Oxytocin and progesterone release from bovine corpus luteal cells in culture: effects of insulin-like growth factor I, insulin, and prostaglandins

The ruminant corpus luteum synthesizes and secretes oxytocin, but little is known of the regulation of these processes in the ovary. In the present work we describe a method for the preparation of cells from the early bovine corpus luteum (1-5 days postovulation) and their maintenance in serum-free culture. The release of oxytocin and progesterone from these cells was increased by the addition of insulin or insulin-like growth factor I (IGF-I), but not by IGF-II. Hormone release (measured between 60 and 84 h of culture) was increased approximately 5-fold (oxytocin) and 2.5-fold (progesterone) by maximally effective concentrations of IGF-I (EC50, 0.27 nM) and insulin (EC50, 1.94 nM). Sustained exposure (0-84 h) to prostaglandins (PGs) caused a dose-dependent reduction in oxytocin release in the presence of IGF-I (PGF2 alpha EC50, 31 nM; rank order of potency, PGF2 alpha greater than PGE2 greater than PGE1), but did not markedly reduce progesterone release. The inhibitory effect of PG on oxytocin production was mimicked by sustained exposure to a protein kinase-C activator (phorbol 12,13-dibutyrate), supporting the proposed role for this enzyme as a mediator of PG action. These data provide the first demonstration that oxytocin release from early bovine corpus luteal cell cultures can be regulated by insulin, IGF-I, and PGs. Since granulosa and/or luteal cells produce and respond to IGF-I and PGF2 alpha, our data indicate functional interaction of these compounds in the regulation of luteal cell activity.     

Secretory activity of bovine ovarian granulosa cells transfected with sense and antisense insulin-like growth factor (IGF) binding protein-3 and the response to IGF-I, GH, LH, oxytocin and oestradiol.

The aim of our in vitro experiments was to examine if IGF binding protein (IGFBP)-3 is involved in control of bovine ovarian secretory activity. For this purpose we performed the transfection of bovine granulosa cells with cDNA sense and antisense constructs increasing or inhibiting IGFBP-3 synthesis. The release of IGFBP-3, progesterone, oxytocin, IGF-I and prostaglandins F (PGF) and E (PGE) by control and transfected cells was compared. The transfected ovarian cells were cultured with and without bLH (100 ng/ml), bGH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and oestradiol-17beta (100 ng/ml). The concentration of IGFBP-3 produced was assessed using ligand and western blotting and secretion of progesterone, oxytocin, IGF-I, PGF and PGE was evaluated using RIA/IRMA techniques. Transfection of cells with the sense IGFBP-3 cDNA construct resulted in the expected increase in IGFBP-3 release, whereas the antisense IGFBP-3 construct induced the expected reduction in IGFBP-3 output. The granulosa cells transfected to overexpress IGFBP-3 had an increase in IGF-I, PGF and PGE release, and a decrease in basal and hormone- or growth factor-induced accumulation of progesterone and oxytocin. The granulosa cells transfected to have reduced IGFBP-3 expression gave primarily significant opposite findings. The present results suggest the involvement of IGFBP-3 in control of bovine ovarian steroid, peptide hormone, growth factor and prostaglandin release. IGFBP-3 is a physiological stimulator of IGF-I and prostaglandin release and an inhibitor of steroid and peptide hormone output.     

Regulation, action and transport of prostaglandins during the periovulatory period in cattle

