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Objective: The purpose of this study was to determine if insulin or insulin-like growth factors (IGF) either alone or in combination with estradiol (E2) could maintain progesterone secretion by rabbit luteal cells in culture., Methods: Progesterone secretion was measured in media of luteal cell cultures incubated in serum-free media alone or containing E2 with or without added insulin, IGF-I or IGF-II., Results: Progesterone production by rabbit luteal cells in culture fell precipitously in serum-free media containing E2, IGF-I, IGF-II, or insulin alone. However, when E2 was combined with insulin, IGF-I, or IGF-II, serum progesterone production was stimulated two- to fivefold above control levels., Conclusion: There is an important synergism between insulin/IGF and E2 in regulating luteal steroidogenesis.

The purpose of this study was to sequentially characterize peripheral chorionic gonadotropin (CG), estradiol (E2), and progesterone (P) profiles during early pregnancy in the baboon (Papio anubis). Ten pregnant baboons were bled sequentially at eighteen time points between Days 8 and 128 of gestation. In addition, blood was obtained at corresponding time points from 5 spontaneously aborting baboons. CG levels were assessed in a mouse Leydig cell bioassay using rhesus pituitary LH as the standard E2 and P levels were measured by RIA. Pregnancy-associated CG activity was detectable by Day 15 (901.4 +/- 275.6 ng/ml), peaked at Day 27 (53,494.1 +/- 14,995.6 ng/ml), and then returned to baseline values by Day 51 (45.4 +/- 8.9 ng/ml). Mean E2 concentrations rose from 28.9 +/- 4.3 pg/ml on Day 8 to 280.6 +/- 145.5 pg/ml on Day 58 and then increased 9-fold to a level of 2436.4 +/- 928.0 pg/ml on Day 72. Mean E2 concentrations ranged between 2065.9 and 3830.9 pg/ml from Day 72 through Day 128. Mean P concentrations ranged from 7.5 to 10.2 ng/ml between Days 8 and 21, rising dramatically to 38.6 +/- 4.6 ng/ml on Day 37 prior to declining to levels that ranged from 11.1 to 17.1 ng/ml between Days 51 and 128. CG levels were low in 3 of 5 animals prior to spontaneous abortion E2 and P concentrations preceding spontaneous abortion were similar to values in uncomplicated pregnancy; however, at the time abortion was detected P concentrations in all 5 animals were 1.3 ng/ml or less.(ABSTRACT TRUNCATED AT 250 WORDS)

Although estradiol is luteotropic in rabbits, corpora lutea develop and secrete progesterone in the absence of estradiol for 4-5 days. This period of estradiol independence is associated with high levels of LH-activated adenylyl cyclase and low levels of estradiol receptor. To determine if progesterone secretion is dependent on LH-stimulated adenylyl cyclase during this period of development, ectopic corpora lutea were established in ovariectomized rabbits. Rabbits were injected daily or twice daily with saline or 50, 100, or 150 IU hCG to desensitize LH-responsive adenylyl cyclase. On day 3, 4, 5, or 7, adenylyl cyclase activity was measured in luteal homogenates, and serum progesterone levels were determined by RIA. Basal and LH-stimulated adenylyl cyclase levels rose through day 5 of pseudopregnancy. Daily treatment with 50 or 100 IU hCG did not alter basal activity, but decreased LH-stimulated adenylyl cyclase activity by approximately 50% on day 3, by 80-90% on day 4, and by 90-95% on days 5 and 7. Serum progesterone levels were not different in hCG- and saline-treated rabbits. When LH-stimulated adenylyl cyclase was completely desensitized on days 3 and 4 of pseudopregnancy with twice daily injections of 150 IU hCG, serum progesterone levels were again, not different in saline- and hCG-treated rabbits. These results demonstrate that progesterone production by ectopic corpora lutea is not altered when LH-stimulatable adenylyl cyclase is desensitized and suggest that this early period of luteal development is independent of LH as well as estradiol.

