Biomedical subjects

B D Murphy

Publications and source records attributed to B D Murphy.

At least 55 records · Page 3Linked to original sources

PubMed1992

Partial characterization of the gonadotropin-releasing hormone (GnRH) gene transcript in the rat ovary.

It has been hypothesized that GnRH or a GnRH-like peptide is produced in the rat ovary, but the presence of GnRH in the ovary has not been unequivocally demonstrated. This study was undertaken to determine whether the GnRH gene is expressed in the rat ovary and to compare the GnRH gene transcripts from the ovary and the hypothalamus. Twelve samples of total RNA from ovaries of individual rats were screened by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of GnRH gene transcripts. Fragments of GnRH cDNA were amplified using pairs of specific primers. GnRH transcripts were detected in all the ovaries examined, and differed from hypothalamic GnRH transcripts in two ways: first, in the ovaries a greater proportion of GnRH transcripts contained intronic sequences; second, the major transcription start utilized in the ovary differed from that used in the hypothalamus. Although fully processed GnRH gene transcripts were detected by RT-PCR in both, ovary and hypothalamus, they were not detected in the ovary by Northern blot. The GnRH probe hybridized specifically to the predicted 0.6 kb transcript in the hypothalamus, and to a 3.3 kb transcript in the ovary. We conclude that in the ovary, most GnRH gene transcripts retain intronic sequences.

Animals↗

PubMed1991

Effects of carbohydrates on the pharmacokinetics and biological activity of equine chorionic gonadotropin in vivo.

The sialylation of eCG was examined to determine its influence on the in vivo metabolism and biological activity of the molecule. Sialic acid was decrementally removed from eCG by incubation with agarose-linked neuraminidase for varying time periods. Pharmacokinetic parameters for the disappearance of 4,000 IU (267 micrograms) of three desialylated eCG preparations (20%, 53%, and 80% sialic acid removed) and control eCG were determined in sheep. The clearance rate of eCG increased (p less than 0.05) with each decrement of sialic acid. The removal of 53% sialic acid enhanced the distribution of eCG into the tissues, compared to control and 20% desialylated eCG (p less than 0.05), presumably because of increased lipid solubility and decreased molecular size. Desialylation to 53% did not alter the elimination half-life of eCG. The removal of 80% sialic acid resulted in the disappearance of eCG from the serum within 1 h, whereas control eCG was still present at 120 h. In vivo trials in rats disclosed that the control eCG preparation increased ovulatory rate at doses of 10-100 IU and ovarian weight at doses of 10-300 IU relative to saline-treated rats (p less than 0.01). The 20% desialylated eCG induced superovulatory and ovarian weight responses, but 100-500 IU were required to achieve the same result as that produced by control eCG. The 53% and 80% desialylated eCG preparations induced a mild superovulatory response (p less than 0.01) but no ovarian weight response. It was concluded that sialic acid was significant to the distribution and disappearance of eCG. The effects of carbohydrate removal on biological activity (e.g., superovulation) are primarily a function of clearance rate rather than tissue-specific phenomena.

Animals↗

PubMed1991

Effects of active immunization against gonadotrophin-releasing hormone on the concentrations of noradrenaline, dopamine, 5-hydroxytryptamine and some of their metabolites in the brain and sexual organs of male rats.

This is an investigation of the effects of active immunization against gonadotrophin-releasing hormone (GnRH) conjugated to keyhole limpet hemocyanin on brain and male sexual organ concentration of catecholamines and 5-hydroxytryptamine. The treatment induces antibodies against GnRH with consequent interference with reproductive function as indicated by the attendant decrease in serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. Rat striatal and mesolimbic dopamine levels were moderately increased by this treatment but no changes were observed in the hypothalamus. GnRH immunization also increased brain 5-hydroxytryptamine concentrations as observed in the hypothalamus, olfactory tubercles and striatum. In the male accessory sexual organs, immunity against GnRH alters the density of noradrenergic concentration. The changes occurred in opposite directions in the different regions investigated; increases in noradrenaline were observed in the prostate, with a reduction being detected in the epididymus and no changes were observed in the vas deferens or the seminal vesicles. 5-Hydroxytryptamine levels increased in the vas deferens but no effects were observed in the epididymus, prostate or seminal vesicle. It is concluded that GnRH immunization with the consequent reductions in circulating FSH, LH and testosterone are associated with changes in neurotransmitter concentration both in the brain and in some of the accessory sexual organs in the male rat.

