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We investigated whether prolactin (PRL) treatments resembling the intermittent PRL surges of estrous cycles could induce luteal regression in hypophysectomized rats. Immature female rats were stimulated to ovulate and form corpora lutea with exogenous gonadotropins, and were hypophysectomized following ovulation. A single s.c. injection of either vehicle (VEH) or PRL was administered to each rat on post-hypophysectomy Day 8 and again on Day 11. The four resulting treatment groups consisted of rats that received two injections of VEH, VEH followed by PRL, PRL followed by VEH, or two injections of PRL. Rats were killed 24 or 72 h following the second injection. Plasma 20alpha-dihydroprogesterone, luteal weight, and total luteal protein were determined. One ovary was sectioned for immunohistochemistry for monocytes/macrophages, apoptotic nuclei, and major histocompatibility class II (MHC II) molecules. No effect of time (following injection) was observed on any endpoint, indicating that PRL does not have an ongoing regressive action. Time groups from within each treatment group were therefore pooled for analysis. Significant declines (P: < 0.05) in plasma concentrations of 20alpha-dihydroprogesterone, luteal weight, and protein per corpus luteum occurred only after two injections of PRL. Numbers of luteal monocytes/macrophages, apoptotic nuclei, and MHC II-positive cells were low in all groups; numbers of luteal monocytes/macrophages increased following two injections of PRL (P: < 0.05). We conclude that PRL has a cumulative regressive effect on the corpus luteum of the hypophysectomized rat. Drawing a parallel with the estrous cycle, we suggest that continued exposure to PRL, over several cycles, is necessary to induce full luteal regression.

The administration of prolactin to hypophysectomized rats results in regression of the corpora lutea, accompanied by immune-inflammatory events such as infiltration of monocytes and macrophages. Recent reports indicate an autocrine role for progesterone during the lifespan of the corpus luteum. In the present study, an inhibitor of 3beta-hydroxysteroid dehydrogenase, Trilostane, was used to investigate the hypothesis that a decrease in luteal tissue steroids precipitates the cascade of immune-inflammatory events leading to luteal regression in prolactin-treated hypophysectomized rats. Immature rats were induced to ovulate by administering eCG-hCG, and hypophysectomized on the day after ovulation (at 32 days of age). Rats were injected s.c. 9-11 days after hypophysectomy with (a) Trilostane (80 mg kg(-1) day(-1)), (b) ovine prolactin (500 mg day(-1)), (c) Trilostane plus prolactin, or (d) vehicle. Plasma and luteal tissue progesterone and 20alpha-dihydroprogesterone ('progestin') were quantified; luteal tissue monocytes-macrophages and apoptotic nuclei were counted, and luteal wet mass was determined. Rats treated with prolactin alone showed the expected markers of luteal regression: decreased plasma progestin, increased numbers of monocytes-macrophages and apoptotic nuclei in luteal tissue, and decreased luteal wet mass; however, progestin concentration in luteal tissue was unchanged. Treatment with Trilostane reduced plasma and luteal tissue progestin, but did not result in an infiltration of monocytes-macrophages or increased numbers of apoptotic nuclei in the corpora lutea, or any change in luteal wet mass. Trilostane in combination with prolactin reduced plasma and luteal tissue progestin and produced the expected markers of regression, with the exception of luteal tissue mass, which remained unchanged. In conclusion, inhibition of steroidogenesis does not initiate luteal regression or augment prolactin-induced luteal regression in hypophysectomized rats. Prolactin-induced infiltration of monocytes-macrophages is not accompanied by a decrease in luteal tissue progestin, at least in the early stages of luteal regression.

