Oxytocin and estradiol concentrations in follicular fluid as a means for the classification of large bovine follicles.

Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F(2)alpha 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol>/=100 ng/ml and oxytocin<65 pg/ml (preovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol<100 ng/ml and oxytocin>/=65 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol<100 ng/ml and oxytocin<65 pg/ml (atretic follicles). Treatment with prostaglandin F(2)alpha significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol: progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF(2)alpha was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles.

The hormonal regulation of cholesterol side-chain cleavage cytochrome P450, adrenodoxin, and their messenger ribonucleic acid expression in bovine small-like and large-like luteal cells: relationship with progesterone production.

The bovine corpus luteum contains two steroidogenic cell types, small and large luteal cells. The present study aimed to examine molecular mechanisms regulating progesterone (P4) production in long term cultures. The content of the side-chain cleavage (SCC) enzymes cytochrome P450scc and adrenodoxin (ADX) and the steady state availability of their mRNAs were determined and compared to P4 production in each of the luteal cell types. Small-like (SLC) and large-like (LLC) luteal cells were obtained by incubating theca interna and granulosa cells with forskolin and insulin. Upon luteinization, LLC expressed 2- to 3-fold higher amounts of both SCC enzyme mRNAs than did SLC. Moreover, 8 days after stimulant removal, LLC retained their P4 production capacity, expressed P450scc and ADX mRNAs, and contained these proteins. Nevertheless, the presence of the luteinizing agents in LLC culture medium was required for maximal expression of SCC enzymes. In the SLC, P4 production, P450scc and ADX content, and their mRNAs showed a much stronger dependence on chronic cAMP (and insulin) stimulation. In SLC, stimulant removal was accompanied by a sharp decrease (95% reduction) in P4 production, P450scc and ADX enzyme content (57% and 90% reduction, respectively), and their mRNAs (90% and 95% reduction, respectively). However, their steroidogenic capacity could be restored by forskolin and insulin replenishment. Interestingly, P4 production by both luteal cells types was reflected better in ADX than in P450scc content. These observations emphasize the contribution of the large luteal cell to P4 output, which may become crucial when hormonal support of the corpus luteum is deficient.

Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theca and granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theca interna cells cultured in the presence of different luteinizing factors--insulin (2 micrograms/ml), forskolin (10 microM), or a combination of the two were examined and characterized. Direct binding of [125I]IGF to the conditioned media was compared to progesterone production under these different treatments. In theca cells, maximal secretion of IGFBPs was achieved using forskolin alone, whereas maximal progesterone production was induced by the insulin+forskolin treatment. In contrast, maximal secretion of both IGFBPs and progesterone in granulosa cells was achieved using forskolin alone. IGFBP species secreted by the two cell types under the different treatments were detected by ligand blotting. Conditioned media from theca cells in serum-free medium collected on the seventh day of culture exhibited three bands of 34, 40 and 44 kDa when treated with insulin or forskolin. The intensity of the 40-44 kDa complex was enhanced and a 21 kDa band appeared when cells were treated with a combination of insulin plus forskolin. Conditioned media of granulosa cells stimulated with insulin or forskolin exhibited 21, 27, 29, 34 and 40-44 kDa bands. Treatment with insulin+forskolin greatly increased the intensity of a 40-44 kDa complex. A similar shift towards high molecular weight binding proteins was observed when these media were analyzed by high-performance liquid chromatography gel filtration. These findings substantiate the secretion of IGFBPs by bovine theca and granulosa cells and show it to be dependent on culture treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidogenic enzyme content and progesterone induction by cyclic adenosine 3',5'-monophosphate-generating agents and prostaglandin F2 alpha in bovine theca and granulosa cells luteinized in vitro.

In vitro luteinization of bovine granulosa (LGC) and theca (LTC) cells was achieved by culturing cells with forskolin (10 microM) and insulin (2 micrograms/ml) for 9 days. This treatment induced the presence of cytochrome P450scc and adrenodoxin in both cell types, but to substantially higher levels in LGC than in LTC. Forskolin dose-dependently stimulated the secretion of progesterone and cAMP after 3 h of incubation in both cell types although LGC were less sensitive to this stimulation than were LTC. Only LTC were responsive to LH, in accordance with their higher LH/hCG binding capacity. Both prostaglandin F2 alpha (PGF2 alpha) and phorbol 12-myristate 13-acetate (TPA) increased progesterone production during 3 h incubation of LGC and LTC, and treatment with staurosporine (a protein kinase C inhibitor) reversed this effect. Neither TPA nor PGF2 alpha alone affected cAMP levels but each acted synergistically with forskolin to increase cAMP accumulation. These results indicate that 1) elevated progesterone output from LGC is related to steroidogenic enzyme level; 2) bovine LH (up to 100 ng/ml) does not provoke a response in LGC due to their low LH/hCG binding capacity; 3) cAMP-protein kinase A and protein kinase C pathways are both involved in progesterone production by LGC and LTC, possibly by enhancing cholesterol transport.

Interaction of estrogen receptors with drugs that affect prolactin secretion.

The competition of bromocriptine and lisuride hydrogen maleate (LIM) with estradiol binding to various tissues was evaluated by the dextran coated charcoal method. Bromocriptine and LIM competitively inhibited the binding of [3H] estradiol to its cytosolic receptors in rat uterine, pituitary and hypothalamic tissue and in DMBA induced mammary tumors. Ki was 2 X 10(-5) M for bromocriptine and 2 X 10(-4) M for LIM. Metoclopramide, dopamine and L-dopa had no significant effect on [3H] estradiol binding. The interaction of bromocriptine and LIM was specific for estrogen receptors. There was no interaction with progesterone receptors from rat uterus and pituitary and with testosterone receptors from rat epididymis and testis. When tested for estrogenity in the immature rat uterus, bromocriptine and LIM induced specific estrogen inducible proteins such as cytosolic estrogen and progesterone receptors. However, they do not affect the uterine/body weight ratio and peroxidase activity. A clear interaction of inhibitors (bromocriptine and LIM) of prolactine secretion, with cytosolic estrogen receptors from various tissues was shown. Some in vivo estrogenic effect was also demonstrated in the immature rat uterine system.