The luteotropic action of estradiol has been studied in the rabbit and rat, and it is proposed that in both species, estradiol may be the "ultimate" luteotropic hormone. The acute dependence of the rabbit corpus luteum upon estradiol is illustrated by the rapid decline in serum progesterone after withdrawal of estradiol (removal of Silastic implant containing 17 beta-estradiol) and by the restoration of serum progesterone to normal values when the estradiol implant is replaced 24 hours later. The identification and characterization of a cytoplasmic and nuclear estrogen receptor in the rabbit corpus luteum and the translocation of the cytoplasmic receptor suggest that estradiol may be acting as it does in other estrogen target tissues. The administration of LH antiserum in pregnant rats causes rapid decreases in luteal estradiol and serum progesterone concentrations, which can be prevented by the administration of low doses of testosterone. This luteotropic effect of testosterone is attributed to an action of estradiol which is formed within the corpus luteum via the luteal aromatase. The rabbit corpus luteum, which has no aromatase, is totally dependent upon estradiol produced by the ovarian follicles and LH is essential to maintain follicular estradiol synthesis. The rat corpus luteum, which is rich in aromatase activity, may produce its own estrogen from an androgen precursor synthesized within the luteal tissue. The essential role of LH may be the stimulation of the synthesis of androgen precursor. As a working hypothesis it is proposed that in these two species LH is necessary to stimulate the synthesis of estradiol which then acts to sustain progesterone secretion.

Previous studies have established that 17 beta-estradiol is the principal luteotropic hormone in the rabbit. However, a direct effect of 17 beta-estradiol on rabbit luteal cell progesterone production has been difficult to show in vitro. The goal of this study was to develop a system in which the effect of estrogen on luteal cell progesterone production could be studied in vitro. To that end, a dissociated rabbit luteal cell preparation was developed using collagenase and the resultant isolated cells were studied using a perifusion system. Optimization of the cell digest procedure revealed that: inclusion of 2% bovine serum albumin in our optimal dissociation medium increased cell yield; and animals killed by cervical dislocation maintained more stable levels of progesterone during a 7-h perifusion compared to animals killed with barbituate-induced euthanasia (euthobarb). When dissociated luteal cells were perifused with medium, stable progesterone output (greater than 80% of initial levels) was observed for 5-6 h, after which medium progesterone concentrations declined. The inclusion of 17 beta-estradiol (10(-8) M) in the perifusion medium maintained progesterone output at control levels for up to 15 h. However, the maintenance of progesterone was not noted until after 5 h of perifusion, suggesting that the effect of estradiol may be time dependent. Thus, this investigation describes a rabbit luteal cell dissociation technique and perifusion system that may be used to examine the mechanism through which estradiol acts to maintain rabbit luteal progesterone production.

We have investigated the role of 17beta-estradiol in the early development of the rabbit corpus luteum. Ectopic corpora lutea were established in all animals by autotransplanting preovulatory follicles beneath the kidney capsule 6.5 to 8 h after mating (day 0). At this time, the rabbits were bilaterally ovariectomized and in some of these rabbits a Silastic capsule containing crystalline 17beta-estradiol was implanted SC. Mean serum concentration of estradiol in rabbits with an estradiol impant was 16.4 pg/ml. In rabbits without an estradiol implant, the estradiol concentration averaged 1 pg/ml despite the presence of transplanted luteinized follicles. Daily blood samples were analyzed for progesterone by radioimmunoassay. Ectopic corpora lutea developed in rabbits with or without estradiol treatment. Serum progesterone concentrations in the two groups increased above castrate values and were not significantly different from one another through day 5. After day 5, progesterone concentrations steadily increased in rabbits treated with estradiol and reached a value of 4.8 ng/ml by day 10. In contrast serum progesterone steadily decreased after day 5 in rabbits without estradiol treatment to a level of 450 pg/ml by day 10. Hysterectomy on day 0 did not prevent this decline in progesterone, indicating that a uterine luteolytic agent was not involved. Total luteal weight on day 10 was positively correlated with serum progesterone concentration (r equals .92; P greater than 0.01). These results indicate that for a period of approximately 5 days afte r ovulation, the development of the rabbit ectopic corpus luteum and the secretion of progesterone are autonomous from estradiol secreted by ovarian follicles. After this time, there is an absolute requirement for estrogen which permits further development of the corpus luteum and the continuation of progesterone synthesis.

Quartered CL from 7-day pseudopregnant rabbits were incubated at 37 degrees C for 0-180 min in the presence of BSA, LH or adrenaline in Krebs-Ringer-bicarbonate buffer. Total progesterone at each time point was quantified in homogenates of tissue plus incubation media and expressed relative to CL protein. Progesterone increased linearly with time during the first 30 min of incubation in the presence of BSA. LH and adrenaline markedly accelerated progesterone accumulations relative to the BSA control. At 10 min, progesterone accumulation in the presence of LH and adrenaline were 2.4 and 5.9 times that in the absence of stimulators, respectively. Both hormones caused concentration-dependent increases in progesterone and the apparent ED50 was 0.75 microgram/ml for LH and adrenaline. The CL obtained from ovaries of 7-day pseudopregnant rabbits are therefore capable of an acute steroidogenic synthetic response to LH as well as adrenaline.

