Immune cell populations in the equine corpus luteum throughout the oestrous cycle and early pregnancy: an immunohistochemical and flow cytometric study

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16-17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF2 alpha administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16-17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.     

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.     

Effect of prostaglandin E2 alpha on the hCG-stimulated progesterone production by human corpora lutea

The effect of prostaglandin PGF2 alpha on the hCG stimulated and basal progesterone production by human corpora lutea was examined in vitro. hCG (40 i.u./ml) stimulated progesterone formation in corpora lutea of early (days 16-19 of a normal 28 day cycle), mid (days 20-22) and late (days 23-27) luteal phases. This stimulation was inhibited by PGF2 alpha (10 micrograms/ml) in corpora lutea of mid and late luteal phases. PGF2 alpha alone did not show a consistent effect on basal progesterone production. The inhibition of hCG stimulated progesterone production by PGF2 alpha at times corresponding to luteolysis indicates a role for that prostaglandin in the process of luteolysis in the human corpus luteum.     

In vivo intra-luteal implants of prostaglandin (PG) E(1) or E(2) (PGE(1), PGE(2)) prevent luteolysis in cows. I. Luteal weight, circulating progesterone, mRNA for luteal luteinizing hormone (LH) receptor, and occupied and unoccupied luteal receptors for LH

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every four hours inhibited luteolysis in ewes. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in heifers, but infusion of estradiol+PGE(2) inhibited luteolysis in heifers. The objective of this experiment was to determine whether and how intra-luteal implants containing PGE(1) or PGE(2) prevent luteolysis in Angus or Brahman cows. On day-13 post-estrus, Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Coccygeal blood was collected daily for analysis for progesterone. Breed did not influence the effect of PGE(1) or PGE(2) on luteal mRNA for LH receptors or unoccupied or occupied luteal LH receptors did not differ (P>0.05) so the data were pooled. Luteal weights of Vehicle-treated Angus or Brahman cows from days-13-19 were lower (P<0.05) than those treated with intra-luteal implants containing PGE(1) or PGE(2). Day-13 Angus luteal weights were heavier (P<0.05) than Vehicle-treated Angus cows on day-19 and luteal weights of day-13 corpora lutea were similar (P>0.05) to Angus cows on day-19 treated with intra-luteal implants containing PGE(1) or PGE(2). Profiles of circulating progesterone in Angus or Brahman cows treated with intra-luteal implants containing PGE(1) or PGE(2) differed (P<0.05) from controls, but profiles of progesterone did not differ (P>0.05) between breeds or between cows treated with intra-luteal implants containing PGE(1) or PGE(2). Intra-luteal implants containing PGE(1) or PGE(2) prevented (P<0.05) loss of luteal mRNA for LH receptors and unoccupied or occupied receptors for LH compared to controls. It is concluded that PGE(1) or PGE(2) alone delays luteolysis regardless of breed. We also conclude that either PGE(1) or PGE(2) prevented luteolysis in cows by up-regulating expression of mRNA for LH receptors and by preventing loss of unoccupied and occupied LH receptors in luteal tissue.     

A possible mechanism of rapid luteolysis in white-footed mice,Peromyscus leucopus

Adult female white-footed mice, Peromyscus leucopus, were exposed to long (LP) or short (SP) photoperiods for 6 weeks (experiment I). Another group of animals was kept for 6 weeks in SP, then injected SC with 30 μg prolactin twice daily for 2, 3, 4, or 6 days (experiment II). Ovaries from the mice in both experiments were weighed and serially sectioned for light microscopic examination of regressing corpora lutea. In experiment I, it was observed that vessels supporting corpora lutea were dilated, and that their endothelium was either undergoing necrosis or it was missing. Pronounced changes of luteal capillaries led to rupture and intraluteal hemorrhage, thus opening the capillary bed. Regressing luteal cells became segregated and seemed to invade the vascular system passively. They were seen as luteal cell thrombi in medullary veins. This luteolytic course termed “rapid luteolysis” was most apparent in SP ovaries. It differed from “retarded luteolysis,” which represents the well-established luteolytic model of auto- and heterophagocytosis. In experiment II, there was a statistically significant decrease in ovarian weight 4 days after prolactin treatment in comparison with saline-treated controls. At the light microscopic level, signs of both rapid and retarded luteolysis were present, but not intensified. It is concluded: (1) The concept of rapid luteolysis represents a reasonable working hypothesis. (2) Prolactin, though luteolytic at the macroscopic level, failed to produce evidence of increased rapid or retarded luteolysis at the light microscopic level.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

