Prostacyclin and steroidogenesis in goat ovarian cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 micrograms/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P4) and estradiol-17 beta (E2) production by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10(-6)-10(-3)M) and indomethacin (100 ng-1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 micrograms/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 micrograms- 1 mg/ml) and dipyridamole (10(-6)-10(-3)M), when added alone or along with AA, did not affect steroid production. Up to 100 micrograms/ml of U-51605 (9,11-azoprosta-5,13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGI2) and its stable analog 6 beta PGI1 (0.01-10 micrograms/ml) produced a dose-dependent increase in P4 and E2 production in all the three cell types. Increase at 1 and 10 micrograms/ml was significant in all cases. 6-keto-PGE1 (an active metabolite of PGI2 in certain systems) produced an increase in steroid production which was significant in theca at greater than or equal to 1 microgram/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGF1 alpha (stable metabolite of PGI2) was without effect in the present system. The lack of effect of PGI2 at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolone. The present results indicate that AA-stimulated steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGI2 though PGI2 itself can positively modulate the steroid production.     

Prostacyclin and steroidogenesis in goat ovari cell types in vitro

Granulosa, theca and corpus luteum cells of the goat ovary were isolated and incubated separately for 6 hours, with or without various modulators. Arachidonic acid (AA, 10 ng to 100 μg/ml), the precursor for prostaglandin synthesis, produced a dose-dependent increase in progesterone (P₄) and estradiol-17β (E₂) productin by all the cell types. Prostaglandin synthetase inhibitors, aspirin (10⁻⁶−10⁻³M) and indomethacin (100 ng−1 mg/ml), produced a dose-dependent decrease in arachidonic acid-stimulated (100 μ/ml) steroid production. Prostacyclin synthetase stimulators, trapidil (1.6 μg− 1 mg/ml) and dipyridamole (10⁻⁶−10⁻³M), when added alone or along with AA, did not effect steroid production. Up to 100 μg/ml of U-51605 (9,11-azoprosta-5, 13-dienoic acid), a prostacyclin synthetase inhibitor, did not inhibit basal or AA-stimulated steroid production. Prostacyclin (PGl₂) and its stable analog 6βPGl₁(0.01–10μg/ml) produced a dose-dependent increase in P₄ and E₂ production in all three cell types. Increase at 1 and 10μg/ml was significant in all cases. 6-keto-PGE₁ (an active metabolite of PGl₂ in certain systems) produced an increase in steroid production which was significant in theca at 1μg/ml concentrations but had no significant effect on granulosa and corpus luteum cells at any dose level. 6-keto-PGf₁ alpha (stable metabolite of PGl₂) was without effect inthe present system. The lack of effect of PGl₂ at lower concentrations was not altered by either differentiation of the cells with FSH and testosterone or addition of steroid precursors, testosterone and pregnenolene. The present results indicate that AA- stimualted steroid production in the goat ovarian cell type is mediated by prostaglandins other than PGl₂ though PGl₂ itself can positively modulate the steroid production.     

Control of steroidogenesis in small and large bovine luteal cells

Evidence was cited to show that: (1) prostacyclin (PGI2) plays a luteotrophic role in the bovine corpus luteum and that products of the lipoxygenase pathway of arachidonic acid metabolism, especially 5-hydroxyeicosatetraenoic acid play luteolytic roles; (2) oxytocin of luteal cell origin plays a role in development, and possibly in regression, of the bovine corpus luteum; and (3) luteal cells arise from two sources; the characteristic small luteal cells at all stages of the oestrous cycle and pregnancy are of theca cell origin; the large cells are of granulosa cell origin early in the cycle, but a population of theca-derived large cells appears later in the cycle. Results of in vitro studies with total dispersed cells and essentially pure preparations of large and small luteal cells indicate that: (1) the recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system is involved in progesterone synthesis in the bovine corpus luteum; (2) activation of protein kinase C is stimulatory to progesterone synthesis in the small luteal cells; (3) activation of protein kinase C has no effect on progesterone synthesis in the large luteal cells; and (4) protein kinase C exerts its luteotrophic effect in total cell preparations, in part at least, by stimulating the production of prostacyclin. The protein kinase C system may cause down regulation of LH receptors in the large cells.     

