Plasma membrane changes during corpus luteum regression

Structural and biochemical changes were examined in the plasma membrane of luteal cells during corpus luteum regression. Structural alterations as indicated by an increase in the liquid-crystalline to gel phase transition temperature and a decrease in plasma membrane fluidity were observed during luteolysis in microsomes and in plasma membranes prepared from prostaglandin F2 alpha-treated rats, when samples were examined by wide angle x-ray diffraction and fluorescence polarization. In addition, a significant increase in activity of the lipolytic enzyme phospholipase A2 appeared during incubation of plasma membrane samples and dispersed luteal cells at 40 degrees C in the presence of 1.0 mM CaCl2. Similar incubation conditions also produced a drop in human chorionic gonadotropin (hCG) binding in luteal samples from prostaglandin F2 alpha-treated rats. These results indicate that during luteolysis there are important structural changes in the plasmalemma of regressing luteal cells. These alterations appear related to an increase in phospholipase A2 activity and a decrease in hCG receptors. These modifications may account for the decrease in function during corpus luteum regression.     

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.     

Function of abnormal corpora lutea in vitro after GnRH-induced ovulation in the anoestrous ewe

Normal and abnormal corpora lutea were recovered from anoestrous Romney Marsh ewes on Days 3, 4, 5 and 6 after treatment with small-dose (250 ng) multiple injections of GnRH followed by a bolus injection (125 micrograms) with (+P) and without (-P) progesterone pretreatment and a study made of their characteristics in vitro. Plasma progesterone concentrations initially rose concurrently in all animals but abnormal luteal function occurred in 70% of the -P ewes and was defined on Day 5 when plasma progesterone concentrations declined relative to those in the +P ewes. All corpora lutea recovered on Days 3 and 4 appeared macroscopically similar and there were no significant differences between the +P and -P groups in terms of luteal weight, progesterone content and binding of 125I-labelled hCG on these days. However, corpora lutea from the -P animals only exhibited a decline in progesterone production in vitro on Day 4 (P less than 0.01), and morphological differences became apparent on Days 5 and 6 when the abnormal corpora lutea from the -P animals also decreased in weight (P less than 0.01) and progesterone content (P less than 0.001). Binding of 125I-labelled hCG increased on Day 5 in the normal corpora lutea only. These results show that, although abnormal luteal function induced by GnRH treatment of anoestrous ewes could not be distinguished from normal corpora lutea before Day 5 by measurement of progesterone in peripheral plasma, a significant decline in progesterone production in vitro occurred on Day 4 in the abnormal corpora lutea. This was followed by significant decreases in weight and progesterone content and a failure to increase 125I-labelled hCG binding. Abnormal corpora lutea are therefore capable of some initial growth and progesterone production, before undergoing a rapid and premature regression from Day 4, which has similar characteristics to natural luteolysis.     

Calcium-regulated plasma membrane rigidification during corpus luteum regression in the rat

Plasma membrane fractions were prepared from ovaries of superovulated rats and examined for structural changes during luteolysis. Using fluorescence polarization, we observed a rapid rigidification in vitro of samples obtained from ovaries undergoing spontaneous or prostaglandin F2 alpha-induced regression. The rigidification, manifested by a 72% polarization increase over 50 min, is calcium and calmodulin dependent, temperature sensitive and protein mediated. This increase in polarization did not appear in fractions from nonregressing ovaries; however, addition of phospholipase A2 caused virtually identical changes in polarization results as in samples prepared from regressing ovaries. These results suggest that calcium-calmodulin-dependent phospholipase A2 plays a role in membrane deterioration during luteolysis.     

The proestrous prolactin surge is not the sole initiator of regressive changes in corpora lutea of normally cycling rats.

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.     