During the estrous cycle, secretion of prolactin is largely restricted to a surge on proestrus. We investigated whether this proestrous prolactin surge initiates regression of the corpora lutea of the preceding cycle. Adult rats were killed prior to the prolactin surge (Proestrus group), following the prolactin surge (Estrus group), after chemical blockade of the prolactin surge with bromocryptine (Estrus+BRC group), and after blockade of the prolactin surge and administration of prolactin (Estrus+BRC+PRL group). Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected out, weighed, and sectioned for immunohistochemistry or cultured for examination of in vitro progestin production. Numbers of luteal monocytes/macrophages, differentiated macrophages, and apoptotic nuclei per high-power field were greater for Estrus and Estrus+BRC+PRL than for Estrus+BRC, which in turn had greater numbers than Proestrus (P< 0.05). In contrast, BRC completely reversed the decline in luteal weight observed between Proestrus and Estrus (P<0.05). Number of major histocompatibility complex II-positive cells was not different between groups (P>0.05). Finally, progestin production by corpora lutea in vitro was lower for Proestrus than for the other groups (P<0.05). The results indicate that the prolactin surge alone is not responsible for initiation of apoptosis or immune cell infiltration in regressing corpora lutea of the estrous cycle, although prolactin increases these markers of regression. Prolactin does cause a decline in luteal weight; however, the corpora lutea retain the capacity for steroidogenesis. We conclude that although prolactin has a role in luteal regression, it is not solely responsible for the initiation of this process.

We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.

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-17beta. 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-17beta and that the presence of macrophages does not preclude the continuation of progesterone production in surviving luteal tissue revitalized after estradiol removal/replacement.

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-17 beta, 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 micrograms, 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). 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 beta-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.

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-1-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 AMP.

The rabbit corpus luteum possesses LH receptors that are coupled to adenylyl cyclase, but paradoxically it does not require LH as a luteotrophic factor for the maintenance of progesterone secretion. This suggests that rabbit luteal cells may not respond physiologically to LH. Therefore, the present study was undertaken to investigate the responsiveness of the rabbit corpus luteum of pseudopregnancy to human chorionic gonadotrophin (hCG) which acts on the same receptor as LH. Pseudopregnancy was induced by injection of 40 IU pregnant mare serum gonadotrophin followed 50 h later by an injection of 40 IU hCG (day 0). On days 7 and 11 of pseudopregnancy, corpora lutea were obtained and incubated for 2 or 5 h in the presence of either 0.1 or 1 microgram/ml hCG or 1 mM monobutyryl cyclic AMP (bcAMP). Neither hCG nor bcAMP stimulated progesterone production by the isolated corpus luteum, despite a sustained high rate of progesterone production by the tissue throughout the incubation period. By contrast, Graafian follicles removed from the same ovaries and incubated under the same conditions responded both to hCG and bcAMP with large increases in progesterone production. To determine whether the cyclic AMP content of the corpus luteum was altered by in vitro exposure to hCG, day 7 and day 11 corpora lutea were incubated for 5 or 15 min with various concentrations of hCG, and cyclic AMP in the tissue was then measured. Even at the highest concentration of hCG tested (10 micrograms/ml), the cyclic AMP content of the corpus luteum was unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

The potential role of macrophages and T lymphocytes in the destruction of the corpus luteum at the end of the luteal phase was investigated by treating pseudopregnant rabbits with the immunosuppressant glucocorticoid methylprednisolone. Eleven specific pathogen-free New Zealand White rabbits were injected with pregnant mares' serum gonadotrophin (40 iu, i.m.), followed 2 days later by human chorionic gonadotrophin (40 iu, i.v.) to stimulate ovulation. The following day (day 1 of pseudo-pregnancy) all animals had an oestradiol-filled Silastic capsule implanted s.c., to ensure that oestradiol, the luteotrophic hormone in this species, would not be limiting. From day 10 of pseudopregnancy, three animals were injected with a low dose of methylprednisolone (2 mg kg-1 per day) until day 20. Three other animals were injected with a higher dose of methylprednisolone (20 mg kg-1 per day) from day 13 of pseudopregnancy until day 19. Five animals served as control, vehicle-injected animals. Blood samples were taken at intervals and assayed for progesterone. Immunofluorescence was used to stain luteal tissue for macrophages, T lymphocytes and class II antigens, and positive cells were counted under high-power magnification. Methylprednisolone treatment reduced (by about 70%), but did not eliminate, the macrophages in the regressing corpora lutea. In contrast, the high dose of methylprednisolone essentially eliminated T lymphocytes, and reduced (by about 90%) the number of cells expressing class II antigen in the luteal tissue. Despite the effects of methylprednisolone on these cells, serum progesterone profiles were not altered by treatment with methylprednisolone, and pseudopregnancy was of normal duration.(ABSTRACT TRUNCATED AT 250 WORDS)