The advantage of the aggregate culture of isolated ovarian cell types over the monolayer culture

Ovarian cells such as theca interna, granulosa and corpus luteum cells were isolated from pig ovaries and cultured in Erlenmayer flasks (25 ml) containing 3.5 ml of culture medium. The media were replaced every second day and frozen to -20°C for later steroid analysis. The reaggregation of cells was completed within 2–3 days and this was then followed by a period of cell migration. During the subsequent 5–6 day period the reaggregates became larger. The best results were obtained in cultures of isolated theca alone and in combination with granulosa cells, as well as of early corpus luteum cells. Granulosa cells did not aggregate as easily or as completely as the corpus luteum cells. All types of cells investigated were able to secrete progesterone into the culture medium. They secreted more progesterone and for a longer time than cells cultured as monolayers. The aggregate culture seems to be a good model to study the secretion of ovarian cells, by creating the tri-dimensional, and thus more physiological, culture system.     

Demonstration of luteinizing hormone/human chorionic gonadotrophin receptor-binding inhibitor in aqueous extracts of frozen human corpus luteum

Aqueous extracts of frozen human corpora lutea were tested for the presence of an inhibitor of luteinizing hormone-receptor site binding (LHRBI) and for the subsequent effect on the stimulatory response of luteinizing hormone (LH) on progesterone synthesis by sheep ovarian cells. In the presence of human corpus luteum extract of normal menstrual cycle (30,000-g supernatant), the binding of 125I human chorionic gonadotrophin (hCG) to granulosa and luteal cells of sheep ovaries was markedly reduced, but the ability of rat testicular LH receptors to bind labelled hCG was less affected. However, extracts of corpora lutea of the first trimester of pregnancy appeared to be less inhibitory on the binding of LH/hCG to ovarian cells and had no effect on the binding of rat testicular cells compared to those of normal menstrual cycle. Addition of both extracts separately inhibited the LH-stimulated in vitro progesterone synthesis by granulosa cell cultures and by incubated sheep corpus luteum slices. These findings provide evidence for the presence of LHRBI in human corpus luteum.     

Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I.

Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents. Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion. The two hormones had different patterns of secretion during the course of incubation. OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9. The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak. Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts. Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein. However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea. These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.     

Localization of follicle regulatory protein in the porcine ovary

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.     

Microvascular endothelial cells of the corpus luteum

The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).     

Immunolocalization of aromatase, 17 alpha-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary

Immunohistochemical localization of cholesterol side-chain-cleavage, 17 alpha-hydroxylase and aromtase cytochromes P-450 was performed in 35 morphologically normal human premenopausal ovaries by using specific antibodies against the enzymes. In well-developed ovarian follicles in the late stages of follicular growth, immunoreactivity of P-450AROM was only seen in granulosa cells while P-450(17 alpha) and P-450SCC activity was confined to theca interna cells, confirming that follicular oestrogen is produced in granulosa cells by the aromatization of androgens derived from the theca interna cells. In the corpus luteum, this functional differentiation is maintained, since immunoreactivity of P-450AROM was exclusively present in luteinized granulosa cells while that of P-450(17 alpha) was present in luteinized theca calls. Immunoreactivity of P-450SCC was present in both types of cells in the corpus luteum.     

In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics

This study was undertaken to investigate whether bovine granulosa and theca interna cells could be luteinized in vitro into luteal-like cells. Granulosa and theca cells were cultured for 9 days in the presence of forskolin (10 microM), insulin (2 micrograms/ml), insulin-like growth factor I (100 ng/ml), or a combination of these agents. During the first day of culture, granulosa and theca cells secreted estradiol and androstenedione, respectively; progesterone rose only after 3-5 days in culture and reached a maximum on the ninth day of culture. Cells incubated in the presence of forskolin plus insulin exhibited morphological and functional characteristics of luteal cells isolated from the corpus luteum. It was found that cell diameter, basal and stimulated progesterone secretion, and pattern of cell replication for both cell types were comparable to those of luteal cells. Numerous lipid droplets and intensified mitochondrial adrenodoxin staining also indicated active steroidogenesis in luteinized cells. After 9 days in culture, stimulants were withdrawn, and the culture proceeded in basal medium for an additional 5 days; elevated progesterone levels were maintained by luteinized granulosa cells (LGC), whereas in contrast a dramatic drop in progesterone production was observed in luteinized theca cells (LTC). On Day 9, cells were challenged for 3 h with LH (10 ng/ml), forskolin (10 microM), or cholera toxin (100 ng/ml), resulting in a 4-fold increase in progesterone secretion by LTC; the same treatments failed to stimulate progesterone in LGC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase in human ovary

