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The corpus luteum produces progesterone, which is essential for the maintenance of pregnancy. In the absence of a viable embryo, the corpus luteum must regress rapidly to allow for development of new ovulatory follicles. In many species, luteal regression is initiated by uterine release of PGF(2alpha), which inhibits steroidogenesis and may launch a cascade of events leading to the ultimate demise of the tissue. Immune cells, primarily macrophages and T lymphocytes, are present in the corpus luteum, particularly at the time of luteolysis. The macrophages are important for ingestion of cellular remnants that result from the death of luteal cells. However, it has also been hypothesized that immune cells are involved directly in the destruction of luteal cells, as well as in the loss of steroidogenesis; this hypothesis is reviewed in the first part of this article. An alternative hypothesis is also presented, namely that immune cells serve to abate an inflammatory response generated by dead and dying luteal cells, in effect, preventing a response that would otherwise damage surrounding ovarian tissues. Finally, the changes in immune cells that accompany maternal recognition of pregnancy and rescue of the corpus luteum are discussed briefly. Inhibition of immune cells in the corpus luteum during early pregnancy may be due to embryonic or uterine signals, or to maintenance of high progesterone concentrations within the luteal tissue.

In hypophysectomized rats, prolactin induces regression of the corpora lutea. Luteal regression is accompanied by infiltration of monocytes/macrophages, declines in luteal mass and plasma progestins, and increased staining for monocyte chemoattractant protein-1 (MCP-1). We investigated whether similar events are induced during the estrous cycle, after the proestrous prolactin surge. Rats were killed on proestrus or on estrus, and one ovary was frozen for immunohistochemical detection of MCP-1, monocytes/macrophages (ED1-positive), and differentiated macrophages (ED2-positive) and for in situ detection of apoptotic nuclei. Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected from the ovaries of additional rats and frozen for the same analyses and for determination of total protein content. In sections of whole ovaries, intensity and distribution of MCP-1 staining were increased in corpora lutea of multiple ages on estrus as compared to proestrus, as were numbers of differentiated macrophages and apoptotic nuclei per high-power field. Sections of isolated corpora lutea showed these increases on estrus, and the number of monocytes/macrophages per high-power field was also significantly increased. Accompanying these inflammatory/immune events, the corpora lutea on estrus showed decreased weight and total protein per corpus luteum, as compared to corpora lutea on proestrus. These changes are consistent with a proposed role for prolactin in the initiation of luteal apoptosis and of a sequence of inflammatory/immune events that accompany regression of the rat corpus luteum during the normal estrous cycle.

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 µg 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°C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 µM). Media were collected and assayed for progesterone content. Secretion of progesterone (x̄ ± SE, n = 4) was stimulated (p < 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 < 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3β-hydroxysteroid dehydrogenase (3β HSD) activity provided identification of cells with steroidogenic capacity. The diameters (x̄ ± SE) for steroidogenic cells increased (p < 0.05) from Days 2 to 9 (Day 2 = 15.2 ± 0.2 µm; Day 5 = 22.4 ± 0.4 µm; Day 9 = 28.3 ± 1.6 µm). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9. Cells collected on Day 9 were separated by elutriation into two size classes (small ≤ 22 µm and large > 22 µm). Secretion of progesterone was stimulated (p < 0.05) in small cells incubated with oLH (100 ng/ml; 14.7-fold increase) or isoproterenol (100 µm; 6.3-fold increase); secretion of progesterone in large cells was not significantly enhanced by either secretagogue. Thus, the corpus luteum of the Day 9 pseudopregnant rabbit contains steroidogenic cells of heterogeneous sizes, and small and large cell fractions respond differently to oLH nad isoproterenol. These results suggest that two populations of steroidogenic luteal cells exist in the rabbit corpus luteum; these populations may differ in the mechanisms whereby the secretion of progesterone is regulated.

Progesterone, 20α-dihydroprogesterone, and estradiol-17βwere measured in serum of pregnant and pseudopregnant New Zealand White rabbits. Neither progesterone concentrations nor progesterone gradients on consecutive days differed between these two reproductive states until several days after implantation. No differences in concentrations of 20α-dihydroprogesterone were found between pregnant and pseudopregnant rabbits during the 28 days following mating. Estradiol-17βprofiles were similar in pregnant and pseudopregnant rabbits during Days 1–6 and Days 12–17 following mating, with the exception of an apparent rise in estradiol-17βconcentration in pseudopregnant does during luteal regression. These data support the concept that an alteration in ovarian function in response to the presence of embryos does not occur until several days after implantation in the rabbit. Furthermore, the available data clearly indicate that regression of the corpus luteum at the end of pseudopregnancy is not caused by withdrawal of estradiol, the luteotropic hormone. Rather, the corpus luteum regresses in the presence of increasing estradiol concentrations accompanying the return to estrus.

