Endocrine regulation of the corpus luteum.
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Biomedical subjects
P L Keyes
Publications and source records attributed to P L Keyes.
Tumor necrosis factor-a (TNF-a) production and localization of macrophages and T lymphocytes in the rabbit corpus luteum.
Utilizing immunocytochemistry numerous macrophages were localized in regressing corpora lutea. In contrast, few macrophages were observed in young corpora lutea. Regressing corpora lutea readily produced TNF-a in vitro in response to lipopolysaccharide, whereas young corpora lutea produced significantly less TNF-a. T lymphocytes were identified in young corpora lutea preceding the appearance of macrophages. These observations suggest that cells of the immune system and cytokines could be important participants in physiological regression of the corpus luteum.
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Relationship between blood flow and steroidogenesis in the rabbit corpus luteum.
Blood flow in the corpus luteum of the pseudopregnant rabbit was measured with tracer-labelled microspheres before and at 1 and 3 h after saline treatment (N = 8) or after inhibition of progesterone synthesis with aminoglutethimide (N = 10). Before treatment luteal blood flow (29.5 +/- 3.9 ml/min.g-1 (mean +/- s.e.m.] was much higher than blood flow to other tissues (ovarian stroma = 2.9 +/- 0.6; uterus = 0.5 +/- 0.1; adrenal gland = 2.6 +/- 0.2 ml/min.g-1). Aminoglutethimide reduced serum progesterone by 60% within 1 h but luteal blood flow was unchanged (26.2 +/- 3.5 ml/min.g-1). At 3 h after aminoglutethimide, serum progesterone remained low and luteal blood flow was slightly reduced to 22.5 +/- 3.4 ml/min.g-1. This reduction was associated with a significant decline in mean arterial blood pressure which resulted in luteal vascular resistance being unaltered by aminoglutethimide treatment. Further analysis of these data indicated that serum progesterone concentration was not significantly correlated with blood flow to the corpora lutea or with blood flow to other tissues. In contrast, mean arterial blood pressure was highly correlated with blood flow to the corpus luteum (r = 0.80; P less than 0.001) but not to the ovarian stroma (r = 0.04), or adrenal gland (r = 0.06). These results indicate that luteal blood flow is not acutely responsive to changes in luteal progesterone production and suggest that luteal blood flow changes passively with changes in arterial blood pressure.
Aminoglutethimide↗
Role of estrogen and the placenta in the maintenance of the rabbit corpus luteum.
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Animals↗
The roles of prolactin and testosterone in the development and function of granulosa lutein tissue in the rat.
The luteotropic roles of prolactin and testosterone (or estradiol formed in luteal tissue) were investigated in hypophysectomized rats with homografts of granulosa lutein tissue. Using this approach, we could determine the effects of prolactin independently of estrogen, since granulosa lutein tissue does not produce estrogen de novo under these conditions. Luteinizing granulosa cells were expressed from the ovaries of immature pregnant mare's serum gonadotropin-primed Fischer 344 rats 6 h after injection of human chorionic gonadotropin. The cells were transplanted beneath the kidney capsule of adult, hypophysectomized, ovariectomized Fischer 344 recipients, which were treated with hormones daily for 12 or 14 days. In rats without treatment (no hormones, n = 3) and in rats treated with only testosterone (Silastic capsule, n = 6), only small amounts of luteal tissue (less than 5 mg/rat) were found and serum progesterone remained at low concentrations (10 ng or less) throughout the experiment. In contrast, in rats treated either with ovine prolactin (300 micrograms/day, n = 10) or with the combination of prolactin and testosterone (n = 12), serum progesterone increased to 43 ng/ml by Day 8. Beyond Day 8, serum progesterone continued to rise in rats treated with the combination of prolactin and testosterone to reach a mean value of 87 ng/ml by Day 14, and mean homograft wet weight was 49 mg/rat; in rats treated with only prolactin, serum progesterone decreased to 25 ng/ml by Day 14 and homograft wet weight was lower (24 mg/rat). Prolactin and testosterone together stimulated more homograft aromatase activity in vivo than did prolactin alone, but the in vitro production of progesterone was not different.(ABSTRACT TRUNCATED AT 250 WORDS)
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Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy.
Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Hydroxysteroid Dehydrogenases↗
Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture.
On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.
20-alpha-Dihydroprogesterone↗
D-Ala6-des-Gly10-gonadotropin-releasing hormone ethylamide: absence of binding sites and lack of a direct effect in rabbit corpora lutea.
Rat ovarian tissue has been shown to contain high-affinity gonadotropin-releasing hormone (GnRH) receptors, and synthetic GnRH analogues have been shown to inhibit steroid production by rat corpora lutea in vivo and in vitro. These results raise the possibility that an ovarian GnRH-like peptide may be involved in normal luteal regression. We have examined binding of D-Ala6-des-Gly10-GnRH ethylamide (D-Ala) to rabbit corpora lutea, and have investigated the luteolytic activity of this analogue in hypophysectomized, pseudopregnant rabbits. Three hypophysectomized estrogen-treated rabbits were injected with 0.25 mg D-Ala s.c. every 6 h for 48 h during mid-pseudopregnancy, and three were injected with vehicle only. Treatment with D-Ala produced no acute changes in serum progesterone, nor was the time of luteal regression altered. Rabbit anterior pituitary tissue was found to contain high-affinity GnRH receptors (Ka = 7.0 X 10(9) M-1; 188.2 +/- 35.6 fmol/mg protein). However, no similar high-affinity GnRH receptors were detected in rabbit luteal tissue from any stage of pseudopregnancy. Some apparent low-affinity binding was observed, but this displaceable binding was subsequently observed in all control tissues tested. Thus, a potent GnRH analogue does not have any detectable direct effect on steroidogenesis in the rabbit corpus luteum, nor are high-affinity GnRH binding sites present in rabbit luteal tissue.
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Do catecholamines play a physiologic role in regulating corpus luteum function in the pseudopregnant rabbit?
In these studies the beta-adrenergic receptor antagonist propranolol was administered to estrogen-treated hypophysectomized pseudopregnant rabbits in vivo, and serum progesterone concentrations were measured to monitor luteal function. In Experiment 1, which was designed to determine an effective dose of propranolol, 1 mg/(kg X h) s.c. propranolol for 3 h (integral of 80 ng/ml in serum) gave an adequate level of beta-adrenergic receptor blockade, i.e., a 1000-fold inhibition of the blood pressure/isoproterenol dose-response relationship. In Experiment 2, "acute" administration of propranolol (P; 1 mg/(kg X h) s.c.) or saline (control, C) for 24 h on Days 7-8, 10-11, and 13-14 of pseudopregnancy did not produce any marked differences in serum progesterone concentrations in P or C animals on any of the days tested, although hourly fluctuations were observed. In Experiment 3, "chronic" (4-day) treatment with propranolol was achieved by the use of propranolol-containing pellets placed s.c. (integral of 200-600 ng/ml in serum), on Days 13-17. Control animals received pellets of vehicle only. Serum progesterone concentrations were very similar in P and C animals throughout the period of treatment (Days 13-17) and on Days 18 and 20. We conclude that endogenous catecholamines play no major role in regulating luteal steroidogenesis or corpus luteum regression in the pseudopregnant rabbit.
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Control of corpus luteum function in the pregnant rabbit: role of the placenta ("placental luteotropin") in regulating responsiveness of corpora lutea to estrogen.
