Control of bovine placental progestin synthesis: calcium dependent steroidogenesis is modulated at the site of the cholesterol side chain cleavage enzyme

We have previously reported that progesterone synthesis in the bovine placenta is regulated by Ca2+ dependent and cyclic nucleotide independent mechanism. In studies conducted to further define the role of Ca2+ in the synthesis of progestins in bovine placental tissue, it was found that both protein kinase C (PKC), as determined by phosphorylation, and cytochrome P-450 side chain cleavage, as determined by Western blot analysis, were detectable in the steroidogenetically active portion of the placentome. To determine the site of action of PKC, fetal cotyledon cells were incubated in media containing 25-hydroxycholesterol in the absence or or presence of 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It was found that TPA significantly (P less than 0.05) increased the conversion of the exogenous cholesterol analog to progesterone. To determine if the TPA could act synergistically with calcium activators, fetal cotyledon cells were incubated with either methyl isobutyl xanthine (MIX), an activator of intracellular calcium, or the calcium ionophore, A23187, which increases extracellular calcium influx, or both of these agents, in the presence or absence of TPA. It was found that TPA synergistically increased the conversion of sterol to progestins induced by submaximal concentrations of either MIX or A23187. In the presence of both compounds, TPA induced an even more dramatic increase in progestin synthesis. In experiments in which cyanoketone, an agent that inhibits the conversion of pregnenolone to progesterone, was added, TPA addition resulted in increased pregnenolone production, indicating that side chain cleavage of cholesterol is the site of action. The data, therefore, suggest that: (a) Ca2+ affects mechanisms regulating placental steroidogenesis; (2) one locus of Ca2+ is the cholesterol side chain cleavage reaction; and (3) PKC found in this tissue has a role in the Ca activated progestin production.     

Contact sites from human placental mitochondria: characterization and role in progesterone synthesis

To understand the functional compartmentalization of human placental mitochondria, we analyzed the composition and steroidogenic activity of contact sites. Several fractions containing contact sites were isolated using osmotic shock treatment and sucrose gradient centrifugation. These fractions contained various proteins and marker enzymes associated with mitochondrial membranes. The fractions containing the cytochrome P450 side chain cleavage system, cholesterol, nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase, porin, and adenosine 5(')-triphosphate-diphosphohydrolase activity showed the capacity to synthesize progesterone. Our observations indicate that all necessary elements and enzymes for steroidogenesis are present and functional in placental mitochondrial contact sites. This organization may facilitate the metabolism of cholesterol delivered to the outer mitochondrial membrane into steroid hormones by the inner mitochondrial membrane cholesterol side chain cleavage system.     

Bovine placental progesterone synthesis: comparison of first and second trimesters of gestation.

We have previously reported that dispersed caruncle cells from cows during the first trimester of pregnancy, in comparison to caruncle cells from cows of more than 90 days of gestation, produce little progesterone (P4) and are refractory to agents that enhance steroidogenesis. To explain this refractoriness of the first-trimester cells, we determined (1) the expression of cytochrome P450 side-chain cleavage (P450scc) and its mRNA, (2) the expression of adrenodoxin, and (3) 3 beta-hydroxysteroid dehydrogenase activity. We first determined P4 and pregnenolone (P5) production by dispersed caruncle cells from the two gestation periods using RIA. It was found that P4 synthesis by bovine maternal caruncle cells was low or undetectable in the first trimester but increased more than 10-fold in the second trimester of gestation. Addition of 25-OH-cholesterol (5 micrograms/ml) to second-trimester maternal cells increased P5 production, but no effect was observed in first-trimester cells. With [3H]P5 used as substrate, analysis of metabolites on thin-layer chromatography indicated that first-trimester maternal cells synthesized a small amount of P4 (3.02% of total radioactivity) compared to second-trimester cells (16.4%). A readily detectable amount of 17 alpha-OH-P5 was produced by the second-trimester cells (5.02%) but not by the first-trimester cells (0.6%). No other metabolites could be characterized (less than 0.5%). Cytochrome P450scc expression and its mRNA and adrenodoxin content were determined by use of Western blot or dot-blot techniques. Proteins and mRNA were detected in maternal tissues of first and second trimesters of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