Impairment of gonadotropin binding occurs during membrane rigidification in plasma membrane samples prepared from regressed rat corpora lutea

The effect upon human chorionic gonadotropin (hCG) binding of a 90-min incubation of plasma membranes prepared from the corpora lutea of control and prostaglandin F2 alpha injected rats was studied. After incubation for 90 min with 1 mM CaCl2 at 40 degrees C, single point hCG binding assays at room temperature revealed a significant decrease in the degree of binding of approximately 50% in membrane samples prepared from regressed corpora lutea. The binding decrease in regressed samples did not occur if the incubation temperature was reduced to 35 degrees C or if calcium ion was replaced with magnesium. Scatchard analyses indicated that the decrease in binding capacity was the result of a loss of gonadotropin receptors rather than an affinity shift. Specific activities of two membrane-bound enzymes (Na+-K+ ATPase, 5'-nucleotidase) did not change in a correlative fashion during the incubation. In previous studies the same in vitro conditions caused a substantial and significant decrease in membrane fluidity, as determined by fluorescence polarization. Thus it appears that the membrane rigidification is of a specific nature and interferes with gonadotropin binding during luteolysis.     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and ultrastructural studies of bovine corpora lutea

Corpora lutea were collected from cows at four stages of the luteal phase and prepared for immunostaining at the light microscope level. Other corpora lutea, which were fully developed, were dispersed by collagenase treatment and freshly isolated and cultured cells were processed for immunostaining. Electron microscopy was carried out on mature corpora lutea and freshly isolated cells. Positive staining for cholesterol side-chain-cleavage cytochrome P-450 (P-450scc), an inner-mitochondrial membrane enzyme considered to catalyse the rate-limiting step in the conversion of cholesterol to progesterone, was observed in all corpora lutea. The intensity of staining was much greater in mature corpora lutea than in young or regressing corpora lutea. Only small and large luteal cells stained positively and cells of the vasculature and other connective tissue elements did not. When cells were cultured and had become flatter, the intensity of immunostaining was observed to be greater in large luteal cells than in small luteal cells which was interpreted to be due, in part, to the greater volume density of mitochondria in these cells. In some cultured small luteal cells the pattern of immunostaining appeared as whorls of strands encircling the nucleus. This pattern was interpreted as a three-dimensional network of mitochondria organized into 'strands', more than one mitochondrion in cross-section, perhaps formed during the process of attachment and elongation of the cells. Further observations made at the electron microscope level, included the presence of close (5-8 nm) contacts with interconnecting septa between small luteal cells in tissue.     

Modulation of LH/hCG receptors and physical state of ovarian membranes in rat pseudopregnancy

Previous investigations have demonstrated that increased ovarian function during pseudopregnancy in the rat may be associated with alterations of the physical state of membranes. Changes in rigidity of membrane lipids were observed during the formation as well as regression of corpora lutea. The effects of cyclooxygenase inhibitors (indomethacin and acetylsalicylic acid (ASA)) and of selected steroids (estradiol, testosterone and dihydrotestosterone) on the functional state of luteinized ovaries were studied. The compounds were administered to the animals in silastic capsules on different days after hCG injection. ASA and indomethacin administration on days 10 and 11 after hCG injection resulted in an increase in the LH/hCG receptor binding activity and rigidity of ovarian membrane lipids, as determined by fluorescence polarization of 1,6-diphenyl-1,3,5 hexatriene (DPH) probe. This effect was apparent within 7 days after indomethacin and ASA treatment. Both estradiol and testosterone significantly increased the ovarian LH/hCG binding activity, however estradiol did not affect the membrane lipid rigidity. Unlike testosterone, the administration of dihydrotestosterone induced a decrease in membrane lipid rigidity and reduced the accessibility of the LH/hCG receptor. Inhibitors of prostaglandin F2alpha (PGF2alpha) synthesis, as the endogenous mediator of luteolysis, were shown to delay the regression of the corpora lutea and to prolong the luteal activity in pseudopregnant rats.     

Changes in rat luteal ultrastructure and P450scc mRNA and protein content after in vivo treatment with a gonadotropin-releasing hormone agonist