The luteotropic effect of testosterone was investigated in pregnant rats by determining some of the early changes in luteal function associated with the action of this hormone after injection of LH antiserum. The injection of LH antiserum on Day 11 of pregnancy caused a decrease in serum progesterone and in luteal estradiol concentration within 6 h, and subsequently led to abortion. Treatment with a low dose of testosterone via Silastic capsule prevented abortion and the decrease in luteal estradiol and rapidly restored serum progesterone to values which were maintained at elevated, though distinctly lower, values than in control pregnant rats. The reduced serum progesterone in testosterone-treated rats was interpreted as the result of increased formation of 20α-dihydroprogesterone, which appeared in high concentrations within 24 h after injection of LH antiserum and remained elevated through Day 15 of pregnancy, the end of the experiment. In LH antiserum-treated rats without testosterone, the increment in 20α-dihydroprogesterone was also apparent within 24 h, but did not compensate for the sustained loss of progesterone secretion which preceded abortion. Corpora lutea retained aromatase activity in rats aborting after administration of LH antiserum, suggesting that this enzyme was not rate-limiting for estradiol formation in these regressing corpora lutea. These, together with other studies, provide further impetus to the idea that estrogens formed in luteal tissue mediate the sustained progesterone steroidogenic effect of LH. However, one important action of LH may be the suppression of 20α-dihydroprogesterone secretion, thus augmenting the secretion of progesterone.

Our findings that the direct luteotropic effect of estrogen was dependent on the presence of prolactin or rat placental lactogen have suggested to us that prolactin may control estrogen receptor content in the corpora lutea of pregnant rats. To test this hypothesis, the cytosolic and nuclear contents of estrogen receptor were measured in the corpora lutea of pregnant rats in which endogenous prolactin was removed by 1) hypophysectomy and hysterectomy on Day 9, or 2) treatment with ergocryptine (1 mg/rat) on Day 6. Within 24 h, nuclear content of estrogen receptor in lutea cells dropped by 70% in hypophysectomized-hysterectomized rats and 75% in ergocryptine treated rats, while cytosol content dropped by 40% and 38%, respectively. Estradiol treatment (100 µg/ day) did not prevent the decrease in cytosolic or nuclear receptor, whereas prolactin (250 µg/ day) or serum containing rat placental lactogen did maintain luteal cell cytoplasmic receptor content. Nuclear content of estradiol receptor was maintained in ergocryptine treated rats by administration of prolactin alone. However, estradiol had to be administered with prolactin in hypophysectomized-hysterectomized rats to achieve the same effect, suggesting that after ergocryptine treatment intraluteal concentrations of estrogen were sufficient to affect the translocation of cytosolic receptor to the nucleus.These studies indicate that the synergistic action of prolactin with estradiol in the luteotropic process in the pregnant rat may involve, in part, prolactin stimulation of luteal receptor for estradiol, a prolactin dependent response not previously reported.

Quantitative light microscopic and electron microscopic radioautography has been used to investigate the paradoxical increase in RNA synthesis in regressing corpora lutea of the pseudopregnant rabbit after withdrawal of 17β-estradiol. On Day 10 after sterile mating and sc placement of a silastic implant containing 17β-estradiol, implants were removed to initiate regression of corpora lutea. In control animals, the implants were left in place. All rabbits were sacrificed on Day 12 and pieces of corpora lutea were incubated in the presence of [3H] uridine. After appropriate chase periods, the tissues were prepared for light and electron microscopic radioautography. In control corpora lutea most of the luteal cells had concentrations of silver grains over their nuclei, whereas only a diffuse labeling was present over endothelial and interstitial cells. The pattern of grain localization in the regressing tissue showed a marked reduction of silver grains over luteal cells, while the labeling of interstitial connective tissue and vascular endothelium was significantly enhanced. The results from electron microscopic radioautography corroborated quantitative data from light microscopic radioautography, and demonstrated further that the cytoplasm of intensely labeled fibroblasts and endothelial cells in regressing corpora lutea had a highly differentiated appearance as evidenced by the extensive rough-surfaced endoplasmic reticulum and many polyribosomes. It is concluded that rabbit luteal cells maintain a differentiated state under the influence of 17β-estradiol, and that the increased RNA synthesis in regressing corpora lutea represents an activation of interstitial and endothelial cells.