5'-p-Fluorosulfonylbenzoyl adenosine inhibits progesterone synthesis in human placental mitochondria

The human placental mitochondria have an ATP-diphosphohydrolase (apyrase) activity. In this paper we characterized the effect of 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) on placental apyrase, and its repercussion on progesterone synthesis and oxygen consumption. Apyrase activity was inhibited by FSBA. Nucleosides tri- and diphosphates protected against FSBA inactivation, but divalent cations did not, indicating that FSBA attaches itself to an ATP-binding site of apyrase. In mitochondria, the inactivation of apyrase by FSBA was associated with inhibition of progesterone synthesis. Also, the oxygen consumption induced by ATP but not by ADP, was inhibited, clearly showing that FSBA exclusively inactivated the apyrase in human placental mitochondria. It is concluded that the apyrase activity is closely related to progesterone synthesis, probably associated with the cholesterol transport between mitochondrial membranes.     

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.     

Regulation of human placental progesterone synthesis in vitro by naturally occurring steroids

A regulatory model of human placental progesterone synthesis is based on studies with isolated placental enzymes. Steroids causing a dose-dependent inhibition are listed in the standing order of their inhibitory potency (I50 (microM)/Ki value (microM)/type of inhibition: c = competitive and nc = non competitive). Cholesterol side chain cleavage enzyme (mitochondria): Mainly regulated by hydroxylated cholesterol derivates. No inhibition was observed by cholesterylesters and by other naturally occurring steroids tested. 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (mitochondria): 6 beta-hydroxyprogesterone (nc), dehydroepiandrosterone (0.32/0.82/c), 20 alpha-dihydroprogesterone (0.38/-/nc), progesterone (0.46/-), estrone (0.56/0.1/c), estradiol (0.1/0.8/c), 17 alpha-hydroxyprogesterone (2.1/-/nc), 17 alpha-hydroxypregnenolone (0.4/-/c), dehydroepiandrosterone sulfate (2.5/-/c), cortisone (5.0/-), cortisol (100/-). 20 alpha-hydroxysteroid dehydrogenase (cytoplasmic): estrone (0.26/0.7/c), estradiol (0.28/0.9/c), pregnenolone (4.4/9.2/c), 5 alpha-pregnan-3 beta-ol-20-one (4.6/-/nc), estriol (5.1/11.5/c); dehydroepiandrosterone (7.2/14.0/c), 5 alpha-dihydrotestosterone (26.0/-/nc), progesterone (33.0/48.0/c), dehydroepiandrosterone sulfate (50.0/23.0/nc), and testosterone (59.0/63.0/c). An autoregulatory mechanism of placental progesterone synthesis is postulated which is in good agreement with data published by others proving that placental progesterone synthesis is independent of the endocrine organs of the mother and the fetus.     

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.     

Brain-derived neurotrophic factor promotes proliferation and progesterone synthesis in bovine granulosa cells

Brain-derived neurotrophic factor (BDNF) is involved in regulating the growth of ovarian follicles, maturation of the oocyte, and development of the early embryo through its receptor, tyrosine kinase receptor B (TrkB). However, it is still unclear as to how BDNF influences proliferation and steroidogenesis of bovine granulosa cells (GCs). In this paper, we confirmed that BDNF and TrkB were expressed in bovine GCs, and that proliferation and steroidogenesis by bovine GCs were reduced by knockdown of BDNF or inhibition of TrkB. With respect to GC proliferation, BDNF enhanced cellular viability and the percentage of cells in the S phase. BDNF also activated both protein kinase B (PKB, also known as AKT) and the extracellular signal-regulated protein kinase 1/2 (ERK1/2)-signaling pathway. Through the AKT-signaling pathway, BDNF increased the expression of proliferation-related genes, including cyclin A1 (CCNA1), cyclin E2 (CCNE2), cyclin D1 (CCND1), and cyclin-dependent kinase 1 (CDK1). However, through the ERK1/2 signaling pathway, BDNF only increased the expression of CCNA1 and CCNE2. Regarding steroidogenesis by bovine GCs, BDNF promoted progesterone (P ₄) synthesis, but had no effect on estradiol; it also activated the AKT-signaling pathway and increased the expression of steroidogenesis-related genes, including steroidogenic acute regulatory protein (STAR) and hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1 (HSD3B1). In summary, our data are the first to show that BDNF promotes the proliferation of bovine GCs through TrkB–AKT and ERK1/2 signaling pathways and increases P₄ synthesis by bovine GCs through the TrkB–AKT signaling pathway.     

