Stimulated P4 Production Research Articles

Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

Evaluation of Triclosan Effects on Cultured Swine Luteal Cells.

Simple SummaryA great concern has been raised against many chemicals, both natural and man-made, that can mimic or interfere with the hormones. Among these, using swine ovarian cells, we were aimed to explore the potential effect of triclosan, an antimicrobial agent widely used in cosmetics and home products. Our results demonstrate that triclosan disrupts cellular function, in particular interfering with hormone production and proliferation, thus suggesting a critical evaluation of its effects.Triclosan is a chlorinated phenolic, used in many personal and home care products for its powerful antimicrobial effect. Several studies have shown triclosan toxicity and the American Food and Drug Administration (FDA) in 2016 has limited its use. It has been recently included in endocrine-disrupting chemicals (EDCs), a list of chemicals known for their ability to interfere with hormonal signaling with particular critical effects on reproduction both in animals and humans. In order to deepen the knowledge in this specific field, the present study was undertaken to explore the effect of different concentrations of triclosan (1, 10, and 50 µM) on cultured luteal cells, isolated from swine ovaries, evaluating effects on growth Bromodeoxyuridine (BrDU) incorporation and Adenosine TriPhosphate (ATP) production, steroidogenesis (progesterone secretion) and redox status (superoxide and nitric oxide production, enzymatic and non-enzymatic scavenging activity). A biphasic effect was exerted by triclosan on P4 production. In fact, the highest concentration inhibited, while the others stimulated P4 production (p < 0.05). Triclosan significantly inhibited cell proliferation, metabolic activity, and enzymatic scavenger activity (p < 0.05). On the contrary, nitric oxide production was significantly increased by triclosan (p < 0.01), while superoxide anion generation and non-enzymatic scavenging activity were unaffected.

Effects of testosterone and prolactin on steroidogenesis in post-ovulatory cumuli oophori and on in vitro oocyte fertilisation in the rat.

The main objective of these studies was to determine the in vitro effects of prolactin (PRL) and testosterone (T) on steroidogenic function in post-ovulatory cumuli oophori containing unfertilised (ufCOCs) or fertilised (fCOCs) oocytes and to determine the differences between ufCOCs and fCOCs. In vivo, progesterone (P4) content was distinctly higher in isolated ampullae containing ufCOCs than in those containing fCOCs. Moreover, the expression of androgen (ARs) and prolactin (PRL-Rs) receptors was distinctly higher in ufCOCs than in fCOCs. Also, in vitro P4 profiles were generally higher in incubated ufCOCs, which had very high secretion rates of this steroid, especially after treatment with PRL+T. Testosterone significantly increased P4 levels only in incubated fCOCs, while the anti-androgen dihydroxyflutamide (2-Hf) markedly decreased P4 levels in both ufCOCs and fCOCs. Among post-incubation ufCOCs fertilised in vitro, the highest fertilisation rate was observed for oocytes in ufCOCs exposed to PRL+T, while those incubated with 2-Hf or T+2-Hf were not fertilisable. These studies establish differences in steroidogenic function and expression of ARs and PRL-Rs between post-ovulatory ufCOCs and fCOCs, with higher concentrations of P4 being observed in the microenvironment of ufCOCs. PRL+T stimulated P4 production by ufCOCs and increased in vitro fertilisation rate.

Hyperglycosylated Human Chorionic Gonadotropin Does Not Increase Progesterone Production by Luteinized Granulosa Cells