Experiments were performed to examine the interaction between estrogen and "placental luteotropin," the two luteotropins thought to be required for corpus luteum maintenance in the pregnant rabbit. Experiment 1 was designed to determine whether estrogen alone was luteotropic in the absence of "placental luteotropin." Pregnant rabbits were assigned to the following groups: Group A, sham hysterectomy; Group B, hysterectomy; Group C, hysterectomy plus estradiol on Day 20; and Group D, hysterectomy plus estradiol on Day 22. "Placental luteotropin" was removed on Day 21 of pregnancy by hysterectomy and estrogen was administered via an estradiol-filled Silastic implant which was placed s.c. before (Day 20, Group C) or after (Day 22, Group D) hysterectomy. Daily blood samples were taken for radioimmunoassay of serum progesterone and estradiol concentrations. Corpus luteum weights were measured at autopsy on Day 27. Hysterectomy caused serum progesterone concentrations to fall rapidly from 13 +/- 1 ng/ml on Day 21 to 2 +/- 1 ng/ml on Day 23, and to 1 ng/ml or less on Days 24-27. In sham hysterectomized rabbits (Group A), pregnancy was maintained and serum progesterone concentrations remained elevated at 9-15 ng/ml throughout (Days 20-27). Estradiol treatment (Groups C and D) did not prevent or reverse luteal regression induced by hysterectomy and serum progesterone concentrations declined in a similar fashion to Group B. Serum estradiol concentrations were 4-8 pg/ml in all groups and did not vary substantially with stage of pregnancy or treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
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Transient development and function of rabbit corpora lutea after hypophysectomy.
The requirement of the pituitary gland and the role of 17 beta-estradiol in the early development of the corpus luteum was investigated in rabbits hypophysectomized the day after sterile mating (day 1). Serum progesterone in hypophysectomized rabbits was normal for 2 days after hypophysectomy. Luteal tissue from hypophysectomized and sham-hypophysectomized rabbits had similar wet weight (4.0 +/- 0.4 vs. 5.3 +/- 0.2 mg/corpus luteum) and similar concentrations of available cytoplasmic estrogen receptor (1.2 +/- 0.2 vs. 1.5 +/- 0.3 fmol/micrograms DNA) and luteinizing hormone (LH) receptor (4.0 +/- 0.2 vs. 6.1 +/- 1.4 fmol/micrograms DNA) on day 4 of pseudopregnancy. Serum progesterone in hypophysectomized rabbits began to decline on day 4 and was undetectable by day 6. Estrogen receptor and luteal weight in hypophysectomized animals also declined after day 4 to low values by day 6, and serum estradiol was undetectable. However, if estradiol was administered by Silastic capsule implanted subcutaneously at the time of hypophysectomy or 3 days after hypophysectomy, serum progesterone, luteal weight, estrogen receptor, and LH receptor were maintained on day 6 of pseudopregnancy. These results indicate that after a preovulatory gonadotropin surge, the function of newly formed corpora lutea is normal for 3-4 days in the absence of pituitary hormones. However, by 4 days after ovulation, estradiol is required to sustain the structural and functional integrity of corpora lutea.
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17 beta-estradiol maintains normal function of corpora lutea throughout pseudopregnancy in hypophysectomized rabbits.
The hypothesis was tested that the action of 17 beta-estradiol is sufficient to ensure normal luteal function in the absence of pituitary hormones. Rabbits were mated to vasectomized males to induce a normal pseudopregnancy and the next day (Day 1) hypophysectomized or sham hypophysectomized. At this time a Silastic capsule containing estradiol was inserted s.c. in all rabbits. In the presence of this implant serum estradiol values in hypophysectomized and sham-hypophysectomized animals were not different; the mean concentration for the two groups was 9 pg/ml. Measurements of serum progesterone throughout pseudopregnancy revealed essentially superimposable curves for hypophysectomized, estradiol-treated and sham-hypophysectomized, estradiol-treated animals. Further, pseudopregnancy was of normal duration in the two groups, about 17 days. Progesterone secretion was acutely dependent upon estradiol in the hypophysectomized animals, as shown by removal of the Silastic capsule on Day 10; progesterone declined from 18 ng/ml of serum to 10 ng/ml within 12 h, and by 24 h after removal of the implant was 0.5 ng/ml. Twelve-hour incubations of corpora lutea, and measurement of progesterone and 20 alpha-dihydroprogesterone in medium, revealed essentially superimposable rates of production of these steroids for tissues from the hypophysectomized and sham-hypophysectomized animals. We conclude that 17 beta-estradiol satisfies the criteria for the primary luteotropic hormone in this species; further, regression of the corpora lutea is normal and predictable in the presence of estradiol. Beyond the first day following ovulation, the secretion of luteinizing hormone (LH) or other pituitary hormones is not required for a normal course of development, maintenance and regression of the corpora lutea.