Follicular production of prostaglandins (PGs) is essential for mammalian ovulation, but the factors that mediate production and the cell-specific action(s) of PGE and PGF2alpha during the ovulatory cascade remain largely unknown. The aims of these experiments were: (1) to investigate the potential role of oxytocin (OT) in ovulatory PG production, (2) to determine cellular and temporal patterns of expression of mRNA for specific PG receptors during the periovulatory interval, (3) to determine cell-specific effects of PGE2 on progesterone secretion, and (4) to investigate the potential for an active transport mechanism that may regulate the effect of PGs during the ovulatory cascade, using cattle as the animal model. Heifers were treated sequentially with PGF2alpha and GnRH to induce luteal regression, a follicular phase and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). In experiment 1, OT increased the secretion of PGE and PGF2alpha by granulosa cells collected from preovulatory follicles (before the LH/FSH surge) and OT production by pieces of follicle wall from periovulatory follicles (after the LH/FSH surge) was regulated by progesterone acting through the progesterone receptor. In experiment 2, levels of mRNA for the PGF2alpha receptor and three PGE receptor subtypes were determined by semi-quantitative RT-PCR in theca interna and granulosa cells from pre- and periovulatory follicles collected at 0, 6, 12, 18 and 24 h post-GnRH. Time- and cell-specific patterns of change in mRNA for PG receptors were observed, suggesting multiple effects of both PGE and PGF2alpha in both theca interna and granulosa cells throughout the ovulatory cascade. Cell-specificity of PG action was confirmed in experiment 3; PGE2 increased the secretion of progesterone by theca interna but not granulosa cells collected from late periovulatory follicles. The results of experiment 4 revealed the expression of mRNA for the bovine PG transporter in theca interna and granulosa cells and its regulation during the periovulatory period, thus revealing the presence of a potential transport mechanism that could regulate cellular distribution of PGs throughout the ovulatory cascade. Taken together, these results provide new insight into mechanisms that regulate the production, distribution and site of action of PGE and PGF2alpha during the ovulatory cascade.     

Paracrine regulation of insulin-like growth factor I (IGF-I) an IGF-II on prostaglandins F2alpha and E2 synthesis by human corpus luteum in vitro: a possible balance of luteotropic and luteolytic effects.

The existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins has been demonstrated as well as the ability of IGF-I to positively affect steroidogenesis in human granulosa cells. Furthermore, we recently showed that IGF-I and IGF-II stimulate progesterone secretion by human luteal cells. As the PGs, PGE2 and PGF2alpha, are classically known to have luteotropic and luteolytic effects, we wanted to determine whether the IGFs could affect the human luteal phase by influencing the PG system. For this reason, human luteal cells were cultured for different times (12, 24, and 48 h) with IGF-I, IGF-II (10-100 ng/mL), and GH (100 ng/mL), and both PGs were assayed in the medium culture. We found that both IGF-I and IGF-II were able to stimulate PGE2 synthesis in a time- and dose-dependent way, whereas they both inhibited PGF2alpha production. GH, too, significantly reduced PGF2alpha synthesis; this effect was IGF-I mediated because it was reverted by increasing dilutions of an anti-IGF-I antibody. On the contrary, no GH effect was observed on PGE2 production. In conclusion, based on these data and on our previous results, we speculate that IGFs could influence luteal steroidogenesis through PG system.     

Effects of prostaglandin F2alpha and E2 on the production of progesterone by human granulosa cells in tissue culture.

Human granulosa cells with differing steroidogenic potentials were cultured in vitro. The effects of prostaglandin F2alpha (PGF2alpha) and PGE2 on the progesterone output and viability of these cells were investigated. Prostaglandin F2alpha either alone or in combination with LH and FSH inhibited the production of progesterone over a wide range of concentrations (1-8000 ng/ml). However, the inhibitory effect of PGF2alpha was 200 times less effective when the cells were exposed to LH and FSH for 6 days before the addition of the prostaglandin. By contrast PGE2, at concentrations from 1 to 500 ng/ml, markedly stimulated the production of progesterone by granulosa cells, and this was not prevented by the addition of PGF2alpha. The degree of inhibition by PGF2alpha or stimulation by PGE2 was related to the biosynthetic capacity of the cells. These studies suggest that PGF2alpha may act directly on the adenylate cyclase system of human granulosa cells by blocking its activation by LH, and they demonstrate that functional regression of the luteal cell can be induced independently of the blood vascular system.     