Animals↗

PubMed1991

Equine chorionic gonadotropin.

Cells from the chorionic girdle of the equine trophoblast invade the maternal endometrium at day 36 of gestation and become established as secretory elements known as the endometrial cups. These structures, which persist for 40-60 days, produce a gonadotropin which can be found in circulation until about day 130 of gestation. This glycoprotein has been identified in the horse and the donkey, with the former having received much better characterization. It consists of 2 noncovalently linked peptide chains; an alpha-subunit of 96 amino acids, which is common to that found in other horse glycoprotein hormones. The beta-subunit of 149 amino acids is identical to horse LH beta. Horse CG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. The alpha-subunit has two and the beta-subunit one N-linked glycosylation site, and the beta-chain has in excess of four O-linked glycosylation sites. The N-linked glycans have some oligosaccharides that are not found on other glycoprotein hormones. The sialic component of glycosylation confers an exceptionally long half-life on CG compared to other glycoprotein hormones. Horse CG has LH-like activity in horse receptor and in vitro bioassays. In spite of the amino acid homology, it has lower LH activity than does horse LH. Its most intriguing, and as yet unexplained, characteristic is its pronounced FSH and LH activity in species other than the horse. Horse CG binds to FSH receptors of virtually all mammalian species, other than the horse, in which it has been tested and will produce biological effects peculiar to FSH. It has similar and potent interaction with LH receptors. The structural basis of this duality is not known but may be related to the region 90-110 of the beta-chain. Horse CG is believed to be constitutively expressed by the trophoblastic cells until the endometrial cups degenerate. The role of CG in equine gestation is not completely understood. It is believed to act as an LH-like hormone to induce supplementary ovulation and/or luteinization of follicles in the mare. It has not been established whether CG or the accessory corpora lutea are necessary for successful horse pregnancy. They may serve as a redundant system to assure that there is sufficient secretion of the primary corpus luteum to maintain pregnancy until the placenta assumes its role as the principal steroidogenic organ of gestation.

Amino Acid Sequence↗

PubMed1991

Protein kinase C, an endogenous regulator of hormone-induced cyclic AMP induction in porcine luteal cells.

LH, in addition to increasing cyclic AMP (cAMP) in ovarian cells, stimulates phosphoinositide hydrolysis producing inositol trisphosphate and diacylglycerol (DG). DG activates phospholipid- and calcium-dependent protein kinase (PKC). In the present study, we have used both PKC activators and inhibitors to examine the interactions of the PKC pathway on hormone-induced cAMP production in porcine luteal cells. Phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-induced cAMP production. A time-course study indicated that the facilitatory effect of PMA was greater when added to incubation tubes following addition LH or forskolin. The non-tumour-promoting phorbol ester 4 alpha-phorbol 12,13-didecanoate, which does not stimulate PKC activation, did not facilitate hormone-induced cAMP induction. PKC inhibitors polymyxin B, sphingosine and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) antagonized the facilitatory effect of PMA on LH-induced cAMP production. The cAMP induction by both LH and forskolin was inhibited in the presence of PKC inhibitors. Polymyxin E, which differs from polymyxin B by a single amino acid and does not inhibit PKC activation, did not inhibit LH- or forskolin-induced cAMP induction. The results of this study provide evidence for a facilitative action of the PKC effector system on hormonally stimulated cAMP production. Furthermore, PKC may be an important endogenous regulator of adenylate cyclase activity in porcine luteal cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Comparative effects of insulin and insulin-like growth factor-1 on follicle-stimulating hormone-induced responses in porcine granulosa cells.