Previous studies have demonstrated that plasma progesterone levels decrease in pregnant rats treated in vivo with a gonadotropin-releasing hormone agonist (GnRH-Ag), without changes in testosterone or estradiol levels in ovarian vein plasma. The objective of this study was to determine the loci of GnRH-Ag disruption of progesterone synthesis by examining luteal mitochondria, lipid droplets, cellular composition, and P450 side-chain cleavage (P450scc) enzyme and mRNA content in the pregnant rat. On Day 7 or 11 of pregnancy, osmotic minipumps containing GnRH-Ag were implanted into 5-7 rats. Sham operations were performed on 5-6 controls at each time period. Five micrograms per day of GnRH-Ag were released for about 24 h, after which corpora lutea and jugular vein plasma were collected. The corpora lutea were prepared for microscopy or analyzed for P450scc enzyme and mRNA content. Plasma progesterone levels were measured by RIA. In those rats treated with GnRH-Ag, progesterone levels had decreased, and within the luteal cells, there was an increase in the number of lipid droplets and a decrease in the number of tubular cristae within the mitochondria. Concomitantly, P450scc enzyme and mRNA content decreased on both Day 8 and Day 12 of pregnancy. Also, GnRH-Ag treatment decreased the ratio of large to small steroidogenic luteal cells on Day 8 of pregnancy, but did not alter cellular ratios on Day 12 of pregnancy. These observations suggest that treatment with GnRH-Ag inhibits progesterone synthesis by decreasing the amount of P450scc mRNA and enzyme content, which may alter the mitochondrial cristae structure on Day 8 and Day 12 of pregnancy. The reduction in tubular cristae and P450scc enzyme in the mitochondria may account for the increase in lipid droplets, as less cholesterol is converted to pregnenolone. An additional mechanism of inhibition may be the reduction in the number of large steroidogenic luteal cells, which appear to be the major source of progesterone in the rat corpus luteum on Day 8 of pregnancy.     

Biochemical changes in lipids during follicular growth and corpora lutea formation and regression in rat ovary

A biochemical study has been made on quantitative and qualitative changes in lipids of small (less than 300 microns), medium (300 to 550 microns) and large (less than 550 microns) follicles and in the fully developed and regressing corpora lutea. The total lipid content increased in the growing follicles and corpora lutea. Phospholipids formed the major component of total lipids in small sized follicles and developed corpus luteum. The cholesterol amount increased with the growth of follicles but decreased in the developed and regressing corpora lutea. Glycerides were the main fraction of total lipids in regressing corpora lutea. Free fatty acids were present in minor quantities in the growing follicles and corpora lutea. The physiological significance of these lipid changes is discussed.     

The expression of androgen receptor, cytochrome P450 aromatase and FSH receptor mRNA in the porcine ovary

The aim of this study was to visualize the expression of androgen receptor, cytochrome P450 aromatase and FSH receptor mRNAs in various structures of porcine ovary. Porcine ovaries were frozen in liquid nitrogen, and 8 microm sections were prepared for in situ hybridization. In the small, medium and large antral follicles as well as in early, midluteal and regressing corpora lutea, mRNAs for androgen receptor, P450 aromatase and FSH receptor were detected. In small antral follicles high levels of mRNAs for androgen and FSH receptors were observed, mainly in the granulosa layer, while mRNA expression for P450 aromatase was negligible. As follicles grew, amount of mRNAs for androgen receptor and FSH receptor decreased, and that for P450 aromatase increased. Small amounts of androgen receptor mRNA were also present in corpora lutea at all examined stages. P450 aromatase mRNA was not detected in early and midluteal corpora lutea. However, regressing corpus luteum showed a weak expression of aromatase mRNA.     

Actions of prostaglandin F2alpha and prolactin on intercellular adhesion molecule-1 expression and monocyte/macrophage accumulation in the rat corpus luteum

Expression of intercellular adhesion molecule-1 (ICAM-1) and the accumulation of monocytes/macrophages are inflammatory events that occur during PRL (PRL)-induced regression of the rat corpus luteum. Here we have compared the ability of prostaglandin F2alpha (PGF) and PRL to induce, in rat corpora lutea, inflammatory events thought to perpetuate luteal regression. Immature rats were ovulated with eCG-hCG and then hypophysectomized (Day 0), which resulted in a single cohort of persistent, functional corpora lutea. On Days 9-11, the rats received twice daily injections of saline, PGF (Lutalyse, 250 microg/injection), or PRL (312 microg/injection) to induce luteal regression. Surprisingly, luteal weight and plasma progestin concentrations (progesterone and 20alpha-dihydroprogesterone) did not differ between PGF-treated rats and controls; whereas both luteal weight and plasma progestins declined significantly in PRL-treated rats. Furthermore, corpora lutea of PGF-treated rats and controls contained relatively minimal ICAM-1 staining and few monocytes/macrophages. In contrast, but as expected, corpora lutea of PRL-treated rats stained intensely for ICAM-1 and contained numerous monocytes/macrophages. In an additional experiment, there was no indication that luteal prostaglandin F2alpha receptor mRNA diminished as a result of hypophysectomy. These findings suggest that prolactin, not PGF, induces the inflammatory events that accompany regression of the rat corpus luteum.     