Mechanism of the rapid antigonadotropic action of prostaglandins in cultured luteal cells

A reproducible method for dissociation and culture of rat luteal cells is described. The concentration of LH required to produce half-maximal stimulation of progesterone secretion was 50 ng/ml. The effects of prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)) on basal and luteinizing hormone (LH)-stimulated progesterone production were examined. Both prostaglandins stimulated basal progesterone production but PGE(2) was about twice as active, showing a 2-fold maximal stimulation at 0.75 muM. When either prostaglandin was incubated simultaneously with LH, a dose-dependent inhibition of progesterone secretion occurred; PGF(2alpha) was 4 times more active than PGE(2), showing 50% inhibition at a concentration of 40 x nM. Thus, both prostaglandins are more active as antagonists than as agonists of LH with respect to progesterone secretion. PGF(2alpha) also inhibited LH-stimulated adenylate cyclase activity and cyclic AMP accumulation. The block in progesterone secretion was reversed by addition of dibutyryl cyclic AMP (1 mM) but not by theophylline (5 mM) alone. These data and the finding that PGF(2alpha) did not affect the specific binding activity of the LH receptor in intact luteal cells indicate that the rapid action of prostaglandins in luteal cells is due to a block of LH-dependent production of cyclic AMP which results in a decrease in progesterone secretion.     

Insulin-like growth factor-I stimulates oxytocin and progesterone production by bovine granulosa cells in culture

The effect of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), fibroblast growth factor (FGF) and nerve growth factor (NGF) on production of oxytocin and progesterone by cultured bovine granulosa and luteal cells was studied. Secretion of oxytocin was stimulated, in a dose-dependent manner, by IGF-I at 48 and 120 h of culture to levels much higher than those after stimulation with LH, FSH, EGF, FGF or NGF. A similar effect of IGF-I was observed for progesterone but, in contrast to oxytocin, secretion of progesterone was not increased by EGF, NGF or FGF. During primary culture, for 4 h, of dispersed bovine luteal cells obtained from corpora lutea between days 4 and 10 of the oestrous cycle, all the growth factors tested failed to stimulate secretion of oxytocin or progesterone. The data suggest the relevance of growth factors (especially IGF-I) for ovarian physiology and their possible importance for differentiation of follicles and luteinization.     

Relationship between the production of prostaglandins and progesterone by luteinizing human granulosa cells.

Luteinizing granulosa cells synthesize high concentrations of progesterone, prostaglandin (PG) E(2) and PGF(2 alpha). The objective of this study was to explore the relationship between prostaglandin and progesterone output from human granulosa cells as they undergo functional luteinization in culture. Granulosa cells were partially purified from ovarian follicular aspirates and cultured at a density of 10(5) cells/ml in serum-supplemented DMEM:Ham's F(12) medium for 0, 1 or 2 days. Cells were then switched to serum-free medium for 24 h before measuring hormone concentrations in this spent medium by specific radioimmunoassays. Over the first 3 days in culture, PGF(2 alpha) and PGE(2) production declined progressively by up to 82+/-3% coincident with a 55+/-11% increase in progesterone output. In subsequent experiments, cells were treated for 24 h on the second day of culture with either 0.01 to 10 microM meclofenamic acid or with 10 microM and 100 microM aminoglutethimide. Meclofenamic acid inhibited synthesis of PGF(2 alpha) and PGE(2) by up to 70+/-9% and 64+/-7% respectively without affecting progesterone output. Likewise, 100 microM aminoglutethimide inhibited progesterone production by 62+/-6% without affecting concentrations of either PGF(2 alpha) or PGE(2). We have concluded that the progressive decline in prostaglandin production and the rise in progesterone output from luteinizing human granulosa cells occur independently of each other.     