The effect of insulin and insulin-like growth factor-1 (IGF-1) on progesterone secretion by porcine granulosa cells and their modulatory effect on follicle-stimulating hormone (FSH)-induced responses were examined. For comparative purposes, growth hormone (GH), previously shown to stimulate IGF-1 secretion, was also included. Granulosa cells from ovarian follicles (3 to 5 mm) were cultured in multiwell plates for the first 48 hours, either in the presence or absence of 1% fetal bovine serum (FBS). Following plating, all cultures were maintained in serum-free media. The addition of only insulin, but not IGF-1 or GH, enhanced progesterone secretion under both culture conditions. When low-density lipoprotein was provided as steroid substrate, a stimulatory effect of insulin on progesterone accumulation was observed with a minimum dose of 10 ng/ml. Granulosa cells cultured in serum-free media from the time of plating secreted less progesterone and were less responsive to FSH compared with cultures plated with 1% FBS. Only insulin, but not IGF-1, enhanced FSH responses to threefold in cells cultured with 1% FBS. However, when cells were cultured in serum-free media from the time of plating, both insulin and IGF-1, but not GH, potentiated the responses to FSH, but insulin was more potent than IGF-1. Insulin-like growth-factor-1 binding studies with granulosa cells indicate the presence of specific high-affinity binding sites (Kd 3.96 nM). A dose of 100 ng/ml of insulin had negligible cross-reactivity with IGF-1 receptors.

Animals↗

PubMed1990

Superovulation of beef heifers with Folltropin: A new FSH preparation containing reduced LH activity.

The optimum superovulatory dose of Folltropin was determined and compared with a standard 28 mg dose of FSH-P in beef heifers. In Experiment 1, mean numbers of corpora lutea (CL) did not differ among the groups treated with 10, 20, 30 or 40 mg Folltropin or FSH-P, and the mean CL number was reduced (P<0.05) only in the 5 mg Folltropin group. Mean numbers of ova/embryos recovered, fertilized and transferable were greater (P<0.05) for the 10, 20 and 30 mg Folltropin groups than for the 5 mg group. The 40 mg Folltropin group and the FSH-P group were intermediate. The percentage of fertilized and transferable embryos did not differ over the dosages used in this experiment. In Experiment 2, mean numbers of CL were greater for the 9, 18 and 36 mg Folltropin groups than for the 4.5 mg group, with the 9 mg group being lower than the 36 mg group (P<0.05). The 18 mg group was intermediate and did not differ. Mean numbers of ova/embryos recovered and fertilized ova were greater for the 9, 18 and 36 mg groups (P<0.05) than for the 4.5 mg group. The percent of fertilized and mean number and percentage of transferable embryos did not differ among treatments. We conclude that Folltropin may be a satisfactory superovulatory replacement for FSH-P and that a dose of 18 to 20 mg Folltropin may be within the optimum superovulatory dosage range for beef heifers. Dosages of Folltropin of more than twice the optimum did not result in deterioration of ova/embryo quality.

Journal Article↗

PubMed1990

Effects of the dam on equine chorionic gonadotropin concentrations during pregnancy.

Breeding trials were designed to determine the influence of the mare on serum concentrations of equine chorionic gonadotropin (eCG) from Day 39 to Day 104 of gestation. Sires were ranked according to mean eCG concentrations found in the groups of randomly selected mares to which they were mated in 1983. Mares were ranked according to their mean eCG concentrations on Days 55, 71 and 85 of gestation (Day 0 = mating), in 1983 and 1985. In the 1986 breeding season, mares that had pregnancies characterized by high eCG levels were mated to sires previously associated with low eCG concentration pregnancies and low producing mares were mated to sires associated with high eCG concentration pregnancies. The highest eCG concentrations (12.8 micrograms/ml serum) were detected on Day 55 of gestation in mares mated to the low-ranked sire (P less than 0.01), indicating an influence of the mare on serum eCG concentration. A comparison of eCG production by individual mares in 1985, 1986 and 1987 showed that mares retained the same rank (P less than 0.001), regardless of the rank of the sire to which they were mated. It was concluded that the influence of the mare predominates in determining eCG concentration. In 1987, the highest ranked sire was mated to the highest eCG producing mares (ranked 1-20) and the second highest ranked sire was mated to mares ranked 21-40. Two distinct subgroups resulted, suggesting that the selection of sires and mares could be used to maximize the production of eCG.

Animals↗

PubMed1990

Evidence for the regulation of 3 beta-hydroxysteroid dehydrogenase messenger RNA by human chorionic gonadotrophin in luteinized porcine granulosa cells.