A potential role of insulin-like growth factor I (IGF-I) in the regulation of steroidogenesis in the rabbit corpus luteum was investigated using a primary culture of luteal cells obtained 3 days after ovulation. Dissociated cells were cultured for 1 day in the presence of medium 199 and 10% fetal bovine serum; thereafter, the cells were cultured in medium 199 containing 0.1% BSA, gentamicin (50 micrograms/ml), and hormones or growth factors, and without serum. IGF-I (human recombinant, 100 ng/ml) was as effective as LH (ovine, 10 ng/ml) in maintaining progesterone accumulation through 4 days of culture. Estradiol (10(-8) M), either alone or in combination with LH or IGF-I failed to stimulate progesterone accumulation, which was not surprising since these cells did not possess estrogen receptors. The stimulation of progesterone by IGF-I was not detectable until 24-36 h after introduction of the growth factor to the cultures, whereas stimulation by LH was observed within 2 h. The steroidogenic effect of IGF-I was not attributable to increased cell number, as DNA values or [3H]thymidine incorporation were unchanged by IGF-I. IGF-I increased functional enzymatic activity, observed as increased progesterone accumulation in the presence of 25-hydroxycholesterol used as exogenous substrate. These data indicate that luteal cells have the capacity to respond to IGF-I, raising the possibility that IGF-I has a role in the regulation of steroidogenesis in the rabbit corpus luteum.

Experiments were conducted to determine whether continuous pituitary hormone support is required for expression of the LH and beta-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 beta-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 beta-adrenergic hormones in CL of estrogen-treated rabbits does not require pituitary hormone support.

To gain a better understanding of the luteotropic action of estrogen, we have investigated the effect of estrogen on the synthesis of the enzyme, cholesterol side-chain cleavage cytochrome P-450 (P-450scc) in the rabbit corpus luteum. Using an established protocol, rabbits were treated with estradiol, and the estradiol was then withdrawn on day 9 of pseudopregnancy, which caused an 88% fall in serum progesterone within 48 h. In other rabbits, estradiol was replaced at 48 h which stimulated a 6.6-fold increase in serum progesterone concentration within the next 24 h. Luteal tissues were incubated with [35S]methionine and homogenized, and a mitochondrial fraction lysate was obtained. Equal trichloroacetic acid-precipitable radioactivity was taken for immunoprecipitation using a well-characterized polyclonal antiserum against bovine adrenal P-450scc. The immunoisolated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radioactivity was visualized by autofluorography. The results indicate that the rate of synthesis of P-450scc in 48 h-estradiol withdrawn animals was markedly reduced, and by 72 h of withdrawal was barely detectable. When estradiol was reintroduced, the synthesis of P-450scc was increased. Despite the prominent changes in P-450scc synthesis, immunoblotting revealed only a minimal (approximately 30%) decrease in relative P-450scc content by 72 h after estradiol withdrawal. Analyses of DNA and protein contents of luteal tissues revealed an increase in DNA per mg luteal tissue, a decline in total tissue protein/DNA ratio, but no change in mitochondrial fraction protein/DNA ratio after estrogen withdrawal. The results indicate that de novo synthesis of P-450scc in the corpus luteum is sensitive to estrogen; however, the estrogen-sensitive rate-limiting step(s) for steroidogenesis are at other sites in the steroid biosynthetic pathway.