In the pseudopregnant rabbit maintenance of the corpus luteum and progesterone secretion are dependent upon the presence of 17β-estradiol. Withdrawal of estradiol causes a rapid decline in progesterone synthesis, followed by a decline in weight of corpora lutea. In order to determine some of the molecular events associated with this phenomenon, RNA and protein synthesis was analyzed in corpora lutea at different periods after estradiol withdrawal. Within 48 to 72 h luteal RNA and protein contents per unit DNA both declined, but no change occurred in luteal DNA content. However RNA synthesis, measured by incorporation of [3H] uridine into RNA, increased during the same period by as much as 100 percent. This increased rate of [3H] uridine incorporation could not be attributed to increased uptake of the radioactive precursor or to the rate of RNA turnover. Cytological observations showed marked regressive changes in luteal cell structure. The compelling evidence for regression of luteal cells after estrogen withdrawal suggests that the paradoxical increase in RNA synthesis might be attributed to activation of stromal cells in the regressing corpus luteum.

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. Corpus luteum weights on Day 27 were significantly reduced in hysterectomized rabbits (Groups B-D) compared to those in Group A. In Experiment 2, corpora lutea were removed from the ovaries on Day 22, 24 h after hysterectomy or sham hysterectomy, and luteal tissue estradiol-17βconcentrations were measured by radioim’ munoassay. Luteal estradiol- 17βconcentrations (pg/mg tissue and pg/corpus luteum) from hysterectomized and sham hysterectomized rabbits were not significantly different from each other. The results of these experiments indicate that “placental luteotropin” has an important luteotropic role in the pregnant rabbit. Furthermore, these studies indicate that physiological concentrations of estradiol (endogenous or exogenous) have no luteotropic action in the absence of “placental luteotropin,” implying that the placenta may regulate the responsiveness of corpora lutea to estrogen in the pregnant rabbit.

The hypothesis was tested that the action of 17β-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 α-dihydroprogesterone in medium, revealed essentially superimposable rates of production of these steroids for tissues from the hypophysectomized and sham-hypophysectomized animals. We conclude that 17β-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.

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 × 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.

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/µg DNA and 0.4 ± 0.1 fmol/µg DNA, respectively, on Day 10 of pseudopregnancy. An i.v. injection of 4 µg 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 µg estradiol on Day 10, unoccupied cytoplasmic estrogen receptor in saline- and estradiol-injected rabbits was 1.0 ± 0.1 fmol/µg DNA and 1.9 ± 0.1 fmol/µg 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 µg of estradiol can readily stimulate progesterone synthesis, and this stimulation is associated with an increase in cytoplasmic receptor.

Serum progesterone was measured by radioimmunoassay on Days 7, 9, 11, 13, 15, and 16 of pregnancy in three groups of rats. 1) rats with two intact ovaries, 2) rats with one intact ovary (unilaterally ovariectomized the evening of proestrus) and 3) rats with ectopic corpora lutea and a single ovary from which in situcorpora lutea were removed on Day 6 of pregnancy. Progesterone concentrations in the three groups were significantly different (P<0.05) from one another on each day of pregnancy with the exception of Day 13 on which progesterone levels in groups 1 and 2 were not significantly different. Mean progesterone concentrations (ng/ml) and total luteal weights (mg) on Day 16 were respectively 126 ng/ml, 59 mg (group 1), 68, 27 (group 2) and 28, 15 (group 3). A significant positive correlation (P<0.01; r = 0.97; n = 21) was found between total luteal weight and serum progesterone concentration on Day 16 of pregnancy.These data indicate that the local environment of the ovary is not a prerequisite for the corpus luteum to secrete physiological quantities of progesterone which are compatible with the maintenance of pregnancy. The levels of progesterone in rats with ectopic corpora lutea are reduced, but this appears to be largely attributable to the reduced quantity of luteal tissue, rather than to a functional impairment.