The effects of hCG on the abundance of messenger RNA for the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-OHSD) were studied in luteinized porcine granulosa cells. There were dose-dependent increases in the mRNA for this enzyme after 8 h incubation with hCG or dibutyryl cAMP. The results suggest that LH-like hormones regulate the expression of the 3 beta-OHSD gene in luteinized tissue, and this genomic regulation is a component of the luteotrophic regulation of steroidogenesis in the ovary.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Regulation of mRNA expression of 3 beta-hydroxy-5-ene steroid dehydrogenase in porcine granulosa cells in culture: a role for the protein kinase-C pathway.

We studied the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granulosa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP accumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3 beta-hydroxy-5-ene steroid dehydrogenase (3 beta HSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the increase in 3 beta HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also inhibited the increase in 3 beta HSD mRNA levels stimulated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu)2cAMP and 8-bromo-cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 3 beta HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 3 beta HSD mRNA levels in porcine granulosa cells in culture.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Interactions between melatonin and prolactin during gestation in mink (Mustela vison).

Immature female mink, 8 weeks of age in July, were treated with implants releasing melatonin. Mating, which induced ovulation, took place during the normal breeding season in the following March. Circulating prolactin and progesterone concentrations did not undergo the expected gestational increases, and no embryos implanted. A similar absence of gestational changes in prolactin and progesterone values ensued in primiparous mink treated with the melatonin implant 2-3 days after the second of 2 matings. Administration of exogenous sheep prolactin (0.5 mg/day) by minipump induced precocious elevation of progesterone concentrations in mated mink. Prolactin administration overcame the effects of melatonin, in that the corpora lutea were activated and embryos implanted, but exogenous prolactin resulted in degeneration of implanted embryos both in the presence and absence of chronic melatonin. The results suggest that melatonin has a single effect in alteration of gestation in mink; i.e. the prevention of prolactin secretion. Hyperprolactinaemia may inhibit embryo development in this species.

Animals↗

PubMed1989

Immunization of cattle against modified peptides of gonadotropin releasing hormone conjugated to carriers: Effectiveness of Freund's and alternative adjuvants.

Two gonadotropin-releasing hormone (GnRH) peptides with a cystein substitution of the first (C1-GnRH) or tenth (C10-GnRH) amino acid were conjugated to ovalbumin and equine serum albumin, respectively, via the sulfhydryl group of the introduced cysteine. Animals were immunized three times at 3-wk intervals with both conjugates in either saline (n = 5), Freund's complete adjuvant (FCA; n = 5), Havlogen (n = 6), Ribi adjuvant system (RAS; n = 5), dimethyl dioctadecyl ammonium bromide (DDA; n = 4), Alhydrogel (n = 5) or Regressin (n = 5). Animals immunized with conjugates in saline or RAS did not produce anti-GnRH titers. The highest anti-GnRH titers were produced by animals treated with FCA. The Alhydrogel and DDA treatments stimulated the production of GnRH antibodies in all animals treated, but titers were lower than in animals immunized with FCA. When vaccines were formulated with Havlogen or Regressin, anti-GnRH titers were low or absent. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were depressed in FCA and in Alhydrogel treated animals. The antisera raised were predominantly directed against either the carboxy- or the amino-terminal end of the GnRH peptide, or directed equally against both, depending on the individual animal. Results suggest that no epitope of GnRH dominates the immune response in cattle and show that the best alternative to FCA is Alhydrogel.

Journal Article↗

PubMed1989

Immunization of sheep against modified peptides of gonadotropin releasing hormone conjugated to carriers.

The efficacy of antigens based on modified GnRH peptides in stimulating the production of antibodies against GnRH in sheep was tested. In the first study cysteine-containing GnRH peptides were conjugated to keyhold limpet haemocyanin (KLH) in 3 different orientations. The 3 conjugates were prepared in an emulsion of Freund's complete adjuvant (FCA) and were injected into 3 groups of 6 castrated male lambs. The 3 vaccines efficiently induced anti-GnRH titers in all the animals treated. The specificity of the GnRH antisera raised varied depending on the orientation of the GnRH molecule in the antigen and on the individual animal. In a second trial designed to evaluate carrier molecules, a cysteine-containing GnRH peptide was conjugated to either KLH, equine serum albumin, ovalbumin or tetanus toxoid. The conjugates were prepared with FCA and injected into intact male lambs. All 4 vaccines stimulated the production of antibodies against GnRH in all the animals treated. The conjugates prepared with equine serum albumin or ovalbumin were the most effective in raising high anti-GnRH titers. In 18 of 20 lambs treated, anti-GnRH titers resulted in a marked atrophy of the testes. We conclude that: 1) the different epitopes of the GnRH molecule are equally immunogenic in sheep; 2) the GnRH antibody response is affected by the carrier used; and, 3) anti-GnRH vaccines based on cysteine-substituted GnRH analogues show potential for use in immunocastration of livestock.