The potential involvement of macrophages, T lymphocytes, and the cytokine tumor necrosis factor (TNF) in regression of the corpus luteum was investigated at different stages of pseudopregnancy and pregnancy by use of immunocytochemical methods and a TNF bioassay. Few macrophages (11 +/- 6 per high power field of 8-microns frozen sections of corpus luteum, Day 10 of pseudopregnancy) were observed until the very end of pseudopregnancy, when the number of macrophages increased greatly (176 +/- 42 per high power field, Day 19 of pseudopregnancy). Pregnancy, of 32 days duration, delayed large-scale macrophage accumulation until 3 days after parturition (154 +/- 30 per high power field). Low TNF activity (approximately 1.0 U/mg protein) was detected in incubations of luteal tissue at all stages; in response to lipopolysaccharide, TNF values in medium increased 10- to 30-fold at times of luteal regression and macrophage accumulation (1 day postpartum and Day 19 of pseudopregnancy). Class II-positive T lymphocytes were observed in luteal tissue, but unlike macrophages, the number of lymphocytes did not increase at the time of regression of the corpus luteum. These data are consistent with the hypothesis that involution of the corpus luteum is promoted through the interactions of inflammatory cells and action of TNF, although the action of TNF has not been determined in this luteal tissue. Through unknown mechanisms, pregnancy postpones the accumulation of macrophages in the corpus luteum, in association with the prolongation of luteal function until the time of parturition.

To explain the high rate of blood flow in the corpus luteum, we hypothesize that luteal blood vessels offer minimal resistance to flow and are incapable of vasomotion. This hypothesis was tested in rabbits at mid-pseudopregnancy by measuring blood flow in the corpus luteum and ovarian stroma with tracer-labeled microspheres at three levels of arterial blood pressure, which was manipulated by constricting the aorta above the ovarian artery. In addition, the distribution of vascular smooth muscle in the ovary was evaluated with morphological and immunocytochemical techniques. Decreases in arterial pressure were paralleled by reductions in blood flow in the corpus luteum, whereas ovarian stromal blood flow was unchanged. Consistent with our hypothesis, there was no change in the low level of vascular resistance offered by blood vessels in the corpus luteum, supporting the view that they are maximally dilated and incapable of autoregulation. Morphologically, the vessels within the corpus luteum appeared as large sinusoidal capillaries without smooth muscle, providing an anatomical explanation for the lack of vasomotor control demonstrated physiologically. The absence of vascular smooth muscle was confirmed with immunocytochemistry using an antibody against the muscle-specific intermediate filament, desmin. The fluorescein-labeled antibody decorated arteries and arterioles within the ovarian stroma and near the capsule of the corpus luteum, but did not decorate vessels in the corpus luteum of pseudopregnancy, providing additional evidence that the vessels of the corpus luteum lack the smooth muscle investment necessary to change vascular caliber. From these findings, we have proposed a novel scheme to explain intraovarian blood flow regulation. Vascular resistance in the ovarian stroma, as in most tissues, is acutely regulated by dilation or constriction of intratissue arterioles. In contrast, vascular resistance within the corpus luteum is modeled as a relatively invariable parameter, fixed at a low level by the morphological characteristics of the luteal vasculature. Therefore, the corpus luteum operates on a linear (maximally "vasodilated") pressure-flow curve, does not actively regulate intratissue blood flow, and is subject to acute regulation of perfusion only through changes in extra-luteal vessels.

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.

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)

The effects of hCG on luteal estrogen receptor and steroidogenesis were examined in 9-day-pseudopregnant rabbits. Twenty-four hours after the injection of ovulatory dosages of hCG (10-100 IU), a dose-related loss of luteal estrogen receptor and in vitro progesterone production was observed. The declining progesterone production was not due to the increased conversion of progesterone to the major metabolite, 20 alpha-dihydroprogesterone. The loss of steroidogenesis induced by hCG was not permanent and could be reversed by estradiol treatment started within 24 h of hCG injection. In those animals with a higher luteal estrogen receptor content at the start of the estrogen treatment, the steroidogenic response was greater. These findings indicate that hCG-induced loss of steroidogenesis is associated with the loss of luteal estrogen receptor. The degree of restoration of progesterone synthesis induced by a 24-h period of estradiol treatment may be related to the content of unoccupied cytoplasmic estrogen receptor in the corpus luteum.