The effect of estradiol valerate on follicular dynamics and superovulatory response in cows with Syncro-Mate-B implants

Two experiments were designed to evaluate the effect of estradiol valerate on follicular dynamics and superovulatory response in cows with Syncro-Mate-B (SMB)implants. In Experiment 1, 5 mg estradiol valerate (E(2)), injected at the same time as superstimulation treatments were initiated, resulted in fewer corpora lutea (CL), ova/embryos collected and fertilized ova (P<0.05) than if E(2) was administered with the SMB implant 7 days earlier. In Experiment 2, 31 beef cows and 26 Holstein cows were placed in one of four treatment groups. Group I (control) cows were superstimulated on Day 9 (estrus=Day 0). On Day 2, cows in Groups II, III, and IV received SMB and cows in Group III received E(2). On Day 9, cows in Group IV received E(2), and all cows were superstimulated with Folltropin. The number of CL did not differ (P>0.19) among groups. However, there were more follicles < 10 mm and fewer fertilized ova and transferable embryos (P<0.02) in Group IV cows. Ovarian ultrasonography revealed that the diameter of the largest follicle in Group III cows declined from Day 2 to Day 7 and subsequently increased until Day 13. In contrast, Groups I, II and IV were characterized by apparently linear growth between Days 2 and 13. Differences (P<0.05) were detected between Days 5 and 9. Mean diameter of the largest follicle was smaller for cows in Group III than for the remaining groups on Day 9. It was concluded that SMB did not adversely affect superovulatory response and that E(2) administration resulted in atresia of the antral follicles in the cows with SMB implants.     

Lipoprotein lipase activity in the bovine corpus luteum during the estrous cycle and early pregnancy.

Lipolytic activity measured at pH 8.6 in bovine corpora lutea exhibited classical properties of lipoprotein lipase (LPL) in terms of serum and heparin stimulation and NaCl inhibition. LPL activity was measured in 23 corpora lutea collected at different stages of the estrous cycle and early pregnancy. The LPL activity in cyclic corpora lutea (mumole FA released/hr/100 mg acetone powder) was low at Days 4-8 of the estrous cycle (3.1 +/- 1.5: mean +/- SE) and at Days 19-20 (1.6 +/- 0.6). However, high activity of the enzyme was found at Days 12-15 of the cycle (11.8 +/- 1.8); these concentrations were significantly (P less than 0.01) elevated over those found at Days 4-8 and 19-20. The enzyme activity began to decline at Days 16-18 of the estrous cycle (5.1 +/- 1.7). Low enzyme activity was found in the corpora lutea removed from two cows at Day 22 of pregnancy. Progesterone concentrations were measured in 16 of the 23 corpora lutea and a good correlation (r = 0.75, P less than 0.01) was found between lipoprotein lipase and progesterone concentrations of the tissue. The data suggest that LPL may be involved in controlling the transfer of fatty acids, including arachidonic, from plasma lipoproteins to luteal tissue.     

Changes in localization of immune cells and cytokines in corpora lutea during luteolysis in murine ovaries

Immune cells, which constitute a significant cell mass in the corpora lutea (CLs), are considered to play critical roles in luteolysis, but the details are not fully understood. We histochemically investigated the changes in distribution and cell density of macrophages and T lymphocytes and in tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma, which can induce apoptosis in the luteal cells in murine CLs during luteal regression. No macrophages or T lymphocytes were observed in functionally healthy CLs. Abundant macrophages and increasing T lymphocytes were demonstrated in CLs at the functional regression stage (early stage of regression). At the structural regression stage (late stage of regression), abundant T lymphocytes but no macrophages were demonstrated in the CLs. A moderate amount of TNF-alpha was detected in all CLs at all stages. No IFN-gamma was detected in either healthy or early regressing CLs, but a large amount of IFN-gamma was detected at the late regression stage. Moreover, in cultured luteal cells, reactivity against Fas-ligand (FasL) was caused by pretreatment with TNF-alpha and IFN-gamma and apoptosis was induced by FasL treatment. These findings support the hypothesis that macrophages initiate T lymphocyte aggregation at the early stage of luteal regression, and then T lymphocytes induce apoptosis on luteal cells, which in turn develop sensitivity against FasL by TNF-alpha and IFN-gamma.     