Elevated activity of 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMG-CoA reductase) was observed in the rabbit ovary and corpus luteum during pregnancy. Based on this study, it was proposed that de novo cholesterol synthesis rather than the uptake of exogenous plasma cholesterol (lipoproteins) was of primary importance in providing steroid substrate for progesterone synthesis by the rabbit luteal cell. Using a perifusion system, the present study challenges this hypothesis by demonstrating that both low- and high-density lipoproteins (at protein concentrations of 100 micrograms/ml and 50 micrograms/ml, respectively) were able to acutely stimulate progesterone production by dissociated rabbit luteal cells. The increase in progesterone synthesis was due to increased cholesterol substrate and not to protein-enhanced progesterone release. The ability of luteal cells to respond to lipoproteins was dependent on both dose- and sequence of treatment, with high-density lipoprotein (HDL) being unable to stimulate progesterone production if preceded by perifusion with low-density lipoprotein (LDL) or HDL. In addition, 17 beta-estradiol appeared to regulate lipoprotein utilization by attenuating the LDL response after 1 h of perifusion. We conclude that lipoproteins may provide cholesterol substrate for progesterone biosynthesis in vitro and that 17 beta-estradiol, in addition to maintaining progesterone production by luteal cells, may also regulate lipoprotein utilization. Thus, maintenance of steady progesterone secretion in response to estradiol supercedes that of LDL-stimulated progesterone secretion by rabbit luteal cells in vitro. This study suggests an interaction between estrogen and lipoproteins that may prove physiologically important in regulating progesterone production by rabbit luteal cells in vivo.

Although estradiol is the established luteotropic hormone in the rabbit, the corpus luteum also contains a luteinizing hormone (LH)-activated adenylate cyclase system and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase, which suggests that LH and cAMP may play a physiological role in regulating luteal progesterone production. The present study examined whether human chorionic gonadotropin (hCG) and cAMP derivatives stimulate progesterone production by dispersed rabbit luteal cells in static and perifusion incubations. Results of this study show that progesterone production by rabbit luteal cells is significantly stimulated (p less than 0.05) by hCG concentrations at or greater than 0.1 IU/ml or by dibutyryl cAMP concentrations at or greater than 5 mM. Both agents produce maximal stimulations of approximately 4-fold. However, neither prostaglandin E2 or F2 alpha at concentrations of 0.1-3.0 micrograms/ml altered progesterone secretion. When luteal cells were incubated with maximal concentrations of hCG and lipoproteins together, the resultant progesterone secretion was additive. This suggests that the effects of hCG and lipoprotein are independent. Both responses could be blocked completely by cycloheximide (10(-4) M), and thus appear to be dependent on protein synthesis. The cholesterol derivative 25-hydroxycholesterol (20 micrograms/ml) partially overcame the steroidogenic block by cycloheximide, suggesting that transport of cholesterol, regardless of its origin, into mitochondria was an essential protein-mediated event in these cells. Inhibition of the side-chain-cleavage enzyme by aminoglutethamide blocked progesterone production by rabbit luteal cells in vitro. Although estradiol may dominate in the regulation of luteal progesterone production physiologically, this study clearly demonstrates that potential mechanisms do exist in the rabbit corpus luteum for cAMP-mediated stimulation of progesterone production in the rabbit.

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.

This study was undertaken to determine the regulation of stage-specific endometrial proteins of the baboon uterus by estradiol (E2) and progesterone (P). Ovariectomized females were either untreated or treated with E2 for 7 or 14 days, or primed with E2 for 14 days, and then treated with E2 plus P or P alone for 7 or 14 days. Steroids were administered via Silastic capsules and, as determined by radioimmunoassay, physiological levels were present in the peripheral serum of each treatment group. Endometrial tissue obtained after steroid treatment was incubated in the presence of [35S]methionine for 24 h at 37 degrees C, and the culture medium was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by fluorography. Steroid treatment resulted in a doubling of the synthesis and release of labeled macromolecules into culture medium compared to the levels observed in long-term ovariectomized animals, together with the appearance of specific proteins whose synthesis required either E2 or E2 +/- P. A single protein (Mr 33,000; pI 7.6) was only observed after E2 treatment, and a family of high molecular weight basic proteins (Mr greater than 200,000) and a group of acidic proteins (Mr 130,000) required both E2 and P. In the absence of E2, P maintained the synthesis and release of three basic proteins (Mr 88,000, 66,000, and 40,000) as well as the other proteins observed in the ovariectomized animals.(ABSTRACT TRUNCATED AT 250 WORDS)