Trophoblast-derived human chorionic gonadotropin (hCG) promotes corpus luteum progesterone (P4) production, and wide ranges of serum P4 levels are noted in various pregnancy outcomes, despite similar hCG concentrations. There are five unique biologically active hCG variants in human pregnancy urine, and previous studies of P4 production in response to hCG have used only preparations containing all isoforms. Understanding exactly which hCG variant is primarily responsible for stimulating corpus luteum steroidogenesis may have great clinical and diagnostic implications, including in the setting of ectopic pregnancy. Our objective was to delineate the role of the standard and hyperglycosylated (H)-hCG isoforms in stimulating P4 production by luteinized granulosa cells. Cell culture, ELISA, and fluorometric-based protein assays were done at Duke University Medical Center. Patients were anonymous oocyte donors. Cultured luteinized granulosa cells were treated with 0.25, 0.5, and 1.0 ng/ml total hCG, which contains all isoforms, purified standard hCG (37.1 kDa), and purified H-hCG (42.8 kDa). P4 produced per total cellular protein (nanograms per microgram) was measured via ELISA and fluorometric protein determination kits. Both total hCG (P = 0.0003) and purified standard hCG (P < 0.0001) stimulated a dose-dependent increase in P4 production. Purified H-hCG did not change the P4 produced per total cellular protein response (P value not significant). Standard hCG stimulated P4 production by cultured granulosa cells and likely supports corpus luteum function via interactions with the LH/hCG receptor. In contrast, H-hCG did not increase P4 production, which indicates a nonsteroidogenic role for this protein during early gestation.

Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture

Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation.

Local expression and distribution of growth hormone and growth hormone receptor in the chicken ovary: Effects of GH on steroidogenesis in cultured follicular granulosa cells

Preovulatory follicular development (PFD) is mainly regulated by gonadotropins (FSH, LH) and steroids, although other intraovarian factors are also involved. We analyzed the local expression of growth hormone (GH) in the hen ovary and the role that this hormone may play on the regulation of steroidogenesis in granulosa cells (GCs). Ovarian follicles from sexually mature hens were studied at different developmental stages. Both GH mRNA (by in situ hybridization) and protein (by immunohistochemistry) were expressed mainly in the GCs, and to a lesser extent in the theca cells of the follicular wall. Sequence of a GH cDNA 690-bp fragment obtained from the follicular wall was identical to that obtained from the pituitary. The growth hormone receptor (GHR) mRNA was also expressed in the follicles. Nine GH variants were observed by SDS–PAGE and Western blotting, but the main isoform showed a MW of 17 kDa, at all developmental stages. Addition of GH (0.1, 1, 10 nM) stimulated the synthesis of progesterone (P4) in primary GCs cultures in a dose-dependent manner (1.5, 2.9, 5.4 times, respectively). GH also stimulated the expression of cholesterol side-chain cleavage enzyme (cytochrome P450scc) mRNA, a rate-limiting enzyme during P4 synthesis (2.9, 4.6, 4.9 times, respectively), whereas the synthesis of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA (a constitutive enzyme) was not changed. Both GH and GHR were co-expressed in GCs cultures. The locally expressed GH present in concentrated (4×, 6×, 8×) conditioned media obtained from ovarian GC cultures stimulated P4 production (1.2, 2.2, 4.4 times, respectively) in additional fresh cultured GCs, and this effect disappeared when the conditioned media were treated with antiserum against GH. These data suggest that locally produced GH may modulate follicular development through autocrine/paracrine effects in the chicken ovary.

Prolactin signalling in porcine theca cells: the involvement of protein kinases and phosphatases.

The hypothesis that protein kinase C (PKC) and tyrosine kinases, as well as serine-threonine and tyrosine phosphatases, are involved in prolactin (PRL) signalling in theca cells harvested from porcine follicles was tested. Theca cells were incubated with PRL for 24 h to stimulate progesterone (P4) production. In addition, treatments included inhibitors of PKC and tyrosine kinases, as well as serine-threonine phosphatase inhibitor and tyrosine phosphatase inhibitor. Prolactin significantly stimulated P4 production by theca cells and all inhibitors suppressed the PRL-stimulated P4 production. After incubation with PRL for 2, 5, 10 or 20 min, theca cells were homogenized and cytosolic and membrane fractions were obtained. This was followed by determination of PKC activity in partially purified subcellular fractions by measuring the transfer of 32P from [gamma-32P] adenosine triphosphatase (ATP) to histone III-S. In unstimulated porcine theca cells the major proportion of PKC activity was present in the cytosol. Incubation of cells with PRL resulted in a rapid, time-dependent increase in the amount of PKC activity in the membrane fraction. Protein kinase C activity in the membrane fraction was maximal after 10 min of cells' exposure to PRL. Protein kinase C activation was assessed also by measuring the specific association of 3H-phorbol dibutyrate (3H-PDBu) with theca cells after treatment with PRL. Prolactin significantly increased 3H-PDBu-specific binding in theca cells. In contrast to PKC, total inositol phosphate accumulation was not affected by PRL in the current study. In summary, PRL stimulated P4 production by porcine theca cells derived from large follicles. The results of the study were consistent with the hypothesis that PKC is one of the intracellular mediators of PRL action in porcine theca cells. Protein kinase C activation does not appear to occur through the action of phosphatidylinositol-dependent phospholipase C. Moreover, the involvement of tyrosine kinases, as well as tyrosine and serine-threonine phosphatases, in PRL signalling in the examined cells is suggested.