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was performed in 55 cases of morphologically normal human ovaries by using a specific polyclonal antibody against purified human placental 3 beta-HSD. In small developing follicles, immunoreactivity was observed only in the theca interna but also became recognizable in the membrana granulosa with development of the follicle. At a late stage of folliculogenesis, the intensity of the 3 beta-HSD activity in the membrana granulosa was nearly equal to that of theca interna in 2 or 3 large follicles examined. One to several layers of theca interna cells just beneath membrana granulosa did not demonstrate any immunoreactivity of 3 beta-HSD or that of cytochrome P-450 17 alpha-hydroxylase. These unstained theca interna cells did not appear to be directly involved in ovarian steroidogenesis and might be designated as 'enzymically inactive theca interna cells.' Marked immunoreactivity was observed in luteinized theca and granulosa cells of the corpus luteum.     

Hammond memorial lecture. New concepts of the control of corpus luteum function

Five new concepts concerning the control of corpus luteum function in the cow have been developed in recent years. Prostacyclin (PGI-2) plays a luteotrophic role. Conversely, products of the lipoxygenase pathway of arachidonic acid metabolism, particularly 5 hydroxyeicosatetraenoic acid (5-HETE), play luteolytic roles. Luteal cells arise from two sources. The small luteal cells are all of theca cell origin; the large cells found early in the cycle (Days 2-6) are mainly of granulosa cell origin. However, a population of large cells found after Day 10 of the cycle are of theca cell origin. Oxytocin of luteal cell origin plays a role in development of the corpus luteum and possibly in its regression. The recently described Ca2+-polyphosphoinositol-protein kinase C second messenger system, as well as the LH-cAMP system, is involved in control of progesterone synthesis in the bovine corpus luteum. Progesterone synthesis in the small theca-derived luteal cells is primarily controlled by the cAMP system. However, elevated intracellular calcium diminishes cAMP-mediated progesterone synthesis in these cells. These findings modify our current concepts of the mechanisms of control of progesterone secretion by the corpus luteum and suggest several new lines of research.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

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.     

Basal lamina of avian ovarian follicle: influence on morphology of granulosa cells in-vitro

Experiments were conducted to determine the influence of basal lamina on the morphology of ovarian granulosa cells in vitro. Pure and intact basal lamina was isolated from the large preovulatory follicles of the chicken ovary and designated basal lamina of avian ovarian follicle (BLAOF). Examination of the isolated basal lamina with electron microscope revealed an ultrastructure that is similar to that of basal lamina in the intact ovarian follicle. Pieces of the intact basal lamina were attached to the bottom of 32 mm culture dishes (BLAOF-coated dishes) in which differentiated granulosa cells isolated from the largest preovulatory follicle or undifferentiated granulosa cells isolated from immature small yellow chicken ovarian follicles were cultured; uncoated dishes served as controls. Granulosa cells incubated on intact basal lamina assumed spherical shape, whereas granulosa cells incubated directly on plastic in control dishes became highly flattened. Interestingly, granulosa cells that attached to plastic close to BLAOF (in BLAOF-containing dishes) became rounded. The storage of BLAOF-coated culture dishes at 4°C for 2 years had no apparent effect on its ability of the matrix material to induce changes in granulosa cell shape. Some components of the basal lamina could be solubilized with guanidine–HCl alone (fraction 1; 90–95% of total protein in BLAOF) with the remaining components solubilized with β-mercaptoethanol containing guanidine–HCl (fraction 2; 5–10% of total protein in BLAOF). Differentiated and undifferentiated chicken granulosa cells became rounded when incubated in fraction 1-pre-coated wells; whereas those incubated directly on plastic in control wells were flattened. Similarly, when fraction 1 of solubilized basal lamina was added as liquid to incubation mixture, it caused both differentiated and undifferentiated granulosa cells to assume spherical shapes. The storage of fraction 1-coated culture dishes at 4°C for 12 or more months had no apparent effect on its ability to influence granulosa cell shape. Fraction 1-induced changes in granulosa cell shape were similar to those observed for complete and intact basal lamina (BLAOF). These findings demonstrate that intact homologous basal lamina (BLAOF) or its solubilized (fluidized) form can induce normal (in vivo) morphology in granulosa cells. It is suggested that BLAOF or its solubilized form can be used to culture cells in experiments designed to examine the influence of the natural basal lamina microenvironment on cellular behavior and function.     