Nuclear and cytoplasmic estrogen receptors have been identified and characterized in the rabbit corpus luteum, and validated methods are described for the measurement of both unoccupied and total estrogen receptor. Binding was specific for the biologically active estrogens. Equilibrium binding analysis of cytoplasmic estrogen receptor yielded saturable, high affinity binding sites with a Kd of 5.4 x 10(-11) M. Two types of binding sites were found in the nuclear fraction: one with high affinity (Kd = 8.9 x 10(-11) M) and low capacity, the other with low affinity (Kd = 2.7 x 10(-8) M) and high capacity. In the presence of 0.4 M KCl, the sedimentation coefficients of nuclear and cytoplasmic estrogen receptors are 3.4S, while the value is 6.8S for cytoplasmic receptor in buffer without KCl. Twenty minutes after an iv injection of 2 micrograms 17 beta-estradiol, the available and total cytoplasmic estrogen receptors were depleted. This depletion was accompanied by concomitant and stoichiometric accumulation of receptor in the nucleus, indicating an apparent translocation of the receptor. In corpora lutea of normal rabbits, approximately 80% of the total nuclear receptor is unoccupied. Evidence is presented to suggest that nuclear receptor sites might be ordinarily occupied with estradiol, but during isolation of the nuclear fraction these sites become available or unoccupied. The identification of nuclear estrogen receptor and the phenomenon of translocation of cytoplasmic receptor to the nucleus suggest a similarity of estrogen action in the rabbit corpus luteum and other estrogen target tissues.

Recent reports of an LH-like hormone in the rabbit preimplantation blastocyst and of elevated serum progesterone levels in the presence of unimplanted blastocysts prompted us to characterized further the biological activity of the presumed gonadotropin. Progesterone was measured by a highly specific radioimmunoassay in sera obtained from pregnant and pseudopregnant rabbits after mating (day 0) to fertile or vasectomized males. On days 3, 4, 5, and 6, which represent the preimplantation period, mean progesterone concentrations (ng/ml +/- SE) were 4.3 +/- 0.7, 5.1 +/- 0.5, 6.8 +/- 1.1, and 9.0 +/- 1.9 in 5 pseudopregnant rabbits and 4.1 +/- 0.4, 6.7 +/- 0.6, 5.9 +/- 0.9, and 7.0 +/- 0.8 in 7 pregnant rabbits. In a separate experiment, serum progesterone concentrations in 6 pseudopregnant and 8 pregnant rabbits were 10.1 +/- 0.7 ng/ml and 13.7 +/- 1.1 (P less than 0.02), respectively on days 11-12. Thus, serum progesterone concentrations were not different in pregnant and pseudopregnant rabbits before the time of implantation (day 7), but were higher in pregnant rabbits after implantation. Blastocysts obtained on day 6 and incubated with a cell suspension of immature rat ovaries failed to stimulate the accumulation of progesterone in medium, in contrast to hCG, which was active even in the presence of blastocysts. Day-6 blastocysts also failed to stimulate the accumulation of testosterone from decapsulated rat testes and of progesterone from rabbit ovarian tissues in vitro. A gonadotropic effect of the conceptus can be observed in the rabbit within 4 to 5 days after implantation. However, we find no evidence for the existence of an LH-like hormone in the preimplantation blastocyst which stimulates the rabbit ovary to secrete progesterone.

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.

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.

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.