Secretion of progesterone and prostaglandins by cells of bovine corpora lutea from three stages of the luteal phase

The secretion of prostaglandins (PGs) by bovine corpora lutea was investigated. Corpora lutea from the early, early-mid and late-mid stages of the luteal phase were dissociated by collagenase treatment and cultured in monolayer in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum. Treatment with either LH (100 ng/ml) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) had no effect on progesterone secretion by early luteal phase cells but stimulated progesterone secretion two- to fourfold by cells from the latter stages. The secretion rates, per microgram cell protein, of 6-keto-PGF1 alpha, PGE2 and PGF2 alpha were substantially greater in cells from the early luteal phase than in those from the latter stages, however, all changes in PG secretion in response to treatments were qualitatively similar between cells from the three stages of the luteal phase. The secretion rate of 6-keto-PGF1 alpha was greater than that of PGE2 or PGF2 alpha and was inhibited by treatment with indomethacin (28 mumol/l) but unaltered by treatment with LH, dbcAMP or butyrate (1 mmol/l). Secretion of PGE2 was inhibited by indomethacin but stimulated two- to threefold by treatment with either dbcAMP or butyrate. Secretion of PGF2 alpha was minimal and not inhibited further by treatment with indomethacin, but was stimulated 10- to 40-fold with dbcAMP. Indomethacin treatment inhibited the stimulatory effect of dbcAMP; butyrate had no effect on PGF2 alpha secretion. Treatment with LH had no effect on any of the PGs measured. In these experiments the secretion of progesterone appeared unrelated to any changes in the secretion of PGs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production and localisation of angiotensin II in the bovine early corpus luteum: a possible interaction with luteal angiogenic factors and prostaglandin F2 alpha

The newly formed corpus luteum (CL) rapidly develops after ovulation and has the features of active vascularisation and mitosis of steroidogenic cells. These stage-specific mechanisms also may contribute to gain the function of prostaglandin F2 alpha (PGF2 alpha)-resistant CL at this stage. Recent studies suggest that the vasoactive peptide angiotensin II (Ang II) regulates luteal function. Thus, this study aimed to investigate (i) the expression of angiotensin-converting enzyme (ACE) mRNA by RT-PCR and the ACE protein expression by immunohistochemistry, (ii) the effects of angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), on the secretion of Ang II, PGF2 alpha, progesterone and oxytocin (OT), and (iii) the effects of luteal vasoactive peptides (Ang II and endothelin-1 (ET-1)) or OT on the secretion of PGF2 alpha, progesterone and OT from bovine early CL (days 3--4 of the oestrous cycle), and evaluate a possible interaction of these substances with PGF2 alpha. The expression of mRNA for ACE was found in theca interna of mature follicle, early CL and endothelial cells from developing CL as well as pituitary and kidney, but granulosa cells of mature follicle were negative. The immunohistochemical analysis revealed that blood capillaries (endothelial cells) were stained for ACE, but luteal cells were negative in early CL. To examine the effects of substances on the secretory function of the CL, an in vitro microdialysis system was used as a model. The infusion of bFGF and VEGF stimulated Ang II and PGF2 alpha secretion as well as progesterone, but not OT secretion in early CL. The infusion of Ang II after PGF2 alpha infusion continued the stimulatory effect on progesterone and OT release within early CL until 3 h thereafter. However, the infusion of ET-1 alone had no effect on progesterone or OT release. The infusion of luteal peptides such as Ang II and OT stimulated PGF2 alpha secretion, whereas the infusion of ET-1 did not. In conclusion, the overall results of this study indicate that a functional angiotensin system exists on the endothelial cells of early CL, and that angiogenic factors bFGF and VEGF upregulate luteal Ang II and PGF2 alpha secretion, which fundamentally supports the mechanism of progesterone secretion in bovine early CL. This idea supports the concept that the local regulatory mechanism involved in active angiogenesis ensures the progesterone secretion in the developing CL in vivo.     

Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production.