Animals↗

PubMed1989

Use of low-density and high-density lipoproteins in undifferentiated porcine granulosa cells.

Granulosa cells aspirated from medium-sized porcine ovarian follicles (3-5 mm) in short-term incubation responded to the addition of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) with increased accumulation of progesterone. HDL was more potent than LDL in enhancing progesterone secretion. When granulosa cells were cultured under serum-free conditions for 72 h, HDL but not LDL exhibited a dose-dependent enhancement of progesterone secretion. Addition of insulin to the cells greatly potentiated the stimulatory effect of LDL on progesterone accumulation, while the response to HDL was only slightly increased. Granulosa cells in culture degraded LDL but not HDL. Addition of insulin enhanced LDL degradation. Exposure of cells in culture to chloroquine, an inhibitor of lysosomal function, completely blocked LDL degradation and LDL-induced stimulation of steroidogenesis. The stimulatory effect of HDL was not affected by chloroquine. We interpret these findings to indicate that granulosa cells derive cholesterol from LDL by means of lysosomal degradation, which is not required for use of cholesterol from HDL. Monensin, a carboxylic ionophore that interrupts recycling of LDL receptors, prevented LDL-enhanced progesterone accumulation but not HDL-induced stimulation. This provides evidence that HDL-induced stimulation of steroidogenesis does not involve LDL receptors. We conclude that HDL present in follicular fluid is capable of providing cholesterol to granulosa cells for steroidogenesis. The stimulatory effect of HDL is not due to the presence of apoprotein E, an HDL component that binds to the LDL receptor. A unique HDL pathway that does not involve LDL receptors and lysosomal degradation may operate in porcine granulosa cells.

Animals↗

PubMed1989

Evidence for differences in low density lipoprotein processing by porcine granulosa and luteal cells in vitro: effect of addition of serum for plating of granulosa cells on lipoprotein metabolism.

The present studies were carried out to compare the low density lipoprotein (LDL) metabolism by freshly isolated immature porcine granulosa cells with that by luteal cells. Furthermore, we have examined the effect of serum used for plating of granulosa cells on lipoprotein degradation and utilization. In incubation studies, addition of LDL as an exogenous substrate had a mild stimulatory effect on progesterone accumulation by granulosa cells, while it exhibited a dose-dependent stimulatory effect on luteal cells. When granulosa and luteal cells were incubated with 125I-labelled LDL, membrane binding of LDL occurred in both cell types, but only luteal cells were capable of internalizing the bound LDL. Granulosa cells in incubation degraded LDL much less in comparison with luteal cells, and the amount varied with the maturity of the cells. When granulosa cells were plated with graded amounts of serum which was withdrawn for 48 h following plating, they exhibited enhanced LDL degradation in a serum concentration-dependent fashion. Addition of serum for plating selectively enhanced utilization of LDL, but not high density lipoprotein (HDL) for progesterone accumulation by the cells in culture. Time-course studies on LDL degradation by granulosa cells following serum withdrawal indicate that the ability of cells to degrade LDL decreased in a time-dependent fashion. Serum withdrawal selectively decreased utilization of LDL but not HDL for progesterone secretion. It is concluded that immature granulosa cells have a limited capability to utilize cholesterol carried by LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Luteotrophic agents and steroid substrate utilization.