Effect of prostaglandin F2 alpha on Na+-K+-ATPase activity in luteal membranes

Slices of rat corpora lutea (CL) incubated with prostaglandin F2 alpha (PGF2 alpha) in Krebs-Hensenleit (K-H) Ringer solution showed a decrease in Na+-K+-ATPase activity after 60 min of incubation. However, PGF2 alpha in vitro did not alter Na+-K+-ATPase activity of isolated luteal membrane fractions. Following PGF2 alpha-induced in vivo luteal regression, reduction of Vmax and elevation of the activation energy above transition temperature of the lipid phase of the membrane occurred without changes in Km, optimum pH and transition temperature. These results suggest that reduction of Na+-K+-ATPase activity after PGF2 alpha treatment may be due to reduction in the number of enzyme molecules or to masking of the active site of the enzyme without any change in enzyme characteristics. In addition, a change in membrane-bound enzyme activity may be an early step in PGF2 alpha-induced luteolysis.     

Biochemical changes associated with the mechanism controlling superoxide radical formation in the aging rotifer

Levels of the superoxide radical (SOR) and lipid peroxides were measured and found to increase during aging in the short-lived rotifer, Asplanchna brightwelli. Life-span was altered by changes in environmental temperature, absence of light, diet restriction, exposure to ultraviolet radiation, and addition of vitamin E to the diet. Each of the conditions that lengthened life-span decreased SOR and lipid peroxide levels, and each condition that shortened life-span increased levels of SOR and lipid peroxides. Additional experiments indicated that on the third day of age, there was a significant increase in Ca2+ uptake and phospholipase A2 activity in membrane samples and an elevation in superoxide dismutase and catalase activity in rotifer homogenates. In addition, SOR concentration was inhibited by the addition of bromophenacyl bromide and indomethacin to membrane samples. By day 5 there was also a significant increase in the lysosomal enzyme, alpha-mannosidase. The results of this study indicate that levels of the SOR and lipid peroxides are coupled to rotifer life-span and that activation of phospholipase A2 may contribute to the elevation of these agents in older animals.     

Expression and cellular distribution of cytosolic phospholipase A2 in the rat ovary

Cellular expression of cytosolic phospholipase A2 (cPLA2) was investigated in the rat ovary in different endocrine states. Its mRNA expression was detected by RT-PCR. The immunohistochemistry identified an intense signal for cPLA2 in oocytes. Granulosa and thecal cells in growing follicles were negative, but turned positive during the periovulatory period, whereas those in atretic follicles were highly immunoreactive. The immunoreactive signal was modest in newly formed corpora lutea (CL) but intensified in functionally and morphologically regressing CL. These results show a broad but specific distribution of cPLA2 in ovarian cell types, and suggest its role in ovulation, CL regulation and apoptotic processes.     

Glucocorticoids stimulate the accumulation of lipids in the rat corpus luteum.

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.     

Menstruation in short-tailed fruit bats (Carollia spp.).

Uterine function was assessed histologically in Carollia spp. removed from a laboratory breeding colony. The uteri of periovulatory bats examined on days 1-3 post coitum varied considerably depending upon whether regressing corpora lutea were present. In females lacking such corpora lutea, the endometrium was shallow and proliferative. In most of the bats having regressing corpora lutea, the endometrium was thicker and necrotic portions of the superficial lamina functionalis were being sloughed off with associated bleeding (i.e. in the form of a true menstrual process). The presence of regressing corpora lutea and well-developed endometria in these animals indicated that short-tailed fruit bats are spontaneous ovulators with a functional luteal phase to their cycles. Menstruation was also observed in some females that were not periovulatory, or that lacked regressing corpora lutea. The endometrium was not vascularized by spiral arterioles, but it did contain distinctive granulocytes similar to those observed in catarrhine primates and other menstruating bats. Most endometrial growth occurred after ovulation and, in pregnant animals, was associated with retention of the embryos in the oviducts for approximately 13-16 days. Carollia perspicillata normally produces one young at a time, after a gestation period of about 4 months, and is a seasonal breeder in the wild. Menstruation probably provides this bat with an efficient mechanism for eliminating a well-developed endometrium from its simplex uterus in the event of fertilization failure or early embryonic loss. This may give the female another chance to establish a pregnancy and produce an infant at a reasonably opportune time during the same breeding season.     

