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.     

Determination of cell type specificity and estrous cycle dependency of monocyte chemoattractant protein-1 expression in corpora lutea of normally cycling rats in relation to apoptosis and monocyte/macrophage accumulation

In regressive corpora lutea, apoptosis of luteal cells, expression of monocyte chemoattractant protein-1 (MCP-1), and accumulation of monocytes/macrophages occur. However, whether these three events are correlated and what cell type expresses MCP-1 have yet to be determined. To clarify these issues, we performed histochemical examinations to determine the localization and the numbers of MCP-1 mRNA-containing cells, apoptotic cells, and monocytes/macrophages in corpora lutea of normally cycling rats. We found that the Mcp-1 gene is expressed in nonapoptotic steroidogenic luteal cells. Corpora lutea that contained MCP-1 mRNA-expressing cells increased in number at estrus together with those containing apoptotic luteal cells. When individual corpora lutea at estrus were analyzed, those with many MCP-1-expressing cells contained few apoptotic cells, and vice versa. These results collectively suggest the following pathway for apoptosis- and MCP-1-dependent regression of the corpus luteum: 1) luteal cells are induced to undergo apoptosis at estrus, and the activation of Mcp-1 gene expression follows in nonapoptotic luteal cells; 2) monocytes/macrophages are chemoattracted by MCP-1 toward corpora lutea containing apoptotic luteal cells; and 3) monocytes/macrophages invade corpora lutea and eliminate apoptotic luteal cells by phagocytosis.     

Phosphatidylserine- and integrin-mediated phagocytosis of apoptotic luteal cells by macrophages of the rat

Corpora lutea disappear from ovaries in the absence of conception. The present study was undertaken to examine the hypothesis that disappearance of corpora lutea is accomplished through apoptosis-dependent phagocytosis of luteal cells. When bone marrow cells expressing green fluorescence protein were transplanted into X-ray-irradiated mice, macrophages derived from donor mice were detected within corpora lutea, suggesting macrophage infiltration into the tissue. Dispersed rat luteal cells underwent spontaneous apoptosis during culture and were phagocytosed by luteal macrophages. Treatment with doxorubicin increased the extent of apoptosis in luteal cells, and those cells were more efficiently phagocytosed than cells left untreated. The phagocytosis was inhibited by liposomes containing phosphatidylserine or a peptide containing the integrin-targeted sequence, and was stimulated by milk fat globule epidermal growth factor 8. These results collectively indicate that apoptotic luteal cells are phagocytosed by macrophages in a manner mediated by phosphatidylserine and integrin.     

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.     

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   

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.     

Functional and morphological characteristics of the first corpus luteum formed after parturition in ewes

The ability of sheep luteal cells from the first corpus luteum formed after parturition (Group F) to secrete progesterone in the presence or absence of LH was compared with that of luteal cells obtained from normal cyclic ewes (Group C). Luteal concentrations of receptors for LH and prostaglandins (PG) F-2 alpha (PGF-2 alpha) and the cellular composition of corpora lutea from Groups F and C were also compared. Luteal cells from Group F secreted less progesterone in either the presence or absence of LH (P less than 0.01). There was no difference in the number of receptors for LH or PGF-2 alpha per luteal cell between Groups F and C (P greater than 0.1), nor was there a difference in the number of large or small steroidogenic luteal cells (P greater than 0.1). It was concluded that, if short-lived corpora lutea are insensitive to gonadotrophins, this response is not mediated by decreased numbers of receptors for LH. In addition, if the first corpus luteum formed post partum in ewes is more sensitive to the luteolytic effects of PGF-2 alpha, this effect is not mediated by an increased number of receptors for PGF-2 alpha or an increased proportion of PGF-2 alpha-sensitive large luteal cells.     

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.     

Immunohistochemical localization of estrogen receptors in the ovine corpus luteum throughout the estrous cycle

Using immunohistochemistry and Western blot analysis we attempted to identify the estrogen receptors in ovine luteal cells at different stages of the estrous cycle. Monoclonal antibody against estrogen receptors was used for immunolocalization of estrogen receptor-alpha in corpora lutea sections. Generally, the most intense cytoplasm staining was present in large luteal cells. On the 6th day of the estrous cycle, weak immunostaining of estrogen receptors was observed in large luteal cells as well as in the connective tissue. Luteal cells from regressing corpora lutea expressed the weakest immunostaining. The most intense immunoreactivity for estrogen receptors was found in sections of corpora lutea collected on the 9th day of the cycle. Both, cytoplasmic and nuclear localization was observed depending on cell types in the ovine corpus luteum. Our studies demonstrated the presence of the estrogen receptor-alpha in the luteal cells and suggested an autocrine/paracrine role of estrogen in the regulation of estrous cycle in sheep.     