Ghrelin ovarian cell expression in patients with polycystic ovary syndrome: an immunohistochemical evaluation.

INTRODUCTION: The influence of ghrelin on different organs has been studied recently, e.g. in the regulation of pituitary hormone release, regulation of energy homeostasis, glucose metabolism and insulin secretion, cell proliferation, and reproductive function. However, the etiology of polycystic ovary syndrome has not been fully explained. The aim of our study was to estimate the presence of ghrelin in polycystic ovaries cells and evaluation of the relationship between ghrelin occurrence and cells proliferation. METHODS: In the present work we have compared ten polycystic ovaries with ovaries without pathology as the control group. We used immunohistochemical method to detect ghrelin. The cells proliferation was evaluated by Ki 67 proliferation index. RESULTS: Ghrelin immunostaining was demonstrated in cytoplasm of ovarian secondary interstitial cells and in atretic corpus luteum. The cell nuclei were ghrelin positive in granulosa, theca layers of follicular cyst in both groups as well as in luteal cells of young corpus luteum in healthy ovaries. Ki 67 immunostaining was observed in granulosa and theca layers of follicular cyst in polycystic and healthy ovaries. CONCLUSIONS: It is possible that local ghrelin expression plays an important role in the direct control of ovarian development and function and ghrelin may participate in patomechanism of PCOS.     

Fibroblast growth factor as an intraovarian hormone: differential regulation of steroidogenesis by an angiogenic factor

The potential role of fibroblast growth factor (FGF) in the regulation of granulosa cell differentiation was investigated because of its recent identification as the corpus luteum angiogenic factor. Treatment of rat ovarian granulosa cells with FGF inhibits the capacity of follicle stimulating hormone to stimulate estrogen production and to induce luteinizing hormone receptors. In contrast, although incubations with FGF can inhibit the estrogen-sensitive component of progesterone synthesis, the presence of FGF with suboptimal concentrations of follicle stimulating hormone significantly enhances the synthesis of progesterone. This capacity to differentially regulate steroidogenesis in the granulosa cell is comparable to the potency of FGF (ED50 = 30 pg/ml, 10(-12) M) in other in vitro assays. The observation that an angiogenic factor, like FGF, can specifically increase the sensitivity of progesterone synthesis and simultaneously inhibit estrogen formation supports the hypothesis that this growth factor plays an important role in the development and maintenance of a functional corpus luteum. As such, FGF may be involved in the local regulation of follicular selection, growth and atresia by simple virtue of its capacity to induce a neovascular response on one hand and by its ability to modulate the differentiated response to gonadotropins on the other.     

Sites of transcription of the Müllerian inhibiting substance gene in mouse testis

The factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle-stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8-fold within a 2-day period. In serum-free medium, human luteinized granulosa cells were able to incorporate ³H-thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, ³H-thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH- and estradiol-treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze-thawing, was stable for several weeks at – 20°C, became unstable at 4°C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight >30,000 dalton. We suggest that an inhibitory growth factor produced by human luteinized granulosa cells could be involved in the differentiation of growing follicles to corpus luteum. © 1993 Wiley-Liss, Inc.     

Activation of PKC delta in the rat corpus luteum during pregnancy. Potential role of prolactin signaling

Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC delta, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC delta in a luteinized granulosa cell model. We also assessed the activation status of PKC delta in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC delta was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC delta phosphorylated on serine 662. PKC delta activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC delta to phosphorylate the PKC delta-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC delta. Similarly, immunoreactivity with the phospho-epitope-specific PKC delta antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or following in vitro activation of recombinant PKC delta. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC delta in luteinized granulosa cells. PRL receptor agonists induced translocation PKC delta from the cytosolic to the Triton-soluble membrane fraction and increased PKC delta activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC delta antibody. Analysis of PKC delta activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC delta antibody revealed that PKC delta activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC delta in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC delta is increasingly expressed and active.