Hormonal regulation of messenger ribonucleic acid expression for steroidogenic factor-1, steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage in bovine luteal cells.

To examine hormonal regulation of genes pertinent to luteal steroidogenesis, bovine theca and granulosa cells derived from preovulatory follicles were cultured with various combinations of forskolin and insulin. On Day 8 of culture, progesterone production was measured, and mRNA levels of steroidogenic factor-1 (SF-1), cytochrome P450 side-chain cleavage enzyme (P450scc), and steroidogenic acute regulatory protein (StAR) were determined by means of semiquantitative reverse transcription-polymerase chain reaction. Notably, the combination of forskolin plus insulin stimulated progesterone production in luteinized theca cells. This was probably a result of a synergistic interaction between forskolin and insulin, observed on both StAR and P450scc mRNA levels. However, in luteinized granulosa cells (LGC), forskolin and insulin each independently were able to up-regulate the levels of P450scc and StAR mRNA levels, respectively. Moreover, insulin alone was sufficient to maintain the high steady-state levels of StAR mRNA in LGC. Both insulin and insulin-like growth factor I enhanced StAR gene expression in LGC. SF-1 was constitutively expressed in bovine luteal cells; its amounts did not vary between the two luteal cell types or with hormonal treatments. In summary, this study demonstrates a distinct, cell-type specific regulation of StAR and P450scc mRNA in the two bovine luteal cell types.

Characterization of messenger ribonucleic acid expression for prostaglandin F2 alpha and luteinizing hormone receptors in various bovine luteal cell types.

LH and prostaglandin F2 alpha (PGF2 alpha) control the life span and function of the corpus luteum (CL). Nevertheless, identification of the various cell types (steroidogenic and nonsteroidogenic) expressing the receptors for these hormones remains controversial. In this study we characterized LH and PGF2 alpha receptor (r) expression in the various luteal cell types using quantitative reverse transcription-polymerase chain reaction. We found, in agreement with previously described functions of PGF2 alpha, that the two steroidogenic cell types, as well as luteal endothelial cells, expressed PGFr. In contrast, LHr was mainly expressed by small luteal cells. A similar pattern of PGFr and LHr expression was observed in steroidogenic cells luteinized in vitro and in cells derived from the mature CL. The expression of these two receptors was inversely affected by increased levels of cAMP (achieved by incubating cells with varying doses of forskolin); LHr expression was down-regulated by 50% in the presence of 10 microM forskolin (p < 0.05), while an increase was observed in PGFr expression. In granulosa-derived luteal cells, maximal expression of PGFr was higher (approximately by 3-fold, p < 0.05) than in the theca-derived luteal cells. PGF2 alpha, mimicking its in vivo effect, markedly down-regulated LHr expression in thecaderived luteal cells, abolishing expression at a concentration of 100 ng/ml. In summary, these studies depict cAMP and PGF2 alpha as major regulators of PGFr and LHr expression in the two steroidogenic cell types. All three major cell types of the CL (steroidogenic and endothelial) express PGFr. LHr mRNA, on the other hand, was detected mainly in small luteal cells. Such broad cellular distribution of PGFr may highlight the significant role played by this prostaglandin in the bovine CL.

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.

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.