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [ 125I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL ( p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner ( p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production ( p < 0.05). Both IGFBP-2 and -3 inhibited [ 125I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.

Effects of gonadotropins and insulin-like growth factors-I and -II on in vitro steroid production by bovine granulosa cells

The objectives of this study were: 1) to develop a bovine granulosa cell (GC) culture system; and 2) to use this system to evaluate the effects of gonadotropins (FSH and LH) and insulin-like growth factors-I and -II (IGF-I and IGF-II) on steroidogenesis of bovine GC derived from small, medium, and large antral follicles (diameters ≤4, 5–8 and &gt;8 mm, respectively). Granulosa cells were cultured (concentration, 5 × 105 cells per well) in serum-free medium for 48 h with variable doses of hormones and growth factors. Concentrations of progesterone (P4) and estradiol-17β (E2) in the media were determined by radioimmunoassay. Basal E2 production by GC from follicles of all sizes decreased with time of culture (P &lt; 0.01) while basal P4 production increased (P &lt; 0.01). Basal E2 and P4 production increased with increasing size of follicles (P &lt; 0.01). Only very low concentrations of FSH stimulated E2 production from medium and large follicles. Follicle-stimulating hormone stimulated P4 production by GC of follicles of all sizes (P &lt; 0.05). Luteinizing hormone inhibited E2 production by GC in medium and large follicles (P &lt; 0.05), suggesting that LH is responsible for the rise in plasma E2 through effects on both theca cells and GC. A dose of 100 ng mL−1 of IGF-I increased E2 production by GC from medium and large follicles (P &lt; 0.05). Progesterone production by GC from all categories of follicles was also stimulated by IGF-I (P &lt; 0.05). Estradiol-17β production by GC from large follicles decreased in response to IGF-II (P &lt; 0.05). The physiological role of IGF-II on steroidogenesis in the bovine ovary remains to be elucidated. In summary, these results demonstrate the development of a serum-free culture system for bovine GC, and that FSH, LH, IGF-I and IGF-II have different effects on steroidogenesis by bovine GC from different size follicles. Key words: Granulosa cells, gonadotropins, Insulin-like growth factors, progesterone, estradiol-17β, cows

Expression and function of a scavenger lipoprotein pathway in porcine luteal cells.