Neonatal superior ovarian nerve transection disturbs the cyclic activity of the female rats

The neural pathway most related with ovarian steroidogenesis has been identified as the superior ovarian nerve (SON). This work constitutes the first study of the effects of early ovarian SON transection, which was performed in rats of 4 days of age (SON-t rats) to magnify the effects of the denervation. The rats were studied at the prepubertal (30 days), peripubertal (41 days) and adult cyclic in dioestrus (60 days) reproductive stages. The SON-t rats showed a delay of vaginal opening, a notable disruption of oestrous cyclicity, and a large number of corpora lutea. In all the stages, the circulating levels of FSH, prolactin and growth hormone were lower in SON-t rats than in controls, whereas LH did not vary. Serum androstenedione levels were higher in SON-t rats at 30 days and lower at 41 days, compared with control animals while no difference was observed at 60 days. Serum progesterone levels did not differ between control and SON-t, but serum oestradiol concentrations were higher in SON-t rats in all of the stages. At the peripubertal stage, there were fewer ovarian beta-adrenergic receptors in SON-t ovaries, associated with a rise in the ovarian content of norepinephrine, but no changes were observed in SON-t rats at 30 and 60 days with respect to the controls. The release of progesterone in vitro from luteal cell in SON-t rats at 60 days was reduced in basal condition and under ovine LH or FSH stimulation, when compared with control animals; while no difference was observed in presence of isoproterenol or androstenedione in the culture medium. In corpora lutea of SON-t rats at 60 days, no change was observed in the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), but the activity of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) was reduced, suggesting abnormal luteolysis in spite of the large number of corpora lutea. The interruption of innervation at an early age by SON transection is very important in the regulation of ovarian development in prepubertal and cyclic rats. The functional changes observed in the ovary suggest a possible alteration in the hypothalamic-hypophyseal axis.     

A light and electron microscopic study of the periparturient bovine corpus luteum

Corpora lutea were obtained surgically from fifteen mature Angus crossbred cows representing three experimental groups of five cows each. Cows in Group A were 180 days of gestation, cows in Group B had recently experienced parturition (相似文献   

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.     

The effect of a single high dose of PGF2α administered to dairy cattle 3.5 days after ovulation on luteal function,morphology, and follicular dynamics

A single treatment with PGF2α is assumed to have no luteolytic effect on cows with corpora lutea < 5 days old. The objective of this study was to determine the effect of a single high dose of PGF2α administered to dairy cattle on the morphology and function of the early CL. The study followed a crossover design with a treatment cycle in which 50 mg of dinoprost were administered 3.5 days postovulation and a control untreated cycle. Ultrasound examination and blood samples were performed during the two consecutive cycles. Corpus luteum (CL) diameter, progesterone concentration, and follicular dynamics characteristics were compared between control and treated cycles. Two of nine cows (22%) developed full luteolysis. The remaining seven cows (78%) had partial luteolysis with a decrease (P < 0.05) in progesterone concentration and CL diameter for two and 12 days post-treatment, respectively. The interovulatory interval of treated cycles (19.7 ± 2.4 days) was not different (P > 0.05) from that of controls (23.8 ± 0.9 days). The transient reduction in progesterone of cows with partial luteolysis had no effect on the proportion of cows with two or three follicular waves, follicle growth rate, or preovulatory diameter (P > 0.05). Two cows developed ovarian cystic degeneration during the PGF2α-induced cycle. In conclusion, the treatment of cows with a high dose of PGF2α 3.5 days postovulation induced some degree of luteolysis in all treated cows. This resulted in partial luteolysis in 78% of treated animals and in full luteolysis in the remaining 22%.     

The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells.

Little information is available on the ultrastructure of macrophages in the corpus luteum or their importance in the regression of luteal tissue. In the present study, the fine structure of activated luteal macrophages during pregnancy and the postpartum period was examined by electron microscopy of guinea pig ovaries fixed by vascular perfusion. In these corpora lutea, macrophages can readily be distinguished from luteal cells. Activated macrophages typically display three prominent inclusions in their cytoplasm: (1) heterophagic vacuoles, (2) distinctive large dense inclusions, and (3) large and small electron-lucent vacuoles. In addition, they contain numerous smaller lysosome-like dense bodies. Activated macrophages in corpora lutea also characteristically show many surface protrusions, such as processes, folds or pseudopodia, which often occur in close contact with nearby luteal cells. Generally, nuclei of macrophages are irregular in shape and display a dense border of heterochromatin, thus differing from those of luteal cells. Macrophages seem to be most abundant in regressing corpora lutea, where they commonly display heterophagic vacuoles containing recognizable luteal cell fragments, evidence that these phagocytes ingest senescent luteal cells. The digestion of luteal cell components in heterophagic vacuoles presumably gives rise to the distinctive large dense inclusions typically seen in macrophages. The findings of this study indicate that macrophages play a central role in luteolysis by phagocytizing luteal cells or their remnants. They therefore appear to bring about the reduction in volume of the corpus luteum that occurs as this tissue regresses. These results taken together with those previously published (Paavola, '78) further indicate that breakdown of the corpus luteum during postpartum luteolysis in guinea pigs involves both autophagy and heterophagy.     