Oxytocin is produced in the granulosa-derived cells of the ruminant corpus luteum where its gene is dramatically up-regulated within days of ovulation. Regulation of these processes is poorly understood but oxytocin release can be increased by insulin, insulin-like growth factor I (IGF-I), and gonadotropins. Here we have assessed interactions between these regulatory systems. Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) caused dose-dependent release of oxytocin from bovine granulosa cells cultured in medium containing 100 ng/ml insulin. The gonadotropins also increased oxytocin mRNA levels and their effects were mimicked by forskolin. The effects of these stimuli on oxytocin and progesterone release were synergistically increased by insulin or IGF-I. Binding studies revealed separate binding sites with characteristics of insulin and IGF-I receptors. Insulin potentiated the effects of hCG and forskolin on oxytocin mRNA levels and release of oxytocin and progesterone in cells from follicles containing greater than 50 ng/ml estradiol. In cells from follicles containing less than 5 ng/ml estradiol these stimuli had little effect on oxytocin release although progesterone release was synergistically increased by insulin and forskolin. The data suggest that gonadotropins regulate oxytocin synthesis and release and that these effects are amplified by insulin or IGF-I acting via their own receptors. Changes associated with maturation of the target cells in vitro appear prerequisite for oxytocin production in response to increased cAMP levels in the presence of insulin or IGF-I.     

Prostaglandin synthesis, metabolism, and signaling potential in the rhesus macaque corpus luteum throughout the luteal phase of the menstrual cycle

Prostaglandins in the corpus luteum (CL) reportedly serve as luteotropic and luteolytic agents. Based mainly on studies conducted in domesticated animals and rodents, prostaglandin E2 (PGE2) is generally considered a luteotropic factor, whereas uterine-derived prostaglandin F2alpha (PGF2alpha) initiates luteolysis. However, the role of prostaglandins in regulating primate luteal structure-function is poorly understood. Therefore, a comprehensive analysis of individual mRNA or proteins that are involved in PGE2 and PGF2alpha biosynthesis, metabolism, and signaling was performed using CL obtained at distinct stages of the luteal life span during the menstrual cycle in rhesus monkeys. Peak levels of proteins involved in PGE2 synthesis (prostaglandin-endoperoxide synthase 2, microsomal PGE2 synthase-1) and signaling (PGE2 receptor 3) occurred during periods corresponding to development and maintenance of the primate CL. Immunohistochemistry studies indicated that large luteal cells express PGE2 synthesizing and signaling proteins. Expression of PGE2 synthesizing and signaling proteins significantly decreased preceding the period of functional regression of the CL, which also coincided with increasing levels of PGF2alpha receptor protein expression within the large luteal cells. Moreover, significant levels of mRNA expression for several aldoketo reductase family members that synthesize PGF2alpha from other prostaglandins were observed throughout the rhesus macaque luteal phase, thus supporting the possibility of intraluteal PGF2alpha production. Collectively, our results indicate that there may be intraluteal synthesis and signaling of PGE2 during development and maintenance of the primate CL, followed by a shift to intraluteal PGF2alpha synthesis and signaling as the CL nears the time of luteolysis.     

Effect of prostaglandins(PGs) on progesterone production by human cultured luteal cells and their ability of PGs production

The present study was designed to investigate whether or not prostaglandins(PGs) were produced by human luteal cells(HLC) and their effects on the luteal cells by monolayer culture. The following results were obtained. Cultured HLC secreted progesterone(P), prostaglandin F(PGF) and prostaglandin E(PGE) into a medium at concentrations of 276.6 +/- 38.6, 1.95 +/- 0.36, 2.44 +/- 0.45 ng/ml/1 X 10(5) cells/day (mean +/- SE), respectively. Cultured HLC was able to convert 14C-arachidonic acid to 14C-PGF2 alpha, 14C-PGE2. These two results indicated that HLC had the ability to produce PGF and PGE. Cultures were carried out in the presence of indomethacin (Ind), PGF2 alpha and PGE2 alone as well as in a combination. P production by HLC was reduced in the presence of Ind. P production in the presence of Ind+PGE2 was more than that in the presence of Ind alone. There was no significant difference in P production between the presence of Ind and Ind+PGF2 alpha. It was concluded that HLC had the ability to produce PGs and that PGE2 significantly stimulated P production in as low concentrations as HLC could produce physiologically while PGF2 alpha did not.     