A pathway(s) for uptake of modified (e.g., acetylated, oxidized) low-density lipoprotein (LDL) moieties has been recently discovered on luteal cells of certain species. The expression and function of this pathway during the life span of the corpus luteum (CL) have not been investigated. Aims of the present study were the following: 1) to determine whether porcine small and large luteal cells take up modified LDL; if so, 2) to compare uptake of native and modified LDL by luteal cell subpopulations during the luteal life span and 3) to compare effects of native and modified LDL on luteal steroidogenesis during the estrous cycle. Collagenase-dispersed luteal cells were prepared from porcine ovaries at various stages of the estrous cycle: early (E, Days 4-6), mid (M, Days 8-10), mid-late (ML, Day 13-14), or late (L, Day 16-17) (estrus = Day 0). Cells were incubated with fluorescent-tagged LDL (Dil-LDL; 0-10 microg/ml) or acetylated LDL (Dil-AC-LDL; 0-10 microg/ml). Fluorescence was analyzed by multiparameter flow cytometry in each of three subpopulations of cells: small (SLC) and large (LLC) luteal cells and nonsteroidogenic cells. The percentage of LLC taking up Dil-LDL remained relatively constant (65-75%) from E to ML cycle and then declined (13.3 +/- 4.1%; p < 0.05); these findings were consistent with previous data. In contrast, the percentage of LLC taking up Dil-AC-LDL gradually increased from E (29.8 +/- 8.5%) to ML (68.3 +/- 5.9%) stage and then declined (17.1 +/- 2.3%; p < 0.05). Similarly, Dil-LDL uptake by SLC was relatively constant (15-20%) from E to M cycle, declining to 2.9 +/- 0.5% at L cycle. Dil-AC-LDL uptake by SLC progressively increased from E (5.4 +/- 2.6%) to ML (24.3 +/- 6.9%) and then fell somewhat (12.9 +/- 6.7%) in L cycle. Few (< 2%) nonsteroidogenic cells labeled for Dil-LDL at all stages, whereas Dil-AC-LDL uptake by nonsteroidogenic cells was similar to that by SLC. Dual-uptake experiments revealed subtypes of LLC that took up either native LDL only or both native and modified LDL. Progesterone (P4) production by E and M luteal cell cultures was dose-dependently increased (p < 0.05) by both native and modified LDL. In contrast, modified LDL suppressed (up to 40-50%, p < 0.05) steroidogenesis by ML and L cultures, whereas LDL had no significant effect. Only native LDL stimulated P4 production by isolated SLC, although both native and modified LDL similarly increased P4 production by LLC. In conclusion, expression of one or more scavenger LDL pathways on porcine luteal cell subtypes is dynamic. Moreover, modified LDL can exert both stimulatory and inhibitory effects on luteal steroidogenesis in vitro. Differential expression and function of a scavenger LDL pathway by subpopulations of cells within the CL may play a novel role in luteal development, function, and/or regression.

Effects of Testosterone and Estradiol-17.BETA. on Progesterone Production by Granulosa Cells of the Hen.

The aim of the present study was to examine the effect of testosterone (T) and estradiol-17β (E2), known as being produced in the theca cells, on the production of progesterone (P4) by granulosa cells of the hen. When granulosa cells isolated from the largest preovulatory follicle (F1) were incubated for 4 hr with increasing doses of ovine luteinizing hormone (oLH), T or E2, P4 production by granulosa cells was increased in a dose-dependent manner by oLH, but not by T or E2. However, when F1 granulosa cells cultured for 48 hr in a monolayer culture system were cultured for an additional 48 hr with increasing doses of oLH, T or E2, not only oLH but also T stimulated P4 production of granulosa cells in a dose-related manner while E2 alone had no effect on P4 production. In addition, when the time course of P4 production by F1 granulosa cells in response to oLH and T was examined for 48 hr during 48 to 96 hr of culture, P4 production with oLH was found to be increased at 4 hr of culture, with the maximal level at 6 hr, while P4 production with T increased significantly from 12 hr of culture, with the maximal response at 36 hr. Furthermore, when F1 granulosa cells were precultured with E2 for various times before 4 hr culture with oLH at 96 hr of culture, the increase in P4 production in response to oLH was only found at a pretreatment time of more than 12 hr.These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action over the long term on P4 production, and E2 in long-term action may enhance the sensitivity to LH for P4 production.

Four-day-old bovine corpus luteum: progesterone production and identification of matrix metalloproteinase activity in vitro.

Little is known about matrix metalloproteinases (MMPs), enzymes that are required for the structural remodeling and angiogenesis that occur in the corpus luteum (CL) during the first several days postovulation. In fact, little attention has focused on early CL function including the regulation of progesterone (P4) production. Thus, the objective of the present study was 1) to investigate the effects of insulin, LH, and dibutyryl cAMP on P4 production and cell numbers and 2) to identify MMPs in the 4-day-old CL, with use of a defined culture system. Cultures were seeded with either 1 x 10(6) or 0.5 x 10(6) cells. All cultures containing insulin had higher P4 levels and cell numbers (p < 0.05) than those without. In cultures containing insulin, basal P4 levels were high throughout the culture period. Furthermore, neither LH nor dibutyryl cAMP stimulated P4 production (p > 0.05) at a seeding density of 1 x 10(6), whereas they stimulated P4 production (p < 0.05) at a seeding density of 0.5 x 10(6) on Days 6 and 8 of culture. In conditioned medium of control cultures seeded with 0.5 x 10(6) cells, substrate gel electrophoresis (zymography) showed two intense bands that migrated at M(r) approximately 97,000 and approximately 65,000-64,000, while two weaker ones migrated at M(r) approximately 88,000 and approximately 64,000-63,000. The molecular weights of the M(r) approximately 97,000 and approximately 88,000 species were consistent with MMP-9 family members, while the molecular weights of the M(r) approximately 65,000-64,000 and approximately 64,000-63,000 species were consistent with MMP-2 family members.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of interactions of follicle-stimulating hormone, luteinizing hormone, and prolactin on progesterone synthesis by rat luteal cells during pregnancy.