Differential inhibition of progesterone synthesis in bovine luteal cells by estrogens and androgens

We investigated the roles of estrogens and androgens in the progesterone biosynthesis of bovine luteal cells. The responsiveness of primary luteal cells to the stimulation of tropic agents was observed in a dose-dependent manner. Estrogens and androgens significantly inhibited tropic agent-induced progesterone secretions, but glucocorticoids did not, which indicated the inhibitions were specific. The failure of exogenous 8-Br-cAMP to prevent these inhibitions suggested that took place at the post-cAMP steps. The immunoblot showed that testosterone remarkably decreased the amount of induced P450scc protein after 6-hour treatment, yet 17beta-estradiol did not. The 3beta-HSD activity assays demonstrated that both 17beta-estradiol and testosterone efficiently blocked induced 3beta-HSD activities. Both inhibitory effects of E2 and T on progesterone synthesis were observed one hour after treatment and accompanied with suppressed 3beta-HSD activities. This study presents that estrogens and androgens specifically inhibit bovine luteal function through different mechanisms.     

Influence of noradrenaline on progesterone synthesis and post-translational processing of oxytocin synthesis in the bovine corpus luteum

Noradrenaline (NA) influences secretory function of the bovine corpus luteum (CL), stimulating secretion of progesterone and ovarian oxytocin (OT). To study whether NA is able to stimulate progesterone synthesis and to affect post-translational OT processing, different doses of NA alone or in combination with different doses of OT were added to bovine CL slices from 8 to 13 d of the estrous cycle. To determine which receptors NA affects, and if dopamine (DA) also affects CL function, we used NA or DA combined with a beta-antagonist (propranolol). The results indicated that NA stimulates both luteal progesterone and OT content; furthermore, it increased the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA), terminal enzymes in synthesis of these 2 hormones. The stimulating effect of NA was inhibited by propranolol and by pre-treatment of CL slices with high OT doses. Post-translational processing of OT synthesis by PGA activation was also stimulated by DA, but this effect was inhibited by beta-receptor blocker. Thus DA acts in CL as a NA precursor. In conclusion, it can be assumed that the noradrenergic system affects CL secretory function on different levels of regulation. Furthermore, a high concentration of OT in CL prevents NA from activating PGA and thus decreases post-translational OT synthesis.     

Inhibition of human placental progesterone and estrogen synthesis in early human gestation by aminoglutethimide in vivo

The inhibitory effect of d,l-aminoglutethimide (AG) on the synthesis of progesterone and estradiol in early human pregnancy (8th–12th week of gestation) was investigated in volunteers; control group (n = 11), AG group [1000 mg AG orally at test begin (n = 6)]. Venous blood samples were taken at the beginning of the test and 0.5, 1, 2, 4, 8 and 24 h thereafter. In controls, no significant changes in serum progesterone and estradiol could be observed during 24 h. In the AG group, a decrease in progesterone and estradiol could be observed within 1 h after the test began; lowest serum steroid concentrations were reached after 4 h. Relative to the initial values taken as 100%, the greatest decrease in progesterone ranged between 37 and 83%, 62 15% (x SD) (n = 6); the greatest decrease in estradiol ranged between 32 and 78%, 51 17% (x SD) (n = 6). Twenty four hours after AG treatment, both steroids reached similar concentrations to those found at test begin. No clinical signs (e.g. uterine bleeding, contractions) for the abortifacient action of AG were observed.In conclusion, a single dose of AG (1000mg given orally) cannot induce a therapeutic abortion in early pregnancy. In accordance with in vitro studies, the inhibitory effect of AG on placental progesterone formation is due to an inhibition of mitochondrial cholesterol side chain cleavage. The decrease in estradiol is thought to be related to an inhibition of placental aromatase.     