Expression and localisation of ephrin-B1 and EphB4 in steroidogenic cells in the naturally cycling mouse ovary

Ephrin receptors and ligands are membrane-bound molecules that modulate diverse cellular functions such as cell adhesion, epithelial–mesenchymal transition, motility, differentiation and proliferation. We recently reported the co-expression of ephrin-B1 and EphB4 in adult and foetal Leydig cells of the mouse testis, and thus speculated that their co-expression is a common property in gonadal steroidogenic cells. Therefore, in this study we examined the expression and localisation of ephrin-B1 and EphB4 in the naturally cycling mouse ovary, as their expression patterns in the ovary are virtually unknown. We found that ephrin-B1 and EphB4 were co-expressed in steroidogenic cells of all kinds, i.e. granulosa cells and CYP17A1-positive steroidogenic theca cells as well as in 3β-HSD-positive luteal cells and the interstitial glands; their co-expression potentially serves as a good marker to identify sex steroid-producing cells even in extra-gonadal organs/tissues. We also found that ephrin-B1 and EphB4 expression in granulosa cells was faint and strong, respectively; ephrin-B1 expression in luteal cells was weak in developing and temporally mature corpora lutea (those of the current cycle) and likely strong in regressing corpora lutea (those of the previous cycle) and EphB4 expression in luteal cells was weak in corpora lutea of the current cycle and likely faint/negative in the corpora lutea of the previous cycle. These findings suggest that a luteinising hormone surge triggers the upregulation of ephrin-B1 and downregulation of EphB4, as this expression fluctuation occurs after the surge. Overall, ephrin-B1 and EphB4 expression patterns may represent benchmarks for steroidogenic cells in the ovary.     

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 interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated from pigs in early and midluteal phase.

The first objective of this research was to define the capacity of corpora lutea of pig to secrete estradiol in the presence of an androgen substrate which was testosterone. The second objective was to define the synergism between gonadotropic hormones such as LH, FSH, and PRL and testosterone as measured by estradiol and progesterone secretion by two types of porcine luteal cells. Luteal cells were collected from newly forming corpora lutea (0-3 days after ovulation) and from mature corpora lutea (8-10 days after ovulation). After dispersion, luteal cells were suspended in medium M199 supplemented with 10% of calf serum and grown as monolayers at 37 degrees C. Control cultures were grown in medium alone while other cultures were supplemented with either testosterone alone at a concentration of 1 x 10(-7) M or with 10, 100, 500 ng LH plus testosterone, 10, 100, 500 ng FSH plus testosterone or 10, 100, 500 ng PRL plus testosterone. After 2 days of cultivation all cultures were terminated and media were frozen at 20 degrees C for further steroid analysis. Testosterone added to the culture medium in the absence of gonadotropins was without effect on estradiol and progesterone secretion by luteal cells collected in the corpora lutea of the early luteal phase. On the other hand testosterone added to the medium significantly increased progesterone and estradiol secretion by cultured luteal cells collected in the midluteal phase of the cycle. No additive stimulatory action of gonadotropins and testosterone on progesterone secretion was observed in cultures of luteal cells from the early luteal phase but this was not the case in cultures of luteal cells from the midluteal phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of growth hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured luteal cells collected from early, mature, and regressing porcine corpora lutea

The aim of the present study was to test the hypothesis that growth hormone (GH) and insulin-like growth factor-I (IGF-I) act at a local level to inhibit luteal cell apoptosis. Luteal cells collected from the corpora lutea at different stages of the luteal phase were cultured for 24 h in M 199 medium supplemented with 5% of calf serum to cause attachment cells to the plastic. After 24 h, the media were changed and various concentrations of GH (10, 100 or 200 ng/ml) or IGF-I (30, 50 or 100 ng/ml) were added to the culture medium. Twenty-four hours later, cells were fixed for morphological assessment of apoptotic cells utilising a Hoechst staining technique. To support morphological observations, measurements of caspase-3 activity in cultured porcine luteal cells were performed. Increased incidence of apoptotic bodies and caspase-3 activity accompanied luteal regression and was associated with a decreased progesterone (P4) secretion by luteal cells. GH stimulated P4 secretion by luteal cells collected from developing (ELP) and mature (MLP) corpora lutea but had no effect on its secretion by cells collected from regressing corpora lutea (LLP). Moreover, it had no effect on the incidence of apoptotic bodies in all types of corpora lutea. However, suppression of caspase-3 activity was observed with 100 and 200 ng of GH/ml in all types of corpora lutea. IGF-I had a stimulatory effect on P4 secretion by ELP and MLP, decreased the incidence of apoptotic bodies and suppressed caspase-3 activity in cultures treated with all doses used. In conclusion, our results indicate that both GH and IGF-1 trigger anti-apoptotic effects either indirectly, by increasing progesterone secretion, or directly, through the inhibition of caspase-3 activity and subsequent prevention of apoptotic body formation.     

Expression of mRNA encoding basic fibroblast growth factor (bFGF) in bovine corpora lutea and cultured luteal cells.