Paracrine actions of oxytocin, prostaglandin F2 alpha, and estradiol within the human corpus luteum.

Human luteal cells are known to interact in an auto- and paracrine fashion using a variety of substances, including prostaglandins (PGs), steroids, and peptides. In cultures of dispersed luteal cells obtained from several animal species prostaglandin F2 alpha (PGF2 alpha) and oxytocin (OXT) inhibit progesterone (P) secretion, indicating a luteolytic effect of these substances. The disadvantage of luteal cell cultures is that the different luteal cell types do not communicate with each other, i.e. auto- and paracrine effects cannot be studied. Therefore, we used a microdialysis tubing, which is implanted in human corpora lutea (CL) kept under short term organ culture conditions. Ringer's solution is pumped through the dialysis tubing, and substances secreted by the luteal tissue can be determined in the effluent fractions. This system also allows topical application of substances with putative intraluteal effects. In the present report we used PGF2 alpha, OXT, and estradiol (E2) to examine the effects of these substances on the respective other hormones and on P release from young human CL. Intraluteal application of PGF2 alpha stimulated OXT, E2, and P release. OXT was stimulatory to E2 and P secretion, an effect that can be blocked by a specific OXT antagonist and by tamoxifen. Elevation of intraluteal E2 concentrations also had marked stimulatory effects on P secretion. From luteal cell culture experiments it is known that PGF2 alpha and OXT have direct inhibitory effects on P production, but both substances stimulate E2 release. It was also shown that E2 counteracts the inhibitory effects on P release. Therefore, the PGF2 alpha- and OXT-induced E2 release may be responsible for the increased P release. This assumption is further substantiated by the observation that intraluteally applied E2 stimulates P secretion, and preexposure of human CL to tamoxifen prevents the OXT-induced stimulation of P, but not E2, secretion. We conclude that in young human CL, PGF2 alpha and OXT have dual effects: direct inhibitory effects on P release and E2-mediated stimulatory effects, which in young CL result in a net stimulation of P secretion.     

Stimulation of glycoprotein and protein synthesis in isolated pig gastric mucosal cells by prostaglandins.

The purpose of this study is to evaluate the effects of different prostaglandin derivatives on protein and glycoprotein synthesis and secretion in isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into acid insoluble macromolecules (AIM). PGE2 and 16,16-dimethyl-PGE2 enhanced the incorporation of the amino sugar into cellular (EC50 8 and 75 nmol/l) and secreted (EC50 30 and 270 nmol/l) AIM in a concentration dependent manner during a 20 hours incubation. After incubation for eight hours or more they also stimulated the incorporation of [3H]L-leucine into cellular AIM. PGF2 alpha was considerably less potent (EC50 greater than 1 mumol/l) than the E-type prostaglandins. Iloprost, a stable prostacyclin analogue, was ineffective.     

The effects of lipoxygenase products on progesterone and prostaglandin production by human corpora lutea

The present study was undertaken to assess the effects of the products of the lipoxygenase pathway on steroidogenesis and the production of prostaglandins (PGs) by human corpora lutea in the midluteal phase. In the first experiment luteal cells were cultured with 5-hydroxyeicosatetraenoic acid (5-HETE) at 10, 100, 500, or 1000 ng/mL in the presence or absence of hCG at 100 ng/mL for 10 days. The addition of 5-HETE dose-dependently inhibited progesterone (P) production by the cultural luteal cells. P production stimulated by exposure to hCG was also reduced significantly in response to 5-HETE. However, 5-HETE had no effect on the production of 6-keto-PGF1 alpha, PGF2 alpha, or PGE2 by cultured luteal cells at any point during the culture period. In the second experiment the reaction products of soybean lipoxidase of arachidonic acid (AA-LIP) were added to cultured luteal cells. Treatment with either AA or LIP alone had no effect on basal P production. The addition of AA-LIP at all concentrations tested reduced P production by cultured luteal cells in the presence or absence of hCG. AA-LIP significantly reduced basal 6-keto-PGF1 alpha secretion in cultured luteal cells on day 2. Although the stimulatory effect of AA on luteal PGE2 production was maintained throughout the entire culture period, the lipoxygenase products of AA did not affect AA-stimulated PGE2 production by cultured luteal cell. These results suggest that the products of the lipoxygenase pathway may be important in the involution of human corpora lutea.     