The corpus luteum of the guinea pig. III. Cytochemical studies on the Golgi complex and GERL during normal postpartum regression of luteal cells, emphasizing the origin of lysosomes and autophagic vacuoles

The postpartum involution of corpora lutea was examined by electron microscope cytochemistry of guinea pig ovaries previously fixed by vascular perfusion, a method which produces optimal preservation of steroid-secreting cells and yet maintains enzyme activity. The intracellular digestive apparatus was identified through the localization of two acid hydrolases, acid phosphatase (ACPase) and arylsulfatase. Other marker enzymes localized were thiamine pyrophosphatase (in Golgi cisternae) and alkaline phosphatase (along plasma membranes). Prolonged osmication was used to mark the outer Golgi cisterna. The results demonstrate that luteal cell regression is characterized by a striking increase in the number of lysosomes and the appearance of numerous, double-walled autophagic vacuoles. Both lysosomes and the space between the double walls of autophagic vacuoles exhibit ACPase and arylsulfatase activity. In contrast to earlier periods, just before and during regression, Golgi complex-endoplasmic reticulum-lysosomes (GERL) is markedly hypertrophied, displaying intense acid hydrolase activity. On the basis of various criteria, GERL is proposed to function in the formation of lysosomes and autophagic vacuoles. Lysosomes seem to develop from GERL as focal protuberances of varying size and shape, which detach from the parent structure. Double- walled autophagic vacuoles, often large and complex in structure, initially are produced as GERL cisternae envelop small areas of cytoplasm. Lytic enzymes, perhaps furnished by the engulfing membranes and trapped lysosomes, presumably bring about digestion of the contents of these vacuoles, producing first aggregate-type inclusions, then, as the contents are further degraded, myelin figure-filled residual bodies. ACPase activity occasionally appears within smooth endoplasmic reticulum tubules and cisternae in advanced regression, possibly suggesting that lytic enzymes utilize this membrane system as an access route to GERL. These data indicate that cellular autophagy is a prominent mechanism underlying luteal cell involution during normal postpartum degeneration of guinea pig corpora lutea. Furthermore they suggest that in regressing luteal cells GERL is responsible for packaging acid hydrolases into lytic bodies.     

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: Tissue culture approach

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P₄) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal cells collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.     

Expression of monocyte chemoattractant protein-1 and distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle.

This study characterizes the expression of monocyte chemoattractant protein-1 (MCP-1) and the relative distribution of immune cell populations in the bovine corpus luteum throughout the estrous cycle. Immunodetectable MCP-1 was evident in corpora lutea of cows at Days 6, 12, and 18 postovulation (Day 0 = ovulation, n = 4 cows/stage). Day 6 corpora lutea contained minimal MCP-1 that was confined primarily to blood vessels. In contrast, relatively intense staining for MCP-1 was observed in corpora lutea from Days 12 and 18 postovulation. MCP-1 was again most evident in the cells of the vasculature, but it was also observed surrounding individual luteal cells, particularly by Day 18. An increase in immunohistochemical expression of MCP-1 on Days 12 and 18 postovulation corresponded with increases in MCP-1 mRNA and protein in corpora lutea as determined by Northern blot analysis and ELISA. Monocytes and macrophages were the most abundant immune cells detected in the bovine corpus luteum, followed by CD8+ and CD4+ T lymphocytes. In all instances, Day 6 corpora lutea contained fewer immune cells than corpora lutea from Days 12 and 18. In conclusion, increased expression of MCP-1 was accompanied by the accumulation of immune cells in the corpora lutea of cows during the latter half of the estrous cycle (Days 12-18 postovulation). These results support the hypothesis that MCP-1 promotes immune cell recruitment into the corpus luteum to facilitate luteal regression. These results also raise a provocative issue, however, concerning the recruitment of immune cells several days in advance of the onset of luteal regression.