The in vitro ability of ovine (o) follicle-stimulating hormone (FSH), (o)luteinizing hormone (LH), (o)prolactin (PRL), and recombinant human FSH (rhFSH) to stimulate progesterone (P4) synthesis by rat corpora lutea on Day 4 of pregnancy was investigated. Dispersed luteal cells (large + small cells) were incubated in the presence of the gonadotropins (1-100 ng) alone or in various combinations (10 ng each) for 4 or 24 hr. Given alone, all the ovine preparations stimulated P4 in a dose-dependent manner with even 1 ng of each hormone significantly enhancing P4 production. Significantly, rhFSH--which is devoid of LH contamination--at 10 and 100 ng also stimulated P4 production, thus clearly establishing for the first time that FSH is a luteotropic hormone in the rat. The combination of oFSH + LH + PRL (10 ng each) significantly stimulated P4 synthesis to a greater extent than the combination of any two hormones or individual hormones at both 4 hr or an additional 24 hr of incubation (P < 0.05). This verified in vitro a previously established in vivo luteotropic complex. One hundred nanamolars of phorbol 12-myristate 13-acetate (PMA) did not affect basal P4 secretion but inhibited cAMP, oFSH, and oLH stimulation of P4. Thus, the luteotropic effects of FSH, LH, and activators of protein kinase A are antagonized by the protein kinase C pathway.

Luteotrophic and luteolytic interactions between bovine small and large luteal-like cells and endothelial cells.

Endothelial cells, the most abundant cell type in the bovine CL, were shown to establish intercellular contact with steroidogenic cells of the CL. Two experimental models were used to study the involvement of endothelial cells in luteal cell function: 1) luteal slices in which the integrity and communication between the different cells were maintained and 2) pure large and small luteal-like cells, cultured separately or co-cultured with endothelial cells. The luteolytic effect of prostaglandin (PG) F2 alpha was examined in these two models. Treatment with PGF2 alpha did not alter P4 secretion stimulated by LH in young (2-4-day-old) CL slices, whereas, in slices from mature (6-12 days old) CL, PGF2 alpha significantly reduced (by 40%) the stimulatory effect of LH on P4 secretion. In pure large luteal-like cells, the effect of forskolin plus PGF2 alpha on P4 secretion did not differ from forskolin given alone after 3 or 24 h of incubation. However, when co-cultured with endothelial cells, PGF2 alpha significantly inhibited forskolin stimulation. Endothelial cells significantly stimulated P4 production from large luteal-like cells only. This effect may be attributed to the action of PGI2 secreted by endothelial cells. In summary, endothelial cells may play an essential role in luteal functions by being involved in both luteotrophic and luteolytic processes.

Luteotropic effects of follicle-stimulating hormone (FSH): I. FSH has in vitro luteotropic and synergistic effects with luteinizing hormone and prolactin on progesterone production by hamster luteal cells during pregnancy.