We have determined which ovarian tissues and their associated hormones must be present in periparturient rats to evoke normal patterns of serum LH and prolactin in the immediate postpartum period. Ectopic corpora lutea were established in rats by autotransplantation of follicles beneath the kidney capsule on the evening of proestrus (day 0). Rats with ectopic corpora lutea (group 1), ectopic corpora lutea and a single ovary from which corpora lutea had been removed (group 2), or a single intact ovary and no ectopic corpora lutes (group 3) were allowed to litter and blood samples obtained by heart punctures at intervals after the onset of delivery. Pups were removed to prevent suckling. All rats delivered on days 22 to 24. Surges of LH (>1.0 μg/ml serum) were detected at 4.5 or 9 h after delivery in rats with ectopic corpora lutes and a follicular ovary (group 2) and in rats with a single intact ovary (group 3). However, no surges of LH were found in rats of group 1, as late as 18 h after delivery; mean LH concentration was 100 ± 17 ng/ml. With one exception, elevated levels of prolactin (>85 ng/ml) were found only in those animals which showed surges of LH. The data can be interpreted to suggest that parturition and associated withdrawal of luteal and placental hormones permit the postpartum gonadotropin surge but do not comprise the necessary stimulus; the stimulus appears to be provided by hormones produced by nonluteal tissues of the ovary.

Macrophages within the corpus luteum are associated with spontaneous luteal regression in a number of species. However, an understanding of the consequences of macrophage recruitment on the functional capacity and responsiveness of the luteal tissue has remained elusive. Here we investigate the temporal appearance of macrophages and their potential impact in corpora lutea of rabbits, in which a rapid fall in progesterone synthesis and premature regression of the corpus luteum are initiated by withdrawal of the luteotropic hormone estradiol-17β. Removal of estradiol implants, placed subcutaneously, induced a significant increase in the average number of macrophages per high-power field (hpf) in corpora lutea (p< 0.05) within 72 h. Replacement of the estradiol implants 48 h after their removal resulted in a marginal rebound of plasma progesterone and a variable number of luteal macrophages (range: 6–160 macrophages/ hpf) among the 11 rabbits. A third experiment revealed that the relative numbers of macrophages within the corpora lutea have no apparent relationship to rates of progesterone synthesis in vitro: progesterone production (ng/mg tissue) did not differ (p> 0.05) between corpora lutea of estradiol-maintained rabbits and those of estradiol-replaced rabbits despite obvious differences in numbers of luteal macrophages (2 ± 1 vs. 42 ± 10 macrophages/hpf, respectively; p< 0.05). We conclude that the entry/recruitment of macrophages into the rabbit corpus luteum is sensitive to the luteotropic hormone estradiol-17β and that the presence of macrophages does not preclude the continuation of progesterone production in surviving luteal tissue revitalized after estradiol removal/replacement.

Experiments were conducted to determine whether continuous pituitary hormone support is required for expression of the LH and β-adrenergic cAMP signal transduction pathways in rabbit CL during pseudopregnancy. Parameters of the LH and catecholamine cAMP signal transduction pathways in CL of estrogen-treated hypophysectomized rabbits were compared to those of pituitary-intact rabbits. Results showed that each of the parameters of the LH and β-adrenergic cAMP signal transduction pathways was retained in CL taken from estrogen-treated pseudopregnant rabbits that had been hypophysectomized for as long as 13 days at levels not significantly different from those of estrogen-treated pituitary-intact rabbits. These included luteal basal, and LH-, epinephrine-, and fluoride-stimulated adenylyl cyclase activities; total luteal cAMP levels; the number and affinity of cAMP-dependent protein kinase regulatory subunit cAMP binding sites; binding activity of the type I and type II regulatory subunits; and the amount of catalytic subunit protein of cAMP-dependent protein kinase. We conclude that expression of the proteins of the cAMP signal pathway for LH and β-adrenergic hormones in CL of estrogen-treated rabbits does not require pituitary hormone support.