The transfection-induced overexpression of IGF-binding protein-4 affects the secretory activity of porcine ovarian granulosa cells and their response to hormones and IGF-I

The aim of our studies was to examine whether IGF-binding protein (IGFBP)-4 is involved in the control of the secretion of various ovarian substances and also the mediation of the effects of several hormones and growth factors on this secretion. For this purpose, we carried out the transfection of porcine granulosa cells with a cDNA sense construct, increasing IGFBP-4 synthesis. We then compared the release of IGFBP-3, progesterone, oxytocin and IGF-I by control and transfected cells cultured with and without porcine LH (100 ng/ml), porcine GH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and estradiol-17beta (100 ng/ml). The concentration of IGFBP-4 produced was assessed using ligand blotting, and the release of progesterone, oxytocin, IGF-I and IGFBP-3 was evaluated using RIA/IRMA techniques. It was observed that GH, IGF-I, estradiol, LH and oxytocin alter the progesterone, oxytocin, IGF-I and IGFBP-3 release by porcine ovarian granulosa cells. Transfection of these cells with an IBFBP-4 cDNA expression construct significantly increased the IGFBP-4 accumulation in cell-conditioned medium. Furthermore, this transfection significantly reduced progesterone, oxytocin and IGFBP-3 release, and increased IGF-I output in cells cultured in the absence or presence of GH, IGF-I, estradiol and LH. The addition of oxytocin, but not of other tested substances, fully or partially prevented the effects of IGFBP-4 overexpression on IGFBP-3, IGF-I, but not on progesterone release. The present results suggested that IGFBP-4, as well as GH, IGF-I, estradiol, LH and oxytocin, is a potent regulator of porcine ovarian steroid (progesterone), nonapeptide hormone (oxytocin), growth factor (IGF-I) and growth factor-binding protein (IGFBP-3) release. IGFBP-4 is an inhibitor of basal progesterone, oxytocin and IGFBP-3 release and a stimulator of IGF-I output by porcine ovarian cells. The action of IGFBP-4 on the ovary can be mediated by (1) inhibition of oxytocin release, (2) suppression of receptor/postreceptor events induced by other hormones and IGF-I and (3) stimulation of IGF-I release.     

Inhibitory action of follicular fluid on progesterone and prostaglandin synthesis in bovine follicles.

The present studies examined the inhibitory effect of mid-cycle and preovulatory bovine follicular fluid (bFF) on the secretion of progesterone and prostaglandin F 2 alpha (PGF 2 alpha) by bovine theca and granulosa cells cultured in tissue culture Medium 199. The inhibitory effect of bFF on the secretion of PGF 2 alpha by incubated and cultured hemipituitary glands of male rats was also studied. Addition of 5, 10 or 20% charcoal-extracted, mid-cycle bFF to the culture medium caused a twofold decrease in the accumulation of both progesterone and PGF 2 alpha by the cultured granulosa cells (P less than 0.01). In contrast, when preovulatory bFF was used, there was no inhibitory effect on secretion of progesterone and PGF 2 alpha. The addition of 10% mid-cycle bFF to the culture medium caused a two- to fourfold decrease in the accumulation of both PGF 2 alpha and progesterone by the cultured theca (p less than 0.008). However, in the presence of 1 microgram LH/ml, the inhibitory effect of mid-cycle bFF on progesterone and PGF 2 alpha secretion was abolished. The secretion of PGF 2 alpha was significantly (p less than 0.03) decreased in male rat hemipituitary glands after 5 h of incubation or 18 h of culture. These findings suggest that bFF from mid-cycle follicles inhibits prostaglandin synthetase as well as luteinization. The inhibition disappeared with bFF from preovulatory follicles.