation of prolactin production by 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

A clonal strain of rat pituitary tumor cells (GH3) that spontaneously synthesizes and secretes prolactin (PRL) and growth hormone (GH) was used as model system to study the mechanism of action of 1,25-(OH)2D3. We have previously demonstrated that these cells possess specific cytosol binding proteins for 1,25-(OH)2D3 (Haug and Gautvik, 1985). When the GH3 cells were incubated in a serum-free, chemically defined medium of low extracellular Ca2+ concentration, 1,25-(OH)2D3 stimulated PRL production in a dose-dependent manner. The stimulation was detectable at 10(-11) M, and the maximum effect (2-fold increase) was observed at 10(-9) M (ED50 = 2 x 10(-11) M). The dose-response curve was bell-shaped, and at 10(-6) M 1,25-(OH)2D3 even suppressed PRL production to about 75% of controls. The stimulatory effect was first seen after 2 days and was maximal after 4 days. On a molar basis 25-OHD3 and 1-OHD3 were at least 100 times less potent than 1,25-(OH)2D3, while 24,25-(OH)2D3 had no effect on PRL production. At an extracellular concentration of Ca2+ as low as 4 x 10(-5) M the stimulatory effect of 1,25-(OH)2D3 was small (1.3-fold). Increasing extracellular Ca2+ to 1.5 x 10(-4) M increased the 1,25-(OH)2D3-induced PRL response to 2.1-fold. In contrast to the biphasic effect of 1,25-(OH)2D3 on PRL production, GH production was decreased to about 60% of controls at 10(-8) M and above. These findings indicate that in serum-free medium the stimulatory effect of 1,25-(OH)2D3 on PRL production is critically dependent on the concentration of extracellular Ca2+.     

The stimulatory effect of estradiol 17-beta on prolactin mRNA is inhibited by anti-calmodulin drugs.

The stimulatory action of estrogens on prolactin (PRL) secretion and synthesis is well known; on the other hand, anti-calmodulin drugs have recently been shown to inhibit prolactin in vitro release induced by estrogens. Based on these data, we decided to evaluate the in vivo effect of anti-calmodulin drugs (trifluoperazine and W7) on basal and estradiol-17 beta stimulated levels of PRL mRNA in anterior pituitary lobes obtained from adult male rats. Total RNA was isolated from pooled pituitaries recovered from animals under the same treatment and, from it, hybridizable PRL mRNA was detected. Estradiol-17 beta consistently stimulated PRL mRNA levels by 3-4 fold. The utilization of either trifluoperazine or W7, invariably inhibited estradiol-17 beta stimulated PRL mRNA. Metoclopramide, a drug with antidopaminergic activity, potentiated the stimulatory effect of estradiol-17 beta on PRL mRNA levels. These results suggest that anti-calmodulin drugs have an in vivo antiestrogenic effect on PRL mRNA levels confirming previous in vitro studies. Although, it is difficult to be conclusive about the mechanism through which these drugs act, one possibility is that the calcium-calmodulin system may be involved.     

Stimulatory action of prolactin on gonadotropin secretion in vitro

The action of prolactin (PRL) on the secretion of gonadotropin was investigated by means of a cell culture system of rat anterior pituitary gland. Anterior pituitary glands were removed from Wistar male rats, enzymatically digested and cultured. Luteinizing hormone (LH) release into medium was increased by adding PRL dose-dependently in the range between 10 ng/ml and 1 microgram/ml. This effect of PRL was further augmented by the presence of either gonadotropin-releasing hormone or estradiol. The intracellular LH concentration was also increased by PRL. PRL also caused an increase in follicle-stimulating hormone release into medium dose-dependently. In conclusion, PRL was shown to stimulate the secretion of gonadotropin at the pituitary level, thus suggesting a paracrine mode of PRL action in the anterior pituitary gland.     

FSH-stimulated follicular secretions enhance oocyte maturation in pigs

Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.