Estradiol-17βwas administered continuously to pseudopregnant rabbits to determine whether this treatment would prevent regression of corpora lutea and prolong the period of progesterone secretion. Estradiol was administered via a Silastic capsule which was placed subcutaneously either on the day of mating (Day 0) or the day after mating to vasectomized males. These estradiol implants maintained serum estradiol at approximately 3-fold normal concentrations. Blood samples were taken on alternate days, beginning on Day 2 and ending on Day 22 or 28 at which time the corpora lutea were removed, weighed and examined histologically. Mean serum progesterone concentrations in untreated pseudopregnant rabbits reached a peak value of 13.4 ng/ml on Day 10 and by Day 18 had fallen to 1.5 ng/ml. Continuous estradiol treatment did not enhance luteal weight or serum progesterone levels which were 14.5 and 2.1 ng/ml on Days 10 and 18, respectively. The inability of estradiol to extend the period of progesterone secretion was not due to altered metabolism of estradiol since the serum concentrations of estradiol were not different on Days 10 and 22 in rabbits with an estradiol implant (∿15 pg/ml). Thus, the corpora lutea regressed in the presence of elevated levels of estradiol which is considered to be the principal luteotropic hormone in the rabbit. In pseudopregnant rabbits which were hysterectomized and treated with estradiol, serum progesterone increased to a peak value of 12.4 ng/ml on Days 10–12 and remained elevated at approximately 11 ng/ml throughout the period of observation (Day 28). Hysterectomized rabbits without estradiol treatment showed a gradual decline in serum progesterone after Days 10–12 to levels of 2 to 3 ng/ml on Day 20 and these levels were maintained through the remainder of the experiment (Day 28). To exclude possible intraovarian influences on progesterone secretion, ectopic corpora lutea were established beneath the kidney capsule. The period of elevated progesterone in rabbits with ectopiccorpora lutea with or without an estradiol implant was similar to that in pseudopregnant rabbits with corpora lutea in situ. This indicates that luteal regression is not caused by intraovarian hormone changes. From these experiments we have concluded that: a) a decline in serum estradiol is not a prerequisite for regression of corpora lutea at the end of pseudopregnancy; b) the ovary is not a previleged site for normal luteolysis; and c) the uterus has a primary role in limiting the life span of the corpus luteum by reducing luteal responsiveness to estradiol.

The hypothesis was investigated that estradiol, the luteotrophic hormone in the rabbit, uncouples luteal progesterone production from regulation by LH/cyclic AMP. Progesterone production by corpus luteum (CL) incubated with vehicle, 3-isobutyl-l-methyl-xanthine (IBMX), hCG, or hCG + IBMX was compared in pseudopregnant rabbits treated continuously with estradiol (estradiol-maintained),withdrawn from estradiol for 24–48 h (estradiol-withdrawn), or withdrawn and then replaced with estradiol for 6 or 24 h (estradiol-replaced). Progesterone production in estradiol-maintained rabbits was not altered by hCG and/or IBMX, but was stimulated significantly in estradiol-withdrawn rabbits. This response was reversed (i.e., abolished) in estradiol-replaced (24 h) rabbits. The loss of responsiveness to hCG was not attributable to impaired accumulation of cyclic AMP: basal and hCG-stimulated cyclic AMP concentrations were similar in luteal tissues of estradiol-maintained and estradiol-withdrawn rabbits. The loss of responsiveness to hCG was also not a consequence of maximal progesterone production: CL of estradiol-replaced (6 h) rabbits were also insensitive to hCG, and this occurred before progesterone production attained a maximal rate. We conclude that a striking feature of the luteotrophic action of estrogen is to uncouple the regulation of progesterone production from cyclic AMR.

The recent characterization of the mitochondrial protein, Steroidogenic Acute Regulatory (StAR) protein, as a rate-limiting protein in steroidogenesis prompted us to investigate whether StAR is expressed in the rabbit corpus luteum and whether the expression of StAR is responsive to estradiol-1 7β, the luteotropic hormone in the rabbit. In rabbits treated continuously with exogenous estradiol through Day 13 of pseudopregnancy (n = 9), immunoblot analysis revealed that luteal expression of StAR was stable, ranging from 8.5 to 9.7 U of corrected integrated optical density. Plasma progesterone concentration (mean SEM) remained elevated in these rabbits (14.3 ± 2.1 ng/ml). In contrast, expression of StAR decreased in corpora lutea of rabbits deprived of estradiol for the last 48 and 72 h of the experiment (4.9 ± 2.2 and 0.3 ± 0.2 U, respectively, n = 3 per group), and was associated with a decline in plasma progesterone (0.8 ± 0.1 and 0.5 0.3 ng/ml, respectively). Replacement of estradiol after 48 h of estradiol deprivation (n = 3) stimulated the reappearance of StAR (10.3 ± 2.6 U) and the restoration of plasma progesterone (10.4 ± 4.9 ng/ml). [35S]Methionine labeling of proteins in rabbit corpora lutea revealed that several isoforms of StAR protein were specifically synthesized in response to estradiol treatment. Collectively, these observations are consistent with a proposed role for StAR in the mediation of the luteotropic effect of estrogen to promote the synthesis of progesterone in the rabbit.