Progesterone maintenance of the placental progesterone receptor and placental growth in ovariectomized rats

These studies examine the trophic effects of progesterone (P) on the progesterone receptor (Rp) and growth of the decidua basalis (DB) and junctional zone (JZ) in the rat placenta. Pregnant rats were ovariectomized (Ovx) in mid-pregnancy and received steroid replacement therapy consisting of implantation of P pellets (25 mg) and injections of estradiol (E), 2 micrograms s.c., daily. Placental protein synthesis, measured by 3H-leucine incorporation in vitro, decreased more than 99% within 24 h of Ovx. However, treatment with P immediately after castration maintained control levels of synthesis. Delay of P treatment for 4 h caused a 60% decline in protein production measured 20 h later (p less than 0.01). Intraperitoneal implantation of a 50-mg pellet of the antiprogestin, RU-38486, in intact pregnant rats decreased protein synthesis by 50% within 6 h and by more than 90% 12 h and 24 h post-implantation (p less than 0.01). Growth of DB and JZ in Ovx rats treated for 48 h with P and/or E was studied both histologically and by changes in protein and DNA content. Rp binding activity was also measured by exchange assay under equilibrium conditions. Only P was able to reverse the effects of Ovx on growth of the DB and JZ. P also maintained Rp levels in the DB above those observed in Ovx and Ovx + E-treated groups (p less than 0.01). The Rp may be a constitutive product in the JZ since binding activity was not altered by Ovx or by steroid treatments. This study shows that P is clearly a trophic hormone of the maternal and chorioallantoic placenta and is essential for placental growth, cellular differentiation, and histological integrity.     

Midtrimester pregnancy interruption and the placental progesterone levels

Placental progesterone contents were studied in 10 patients therapeutically aborted at midtrimester by intraamniotic infusions of hypertonic saline, and from 14 patients (12–20 weeks) aborted by intraamniotic instillation of prostaglandin F_2∝. The mean S.E. of the progesterone was 1.99± 0.07 ug/g. placental tissue in the first group, while with prostaglandin abortion the placental progesterone was 1.45± 0.09 ug/g. tissue, which is significantly lower than the results in the first group (P=0.001). The possible mechanism of action of prostaglandin as an effective abortifacient is discussed.     

Native and recombinant bovine placental lactogens

The bovine placenta produces a wide variety of proteins that are structurally and functionally similar to the pituitary proteins from the GH/PRL gene family. Bovine placental lactogen (bPL) is a 200-amino acid long glycoprotein hormone that exhibits both lactogenic and somatogenic properties. The apparent molecular masses of purified native (n) bPL molecules (31-33 kDa) exceed 23 041 Da, which is the theoretical molecular mass of the protein core. At least six isoelectric variants (pI: 4.85-6.3) of bPL were described in cotyledonary extracts and three different bPL isoforms (pI: 4.85-5.25) were found in fetal sera. The bPL molecules that are detected in higher concentrations in peripheral circulation exhibit a more acidic pI than those present in placental homogenates. This may reflect an important glycosylation process occurring just prior to the bPL secretion. The bPL mRNA is transcribed in trophectoderm binucleate cells starting from Day 30 of pregnancy until the end of gestation. In mothers, bPL is involved in the regulation of ovarian function, mammogenesis, lactogenesis, and pregnancy stage-dependent adaptation of nutrient supplies to the fetus. Due to the higher fetal, compared to maternal concentrations of circulating hormone, it has been suggested that bPL primarily targets fetal tissues.