A radiolabelled cRNA was synthesized using a 1.4 kb cDNA complementary to mRNA encoding bovine basic fibroblast growth factor (bFGF) as a template, and used as a probe to investigate the expression of mRNA encoding bFGF in bovine ovarian tissue, and luteal cells in primary culture. Northern analysis of poly(A +)RNA prepared from follicles and corpora lutea of various stages revealed a major mRNA species of 7 kb in corpora lutea of all stages, the amount of which was higher late in the luteal phase. No hybridizable message was detectable in follicles of any size. When luteal cells were established in primary culture, expression of the 7 kb mRNA species was maintained. This expression was increased markedly when cells were treated with LH/hCG or Bt2cAMP. Prostaglandin F-2 alpha treatment caused a marked decrease in the basal content of this 7 kb mRNA, and also severely impaired the ability of LH to stimulate this expression.     

Luteal morphology, atresia, and plasma progesterone concentrations during the reproductive cycle of two oviparous lizards, Crotaphytus collaris and Eumeces obsoletus

From ovulation to oviposition, the corpora lutea of the oviparous lizards Crotaphytus collaris and Eumeces obsoletus exhibit three stages of luteal development: 1) luteogenesis, 2) luteal maturity, and 3) luteal regression. Each stage exhibits distinct characteristics, involving changes in: 1) luteal volume, 2) nuclear diameter of cells within the luteal cell mass, and 3) thecal development. Plasma progesterone concentration is greatest during luteogenesis and is positively correlated with ovarian atresia, although atresia occurred throughout the period of gravidity. These data suggest that in these two species, the corpora lutea secrete high amounts of progesterone immediately following ovulation and exhibit morphologically distinct stages of growth and regression.     

Localization of some steroidogenic enzymes in the ovary of the rabbit

The distribution of delta 5-3 beta-HSD, peroxidase and cytochrome oxidase in immature, sexually mature and pregnant rabbit ovary has been studied histochemically. Corpora lutea are found only in pregnant rabbits. delta 5-3 beta-HSD is present in the theca interna of mature follicles, corpora lutea and interstitial gland cells but is absent in the granulosa cells of both developing and mature follicles. The granulosa cells of mature and developing follicles, hypertrophied theca interna and the luteal cells all show intense cytochrome oxidase activity. Peroxidase is present in the corpora lutea only. It is suggested that delta 5-3 beta-HSD in the theca interna and interstitial gland cells is the enzyme responsible for steroid synthesis in the ovaries of immature as well as sexually mature rabbits, while peroxidase and delta 5-3 beta-HSD present in the corpora lutea together regulate luteal steroidogenesis during pregnancy. The intense cytochrome oxidase activity together with peroxidase and delta 5-3 beta-HSD confirms the observations that this tissue is a site of intense oxidative activity.     

Luteal inadequacy during the early luteal phase of subfertile cows

A study was made of early luteal function (up to Day 6) in cyclic and pregnant heifers and also in older, subfertile cows. There were no differences in vivo or in vitro between cyclic and pregnant heifers, indicating no luteotrophic effect of the embryo at this stage, but the increase in postovulatory peripheral progesterone concentrations was delayed (P less than 0.01) and occurred more slowly (P less than 0.001) in the subfertile cows than in the heifers. The corpora lutea of the subfertile cows were heavier (P less than 0.001) than those of the heifers on Day 6. Basal progesterone production by dispersed luteal cells was similar between heifers and subfertile cows, but there was a difference (P less than 0.001) in the pattern of response to exogenous LH and PGE-2. Cells from subfertile cows were less sensitive to the stimulatory effects of PGE-2 and although LH increased (P less than 0.001) progesterone production by all cells, this stimulation by a low dose of LH was inhibited by PGE-2 in luteal cells from subfertile cows. This effect did not occur in the luteal cells from heifers. These results indicate the possibility that luteal inadequacy, due to a diminished response to circulating luteotrophic hormones, may contribute to embryo mortality in subfertile cows.     

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.     

Pregnant mouse corpora lutea: immunocytochemical localization of relaxin and ultrastructure

Relaxin was localized in corpora lutea of pregnant mouse ovaries by using the unlabeled antibody peroxidase-antiperoxidase technique and a highly specific rabbit antirat relaxin serum. Relaxin immunostaining was first observed in luteal cells located at the periphery of corpora lutea on Day 10 of gestation. The number of relaxin immunostained cells and the intensity of the stain gradually increased to reach a maximum between Days 16 and 18 of gestation. While a few luteal cells were specifically stained for relaxin on Day 1 postpartum, no luteal cells were stained on Day 2 postpartum. Ultrastructural studies of luteal cells from pregnant mouse ovaries revealed the presence of a distinct electron-dense, membrane-bound granule population, which was first observed on Day 12 of gestation. The granules increased in number to reach a maximum between Days 16 and 18 of gestation, and were absent by Day 2 postpartum. The appearance and disappearance of this granule population closely paralleled the relaxin immunostaining in the luteal cells. We suggest that the granules may be the subcellular sites of relaxin storage in the pregnant mouse ovary.     

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.