The cells of the CL are derived from precursors in the ovarian follicle. For their existence and function, they are dependent on a combination of various substances, collectively described as luteotrophins. These luteal stimulatory agents, including luteinizing hormone and prolactin from the pituitary, chorionic gonadotrophin from the placenta, and insulin from the pancreas, are classified as endocrine luteotrophins because they are produced at a distance from their target organ, the CL. Paracrine and autocrine luteotrophins are substances produced by the ovary with specific stimulatory effects on the CL: oestrogens and insulin-like growth factors meet this criterion. The CL produces progesterone from cholesterol which can be synthesized de novo or be derived from cholesteryl esters stored in the CL or from free and esterified cholesterol from lipoproteins in circulation. Cholesterol appears to be transported within the luteal cell by means of the cytoskeleton, by a specific, labile intracellular protein and/or by a specific sterol carrier protein. In this review, we have presented evidence to indicate that the luteotrophins have effects on all forms of cholesterol available to the CL. A major mechanism of action of luteotrophins appears to be the management of cholesterol supplies. LH, acting through a cAMP second messenger system, initiates the process of progesterone synthesis by induction of the cleavage of the side-chain on free cholesterol to produce pregnenolone. LH also acts to hydrolyse cholesteryl esters to produce free cholesterol. HDL metabolism appears to be affected by LH, hCG and cAMP. There is good evidence to suggest that luteal cell binding of HDL is regulated, in part, by LH. All phases of the process of utilization of LDL-cholesterol, including the availability of the LDL receptors, internalization of the LDL receptors and ligands into the luteal cell and degradation of LDL, are regulated by LH and cAMP. These agents induce an increase in mRNA for the LDL receptor. These processes may be, in part, controlled by an intracellular negative feedback mechanism for which cholesterol is the effector. However, in experiments where intracellular cholesterol was elevated by hydroxylated cholesterol or by inhibition of the conversion of cholesterol to pregnenolone, it was demonstrated that LH can override the cholesterol negative feedback mechanism. PRL plays an active role in cholesterol homeostasis in the luteal cell. The available evidence suggests that it is not involved in cholesterol side-chain cleavage, and enhances rather than reverses the formation of cholesteryl esters.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Immunological approaches to contraception in dogs.

The demand for safe, effective and cost-efficient means of pet population control has generated interest in the development of alternatives to surgical gonadectomy. The purpose of this review is to discuss the potential of immunological approaches to fertility control and to outline recent developments that may aid their design. Among the most promising candidates for immunoneutralization is gonadotrophin releasing hormone (GnRH). We have developed a reliable and reproducible GnRH-protein conjugate antigen and demonstrated that immunologically induced infertility is possible in dogs.

Adjuvants, Immunologic↗

PubMed1988

Studies on the mechanism of action of prostaglandin F2 alpha induced luteolysis in rats.

The effects of prostaglandin F2 alpha (PGF2 alpha) administration on the utilization of low density lipoprotein (LDL) and progesterone secretion were examined in dispersed luteal cells from rat ovaries. Immature rats were rendered pseudopregnant with administration of pregnant mare serum gonadotropin and human chorionic gonadotropin. Animals were sacrificed at different times after PGF2 alpha (5 mg/kg) or vehicle administration on day-5 of pseudopregnancy. Administration of PGF2 alpha in vivo decreased human chorionic gonadotropin (hCG) binding to luteal cell membranes in vitro but enhanced binding of LDL. Utilization of labelled cholesterol for steroid synthesis from reconstituted LDL [(3H)-CL-LDL] by dispersed luteal cells was enhanced following PGF2 alpha administration. This suggests that the LDL pathway is not suppressed during prostaglandin induced luteolysis. Progesterone and total progestin secretion in response to N6-2'-0-Dibutyryladenosine 3'5'-cyclic monophosphate (cAMP) was decreased at 2, 4 and 24 hours following PGF2 alpha administration demonstrating a post-cAMP defect in steroidogenesis. Addition of the hydroxylated sterols, 20 or 25-OH cholesterol as substrate stimulated progesterone secretion in vehicle treated rats in a dose dependent fashion with 20-OH cholesterol being more potent. Progesterone secretion in response to stimulation with luteinizing hormone (LH) and cAMP from vehicle treated rats was less than that observed with 20 or 25-OH cholesterol, indicating that endogenous substrate may be a limiting factor in steroid synthesis. The maximal capacity of luteal tissue to produce progestins following PGF2 alpha administration was determined with 20-OH cholesterol as the substrate. The results suggest that the post-cAMP defect at 4 hours following PGF2 alpha administration may be due to failure of the cells to mobilize endogenous cholesterol. However at 24 hours following PGF2 alpha administration the decreased ability of luteal cells to convert cholesterol to pregnenolone may contribute to decreased progesterone synthesis.

Animals↗