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Control of corpus luteum function in the pregnant rabbit: role of estrogen and lack of a direct luteotropic role of the placenta.
The corpus luteum is essential for pregnancy maintenance in the rabbit and appears to require two luteotropins: estrogen from ovarian follicles and a placental luteotropic factor. We have investigated the role of the placental luteotropic factor in maintaining corpus luteum function in the pregnant rabbit in the absence of estrogen. In Exp 1, follicular estrogen was withdrawn on day 21 of pregnancy by ovulating follicles with 10 IU hCG. In Exp 2, estrogen was withdrawn in hypophysectomized pregnant rabbits on day 21 by removing an estradiol (E2) implant. In the presence of this estrogen implant, luteal function and pregnancy are maintained after hypophysectomy, performed on day 4 of pregnancy. In both experiments, fetoplacental viability was ensured by treating the rabbits with medroxyprogesterone acetate (MPA). In both Exp 1 and 2, withdrawal of estrogen on day 21 of pregnancy caused a dramatic decline in serum progesterone concentrations by day 22. Serum progesterone concentrations remained low, and corpora lutea regressed, although viable fetuses were maintained with MPA. In animals not receiving MPA, estrogen withdrawal caused the loss of luteal function, followed by abortion on days 23-24. In contrast, estrogen replacement (via E2 implant) on day 22 in Exp 1 was fully capable of restoring serum progesterone concentrations to pretreatment values on days 24-27 in MPA-treated rabbits. In rabbits not receiving MPA, estrogen replacement also restored serum progesterone concentrations and prevented abortion. These results provide further evidence that estrogen is essential for normal luteal function in the pregnant rabbit. In the absence of estrogen, the rabbit placenta maintained by the progestagen MPA has no direct luteotropic activity.
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Contrasting effects of oestradiol-17 beta and human chorionic gonadotrophin on steroidogenesis in the rabbit corpus luteum.
On Day 10 of pseudopregnancy, rabbits were given an i.v. injection of hCG (10-20 i.u.) that was sufficient to cause new ovulations and the loss of follicular oestradiol secretion. There was an immediate 3-4-fold rise in serum progesterone which returned to near prestimulation values (approximately 27 ng/ml) within 12 h in the presence of an implant containing oestradiol-17 beta. In the absence of oestradiol, serum progesterone continued to decline to reach low values (approximately 4 ng/ml) within 24 h and the original corpora lutea subsequently regressed. The administration of oestradiol 24 h after injection of hCG, when progesterone secretion was low, arrested any further decline in progesterone and then restored serum progesterone to normal values. This steroidogenic effect of oestradiol in vivo was a function of enhanced luteal steroidogenesis; corpora lutea removed and incubated for 12 h produced progesterone at high, linear rates, whereas the corpora lutea from animals that did not receive oestradiol produced low or insignificant quantities of progesterone in vitro. We conclude that hCG at these doses is compatible with continued responsiveness of the corpora lutea to oestrogen and that hCG produces its luteolytic effect primarily by ovulating follicles, thus stopping the secretion of the luteotrophic hormone, oestradiol.
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Relationships between estrogen receptor and estradiol-stimulated progesterone synthesis in the rabbit corpus luteum.