The effect of a single high dose of PGF2α administered to dairy cattle 3.5 days after ovulation on luteal function, morphology, and follicular dynamics

A single treatment with PGF2α is assumed to have no luteolytic effect on cows with corpora lutea < 5 days old. The objective of this study was to determine the effect of a single high dose of PGF2α administered to dairy cattle on the morphology and function of the early CL. The study followed a crossover design with a treatment cycle in which 50 mg of dinoprost were administered 3.5 days postovulation and a control untreated cycle. Ultrasound examination and blood samples were performed during the two consecutive cycles. Corpus luteum (CL) diameter, progesterone concentration, and follicular dynamics characteristics were compared between control and treated cycles. Two of nine cows (22%) developed full luteolysis. The remaining seven cows (78%) had partial luteolysis with a decrease (P < 0.05) in progesterone concentration and CL diameter for two and 12 days post-treatment, respectively. The interovulatory interval of treated cycles (19.7 ± 2.4 days) was not different (P > 0.05) from that of controls (23.8 ± 0.9 days). The transient reduction in progesterone of cows with partial luteolysis had no effect on the proportion of cows with two or three follicular waves, follicle growth rate, or preovulatory diameter (P > 0.05). Two cows developed ovarian cystic degeneration during the PGF2α-induced cycle. In conclusion, the treatment of cows with a high dose of PGF2α 3.5 days postovulation induced some degree of luteolysis in all treated cows. This resulted in partial luteolysis in 78% of treated animals and in full luteolysis in the remaining 22%.     

Selenium Levels and Glutathione Peroxidase Activity in Blood,Plasma and Reproductive Organs in Dairy Cows

Selenium levels and glutathione peroxidase activity were determined in blood and reproductive organs in 12 Norwegian dairy cows at different stages of the oestrus cycle. Blood samples were collected before slaughter, and samples from genital organs were obtained as soon as possible after slaughter. Blood and plasma selenium levels were significantly correlated with selenium levels in follicular fluid, and in ovarian and uterine tissues, but not with the levels in corpora lutea. The activity of blood glutathione peroxidase was significantly correlated with that in ovarian and uterine tissue, but not with activity in corpora lutea and follicular fluid. No effect of stage of oestrus cycle on selenium content or glutathione peroxidase in reproductive tissue was observed.     

Beta-adrenergic receptor concentration and subtype in the corpus luteum of the adult pseudopregnant rat

Luteal beta-adrenergic receptor concentration and subtype were determined in adult pseudopregnant rats during and after the period of the functional luteal phase. The specific beta-adrenergic receptor ligand (-)-3-[125I]iodocyanopindolol ([125I]ICYP) was used to determine the receptor concentration in corpora lutea of adult pseudopregnant rats. A 3-fold increase in beta-adrenergic receptor concentration was seen during the first 2-3 days of pseudopregnancy, whereafter the receptor concentration declined. During the functional luteal regression period (Day 12-15) the receptor levels were still low. In regressed (Day 16-22) corpora lutea a temporary increase in beta-receptor concentration was seen which may represent some role for beta-adrenergic mechanisms in the regulation of morphological regression in the corpus luteum. To determine the beta-adrenergic subtype, competition of [125I]ICYP-binding with selective beta 1- and beta 2-adrenergic antagonists was assessed in corpora lutea of different ages and in rat heart and uterus. The beta-adrenergic receptors in corpora lutea of adult pseudopregnant rats were shown to be solely of the subtype beta 2, regardless of the luteal age.     

Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicles at the end of the bovine estrous cycle

In cows, electrocauterization of large follicles at the end of the luteal phase, lengthened the life span of corpora lutea. Injection of 5 mg of estradiol valerate or of 1 mg of an analogue of prostaglandin F(2alpha) induced luteolysis; however, the injection of estrogen was associated with precocious estrus without either ovulation or corpus luteum growth. Injection of both estradiol valerate and prostaglandin analogue gave the same results as estradiol valerate alone. Deferred luteolysis, observed after electrocauterization of large follicles, seemed to be due to the withdrawal of estrogens and the consequent lack of prostaglandin F(2alpha) production.