Monocyte chemoattractant protein-1 (MCP-1) is a potential mediator of the recruitment of monocytes/macrophages into the regressing corpus luteum (CL). We investigated whether the luteolytic effect of prolactin in the rat is associated with the expression of MCP-1 and an invasion of monocytes/macrophages. Ovulation was induced in immature female rats by injection of eCG (5 IU, s.c.) at 30 days of age. All rats were hypophysectomized 3 days later. Rats received injections of ovine prolactin (250 µg, s.c.) at 12-h intervals on Day 9, 10, and 11 posthypophysectomy; controls received injection of vehicle. Rats were killed by decapitation 24, 48, or 72 h after the first injection of prolactin or vehicle. In rats treated with prolactin, immunoreactive MCP-1 was detected in the CL at 24 h after the first injection, and a consistent level of staining was reached by 72 h with immunodetectable MCP-1 diffused throughout individual CL. The number of monocytes/macrophages in the CL (mean ± SEM) increased significantly after prolactin treatment, from 3.1 ± 1.8 at 24 h to 49.3 ± 8.2 at 72 h (p< 0.05), and the number of monocytes/macrophages was different from that in control, vehicle-treated rats at 72 h (10.3 ± 4.1; p< 24 and 72 h in prolactin-treated rats (p< 0.05).As a functional correlate of luteal regression, plasma progestins (chiefly, 20α-dihydroprogesterone) declined significantly between 24 and 72 h in prolactin-treated rats (p < 0.05). It is concluded that a potentially important component of the luteolytic effect of prolactin in the rat is the expression of MCP-1 and invasion of monocytes/macrophages into the CL.

The known accumulation of macrophages in corpora lutea (CL) at the time of luteal regression prompted us to investigate whether the chemoattractant protein monocyte chemoattractant protein-1 (MCP-1) is expressed in the rat CL. On the day of confirmed mating (Day 0 of pregnancy), regressing CL from the previous (nonfertile) estrous cycle contained immunodetectable MCP-1 and numerous monocytes/macrophages, whereas the newly formed CL of pregnancy, within the same ovary, contained little MCP-1 and few monocytes/macrophages. MCP-1 diminished in the regressing CL on Days 3 and 9 of pregnancy, although numerous monocytes/macrophages remained. The CL of pregnancy on Days 3 and 9 of pregnancy contained minimal MCP-1 and relatively few monocytes/macrophages. By Days 17 and 21 of pregnancy, however, prior to parturition and prior to an accumulation of monocytes/macrophages, expression of MCP-1 increased in the CL of pregnancy. Northern blots revealed a resurgence of luteal MCP-1 mRNA on Day 21 of pregnancy: 3805 ± 1077 on Day 21 vs. 1059 ± 177 on Day 9 (p< 0.05; expressed as densitometric units relative to β-actin). In conclusion, the expression of MCP-1 in the rat CL in association with, or preceding, the appearance of monocytes/macrophages at the time of luteal regression is consistent with the known role of MCP-1 as a potent chemoattractant for monocytes/macrophages. This suggests that MCP-1 might have a prominent role in the immunological process of luteal regression.

Ectopic corpora lutea were established in rats by autotransplantation of follicles under the kidney capsule on the afternoon of proestrus. These ectopic corpora lutea were subsequently tested for their capacity to secrete the steroids required for the maintenance of pregnancy after implantation. Following ovariectomy on days 7, 9, or 11 of pregnancy (day 0 is proestrus, the day of follicle autotransplantation) 16 of 23 rats aborted. Of the 7 animals that remained pregnant, 5 were observed to have one or more large ectopic follicles on the kidney surface, adjacent to ectopic corpora lutea. In another group of animals (4), the corpora lutea in situwere excised on day 7 leaving the remaining ovarian constituents intact. All animals remained pregnant, demonstrating the capacity of ectopic corpora lutea to secrete progestational steroids. Administration of estradiol-17β(0.1 μg per day in oil, sc) maintained pregnancies in 8 of 9 rats ovariectomized on day 7; with one exception, the administration of LH (NIH-S-14) and/or prolactin (NIH-S-8; -S-9) failed to prevent abortions. These data indicate that secretions from corpora lutea are necessary but not sufficient for the maintenance of pregnancy following implantation. Follicular steroids, presumably estrogens, are required together with progesterone from corpora lutea to effect a suitable hormonal environment for the conceptus prior to day 12 of pregnancy. The requirement for estrogens derived from nonluteal ovarian tissues is transient, because pregnancies were maintained following ovariectomy on days 13 or 14 in animals with ectopic corpora lutea.