17-beta-Estradiol, which is luteotropic in rabbits, was administered during pseudopregnancy via polydimethylsiloxane (Silastic) implants to determine the effects on serum progesterone concentrations. Implants which released estradiol at a rate of approximately 2 mug/day were place beneath the skin the day after sterile mating and ovulation (day 0). Blood (3 ml) was obtained from the marginal ear vein on days 3, 6, 9, 10, 11 and 12. Serum estradiol levels, determined by radioimmunoassay, were 2- to 3-fold higher in estradiol-treated rabbits (11.7 plus or minus 1.2 pg/ml) than in untreated pseudopregnant controls (5.9 plus or minus 1.4 pg/ml). Weights of corpora lutea in treated and control rabbits were not different at the conclusion of the experiment on day 12. Serum progesterone concentrations, also determined by radioimmunoassay, were not significantly different between treated and control animals. However, when estradiol implants were removed from other rabbits on day 10, a rapid decline in serum progesterone occurred, from 14.0 plus or minus 2.4 to 2.6 plus or minus 0.8 ng/ml 24 h later. By comparison, serum progesterone concentrations in rabbits with estradiol implants left in place and in untreated rabbits on day 12 were similar (similar to 12 ng/ml). The premature decline in serum progesterone was accompanied by a decrease in the wet weight of corpora lutea. Other experiments revealed: 1) a precipitous fall in serum estradiol to basal values within 2 h after estradiol implants were removed, preceding the decline in serum progesterone by approximately 6 to 10 h; 2) reduced levels of estradiol in ovarian venous blood, but elevated levels of estradiol in peripheral arterial blood of rabbits with estradiol impants. The inability to elevated estradiol to increase serum progesterone or weights of corpora litea suggests that the luteotropic effect is maximal when estradiol is present at physiological concentrations. Following the continuous administration of estradiol, ovarian secretion of estradiol appears diminished and the corpora lutea become dependent upon the exogenous estradiol for luteotropic support. Although the ovaries continue to release measureable quantities of estradiol, this is inmeasurable quantities of estradiol, this is insufficient to prevent regression of corpora lutea when exogenous estradiol is rapidly withdrawn from the circulation.

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.

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.

Ovarian follicles removed from immature rats (preantral follicles) and immature rats treated in vivo with follicle stimulating hormone (FSH) (antral follicles) released progesterone in vitro in response to either human chorionic gonadotropin (hCG), hFSH, or DBcAMP in a time- and concentration-dependent fashion. Antral follicles produced approximately 20 times more progesterone than preantral follicles in response to both FSH and hCG at 10(-7) M and approximately 5 times more progesterone in response to 8 X 10(-3) M DBcAMP. After in vitro incubations, follicles were transplanted beneath the kidney capsules of recipient rats to assess their ability to luteinize after hormonal stimulation. Only antral follicles incubated with hCG, hFSH, and DBcAMP formed ectopic corpora lutea. Adenylate cyclase activity in preantral and antral follicle granulosa cells increased in response to both 10 mM KF and 10(-6) M hFSH with no major differences observed between membranes prepared from preantral or antral follicle granulosa cells. These results demonstrate that follicular maturation is associated with major changes in the ability of the granulosa cells to produce progesterone and luteinize in response to hormonal stimulation and that these changes may be, in part, independent of a functional hormone-responsive adenylate cyclase system.

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.

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.

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.

We tested the hypothesis that estrogen and hCG can modify blood flow in the rabbit corpus luteum. Radioactively labeled microspheres were used to measure luteal blood flow in pseudopregnant rabbits in which estrogen had been withdrawn to initiate premature luteal regression and in pseudopregnant rabbits injected with hCG. Removal of estradiol-filled Silastic capsules on day 10 of pseudopregnancy caused an 80% decrease in the serum progesterone concentration within 24 h. Despite the decline in progesterone secretion, luteal blood flow remained at very high levels and was not different from that in control rabbits treated continuously with estradiol. Replacement of estradiol-filled capsules for 3 h did not change the high rate of blood flow to the corpus luteum, but blood flow in the uterus, vagina, and ovarian stroma was increased. The injection of hCG (10 IU, iv) on day 10 of pseudopregnancy caused a 3-fold increase in blood flow to the nonluteal portion of the ovary and a 3-fold increase in the serum progesterone concentration, but luteal blood flow did not change. We conclude that the acute actions of estradiol or hCG in the rabbit corpus luteum are not mediated by changes in luteal blood flow. Further, the results suggest that the luteal vasculature is regulated differently from the vasculature of other estrogen-responsive tissues and that blood flow in the nonluteal tissues of the ovary can be regulated independently of blood flow in the corpus luteum.

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.

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.

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.