This study was designed to evaluate the in vitro ability of FSH, LH, and prolactin (PRL) to stimulate progesterone (P4) production by enzymatically dispersed CL cells from pregnant hamsters. In light of previous in vivo findings [1], we were especially interested in determining whether FSH is a luteotropin. The CL were collected and pooled on Days 4, 8, 12, and 16 of gestation (Day 1 = sperm-positive vaginal smear). After enzymatic dissociation, combined large and small luteal cells (LC+SC) were incubated in the presence of 10 ng ovine (o) FSH, oLH, and oPRL, alone or in various combinations, for a total of 144 h with the first medium change at 24 h and other changes every 48 h thereafter. FSH and LH alone significantly increased P4 production on Days 4, 8, and 12, while PRL alone increased P4 only on Day 12 (p < 0.05). The combination oFSH+oLH+oPRL significantly stimulated P4 production on Day 4 to a greater extent than the combination of any two hormones (p < 0.05). Ovine FSH+oLH enhanced P4 production on Days 12 and 16 at 48, 96, and 144 h of incubation, to an extent greater than either hormone alone (p < 0.05). When recombinant human FSH (r-hFSH), which is devoid of LH activity, was added (1-100 ng) to dispersed luteal cells from Day 4 pregnant hamsters, a dose-response increase in P4 was evident (p < 0.05); even 1 ng r-hFSH stimulated P4 production at 96 h (p < 0.05). On Day 2 of the cycle, oFSH or oLH, but not oPRL, also significantly stimulated P4 production (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and Preliminary Characterization of Luteotropic Activity in the Rabbit Placenta1

The corpus luteum of the rabbit requires estradiol (E2) and the conceptus to maintain progesterone (P4) during the second half of pregnancy. The factor produced by the fetal placenta that is responsible for maintaining P4 production has not yet been identified. Therefore, the goal of these experiments was to determine whether the rabbit placenta contained luteotropic activity and to begin preliminary characterization of this factor. A bioassay system utilizing corpora lutea explants incubated with placental extracts and other treatments was developed and validated. Neither pooled placental extracts (200 micrograms) nor E2 alone affected P4 production (p > 0.05). However, the combination of placental extracts plus E2 significantly stimulated P4 production approximately 1.5-fold (p < 0.05). Skeletal muscle (200 micrograms) + E2 also had no effect on P4 production, indicating that the effect of placental extracts was tissue-specific. Carboxymethyl-cellulose ion-exchange chromatography of placental extracts yielded four separate fractions, which were examined individually at three doses for activity in the bioassay system. The first and most acidic fraction (Fraction 1) significantly stimulated P4 production at 10 and 100 micrograms when incubated in combination with E2 (p < 0.05). Both heat and trypsin treatment abolished the ability of placental Fraction 1 to stimulate P4 production in the presence of E2. Size fractionation of Fraction 1 using dialysis membranes with 3500, 6000-8000, and 12,000-14,000 molecular weight cutoff points had no effect on bioactivity of Fraction 1. These results indicate that the rabbit placenta contains a potential luteotropin with the characteristics of an acidic, heat-sensitive protein greater than 12,000-14,000 in molecular weight.

Steroidogenic Response of Rat and Pig Luteal Cells to Estradiol-17β and Catecholestrogens in Vitro1

The corpora lutea of several species contain estrogen receptors, but the role of estrogens in luteal function is unclear in most species. In this study, we investigated the direct effect of estradiol-17 beta (E2) and catecholestrogens (2-OHE2 or 4-OHE2) on rat and pig luteal steroidogenesis using in vitro cultures of small (SLC) and large (LLC) luteal cells prepared by elutriation. SLC and LLC were cultured at 37 degrees C for 36 h in serum-free media and treated with E2, 2-OHE2, or 4-OHE2; LH; forskolin (FORS); dibutyryl cAMP (dbcAMP); or combinations thereof. In the rat, E2 (2.5-10 micrograms/ml) inhibited progesterone (P4) production by both cell types dose-dependently. P4 production by rat SLC increased with increasing dose of 4-OHE2 up to the 2.5-microgram dose, then decreased to near control level at the 10-microgram dose. In LLC, P4 production in the presence of 4-OHE2 decreased initially (up to 2.5 micrograms/ml 4-OHE2), then increased at the 10-microgram dose. LH, FORS, and dbcAMP stimulated P4 production by SLC and LLC. For SLC, the stimulatory effects of LH and 4-OHE2 (2.5 micrograms) were comparable but lower than those of FORS and dbcAMP. For LLC, the effects of 4-OHE2 (10 micrograms), LH, and FORS were comparable but lower than those of dbcAMP. In time-course experiments, E2 inhibition of P4 production was observed at 36 and 72 h but not 6 h of culture for SLC and at all time points for LLC.(ABSTRACT TRUNCATED AT 250 WORDS)