The effects of estradiol on luteal estrogen receptor and steroidogenesis were examined on Days 9 through 11 of pseudopregnancy. In normal pseudopregnant rabbits, unoccupied cytoplasmic and total nuclear estrogen receptor were 2.6 +/- 0.4 fmol/microgram DNA and 0.4 +/- 0.1 fmol/microgram DNA, respectively, on Day 10 of pseudopregnancy. An i.v. injection of 4 micrograms of estradiol caused the translocation of cytoplasmic receptor and a 6.6-fold increment in total nuclear receptor within 15 min, which was followed by rapid processing of the nuclear receptor. Both cytoplasmic and nuclear estrogen receptor returned to normal values within 24 h, and during this period, serum progesterone did not change significantly. Withdrawal of an estradiol implant from animals on Day 9 of pseudopregnancy initiated a marked decline in serum progesterone within 24 h. Following an injection of saline or of 4 micrograms estradiol on Day 10, unoccupied cytoplasmic estrogen receptor in saline- and estradiol-injected rabbits was 1.0 +/- 0.1 fmol/microgram DNA and 1.9 +/- 0.1 fmol/microgram DNA, respectively, on Day 11 of pseudopregnancy. Associated with the increase in cytoplasmic receptor there was an increase in serum progesterone (8.2 +/- 1.5 ng/ml), in contrast to saline-injected animals in which serum progesterone continued to decline (1.6 +/- 0.4 ng/ml). Despite the significant differences in cytoplasmic receptor and in progesterone synthesis, total nuclear estrogen receptor was not different in these animals. These data suggest that in corpora lutea already secreting progesterone at high rates during midpseudopregnancy, the rapid translocation of available estrogen receptor does not cause further stimulation of progesterone synthesis. However, if steroidogenesis is first reduced experimentally, then an injection of 4 micrograms of estradiol can readily stimulate progesterone synthesis, and this stimulation is associated with an increase in cytoplasmic receptor.
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A method for transplantation of luteinizing granulosa cells: evidence for progesterone secretion.
A method is described to enable the investigation of luteal tissue that is formed in the absence of ovarian theca lutein cells. Immature rats were given a s.c. injection of pregnant mare's serum gonadotropin (PMSG) followed 48 h later by an i.v. injection of human chorionic gonadotropin (hCG). Both ovaries were removed 8 to 10 h after the injection of hCG, the preovulatory follicles punctured, and the granulosa cells expressed into medium using gentle pressure with a dissecting scalpel. The cells were centrifuged at low speed (50 X g) for 5 min, resuspended and recentrifuged. The cells were aspirated into PE 90 polyethylene tubing, the tubing sealed at one end and then placed into a microcapillary hematocrit tube. The cells were packed by centrifugation in a microhematocrit centrifuge for 2 min, after which they were autotransplanted by injecting them beneath the kidney capsule. Progesterone, determined in blood samples obtained 4 through 12 days after transplantation, was elevated on most days compared to ovariectomized controls without cell transplants. In a second experiment, serum progesterone in ovariectomized-adrenalectomized rats with autotransplants was also elevated (4 to 6 ng/ml) compared to controls without cell transplants in which progesterone was about 0.1 ng/ml. Morphology of transplants revealed luteal cells with considerable vascularity and with the typical appearance of cells observed in in situ corpora lutea. This procedure, with modifications to include homotransplants in inbred strains, holds promise for investigation of the endocrine characteristics of granulosa lutein cells devoid of cells of thecal origin.
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Restoration of the LH surge and ovulation by insulin in alloxan-diabetic immature rats treated with pregnant mare's serum gonadotrophin.
Immature alloxan-diabetic rats injected with pregnant mare's serum gonadotropin (PMSG) do not ovulate and the LH surge is absent. In the present studies we have examined the effects of several insulin treatments on the LH surge and ovulation in alloxan-diabetic rats. Rats were male diabetic by injection of alloxan on day 24 of age. Only those rats with fasting blood glucose concentrations exceeding 180 mg/100 ml on day 237 were considered diabetic. PMSG was injected on day 30. Rats received insulin either by injection (2, 3, 4 or 6 IU/100 g/day); Ultralente B.I.D.) or by subcutaneous implants (Alzet osmotic minipumps; 1.8 or 2.4 IU/day). None of the diabetic rats without insulin treatment ovulated. Some of the animals in each insulin treatment group ovulated, however, the percentage of animals ovulating was highly variable from experiment to experiment when the insulin was given by injection. When insulin was administered by osmotic minipump, the results were more consistent, with at least 60% of the rats ovulating in each experiment. LH surges were found on the afternoon of presumed pro-oestrus (day 32) in diabetic insulin-treated rats which ovulated. In confirmation of previous results, rats without insulin treatment did not have LH surges. Although the site of insulin action has not been determined, these data indicate that the LH surge mechanism in the immature PMSG-treated rat is insulin-dependent.
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