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The corpus luteum is an endocrine gland that synthesizes the steroid hormone progesterone. luteinizing hormone (LH) is a key luteotropic hormone that stimulates ovulation, luteal development, progesterone biosynthesis, and maintenance of the corpus luteum. Luteotropic and luteolytic factors precisely regulate luteal structure and function; yet, despite recent scientific progress within the past few years, the exact mechanisms remain largely unknown. In the present review, we summarize the recent progress towards understanding cellular changes induced by LH in steroidogenic luteal cells. Herein, we will focus on the effects of LH on inter-organelle communication and steroid biosynthesis, and how LH regulates key protein kinases (i.e., AMPK and MTOR) responsible for controlling steroidogenesis and autophagy in luteal cells.

Our goal was to develop an objective computer-assisted volumetric method of assessing vascular flow from colour Doppler ultrasound data of ovarian structures recorded by free-hand movement. We hypothesized that a vascularity index (ratio of the region of blood flood to the region of ovarian structure) obtained from the three-dimensional volumetric analysis would be more precise (less variable) than conventional two-dimensional analysis of single images in estimating the functional status of the preovulatory follicles and corpus luteum. Doppler ultrasound cineloops of water buffaloes (Bubalus bubalis; n = 22) ovaries were recorded daily from 12 h before GnRH treatment to four days after ovulation. Cineloops were processed using Fiji and Imaris software packages for segmenting the area (two-dimensional analysis) and the volume (three-dimensional analysis) occupied by the blood-flow and associated tissue to calculate the vascularity index. For volumetric measurement, all images in a cineloop were used (i.e., no a-priori selection of images) while for two-dimensional analysis, three images from the region with apparent maximum vascularity were selected. The volumetric method was verified with theoretical ellipsoidal volume of the follicle (r = 0.96 P < 0.01) or corpus luteum (r = 0.58 P = 0.02). The variability in the follicular vascularity index among animals was lower using the volumetric method than two-dimensional analysis (0.018 ± 0.002 vs 0.030 ± 0.005, P < 0.01), while the variability for CL vascularity was similar between methods (P = 0.23). An increase in the follicular vascularity index was detected at 12 h after GnRH treatment using both methods (two-dimensional: 0.030 ± 0.008, P < 0.01; three-dimensional: 0.016 ± 0.006, P < 0.02). Buffaloes that ovulated tended to have a greater increase in 3D vascularity index than non-responding buffaloes (P = 0.06); the two-dimensional method was not able to detect these changes. Using the three-dimensional method, a moderate positive correlation (r = 0.59; P = 0.02) was evident between the follicular vascularity index at 14-16 h after GnRH treatment and follicular diameter. In conclusion, an objective volumetric method for assessing relative ovarian blood flow changes was developed using Doppler ultrasound cineloops recorded by free-hand movement. The 3-dimensional method eliminates the need for a-priori selection of images and is more precise as a result of decreased technical variability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate colocalization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F 2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on (1) the spatial distribution of lipid microdomains, (2) lateral mobility of FP receptors, and (3) lateral mobility of FP receptors in the presence of PGF 2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose-dependent manner (P < 0.01). In experiment 2, single particle tracking was used to examine the effects of fish oil treatment on lateral mobility of FP receptors. Fish oil treatment increased microdiffusion and macrodiffusion coefficients of FP receptors as compared to control cells (P < 0.05). In addition, compartment diameters of domains were larger, and residence times were reduced for receptors in fish oil-treated cells (P < 0.05). In experiment 3, single particle tracking was used to determine the effects of PGF 2α on lateral mobility of FP receptors and influence of fish oil treatment. Lateral mobility of receptors was decreased within 5 min following the addition of ligand for control cells (P < 0.05). However, lateral mobility of receptors was unaffected by addition of ligand for fish oil-treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF 2α ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Galectin-1 and galectin-3, beta-galactoside-binding lectins, are predominantly expressed in the regressing corpus luteum (CL) of mouse ovary. This study revealed the expression patterns and cellular localizations of galectins during CL formation and regression by ISH and IHC. Galectin-1 mRNA expression temporarily increased in active CL, preceding the expression of progesterone degradation enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which represents functional luteolysis. The expressions of both galectin-1 and galectin-3 remarkably increased in the structurally regressing CL, which vigorously expressed 20alpha-HSD and contained abundant apoptotic luteal cells. Ultrastructurally, galectin-1- and galectin-3-immunoreactive cells were identified as fibroblasts and infiltrating macrophages, respectively. In addition, some populations of luteal cells themselves expressed galectin-3 in regressing CL and formed unique demarcation membranes in the cytoplasm, showing a non-typical apoptotic feature. Ovary of adult mice with repeated estrus cycles contained CL of three different generations. Among them, the old CL formed during previous estrus cycles consisted of galectin-3-positive luteal cells. The galectin-3-positive old CL was resistant to apoptosis and seemed to be eliminated by a mechanism different from apoptosis. The stage- and cell-specific expression of galectin in CL suggests its differential contribution to luteolysis, and this expression may be mediated by major regulatory molecules of CL function, prolactin and/or prostaglandin F2alpha.

There is increasing evidence that the corpus luteum has an important role in regulating its own demise. A series of experiments was performed to study the effects of luteal concentrations of progesterone on the functions of steroidogenic luteal cells. In the first experiment, steroidogenic small luteal cells (SLCs) were separated from endothelial cells, and it was determined that it was the SLCs that contained receptors for oxytocin. Treatment with progesterone (95 muM) for as little as 1 h decreased (P < 0.05) the percentage of SLCs responding to oxytocin (10 muM) with an increase in intracellular concentrations of calcium, and this effect continued for the duration of the experiment. In a second experiment, the response to oxytocin was increased (P < 0.05) by 3 h (but not 1 h) following progesterone removal, with a further increase by 16 h. The ability of 1 muM prostaglandin F(2 alpha) (PGF(2 alpha)) to increase intracellular concentrations of calcium was also decreased (P < 0.05) by progesterone treatment. By 3 h following removal of progesterone, the percentage of steroidogenic large luteal cells (LLCs) responding to PGF(2 alpha) was increased and not different from that observed in cells 16 h after progesterone removal. Finally, cyclodextrins (methyl-beta cyclodextrin [M beta CD]) were used to remove cholesterol from the plasma membrane of luteal cells, and M beta CD loaded with cholesterol was used to put cholesterol back into the plasma membrane of progesterone-treated cells. Treatment with M beta CD reduced (P < 0.05) the responsiveness of SLCs to oxytocin and LLCs to PGF(2 alpha). Use of cholesterol-loaded M beta CD returned the responsiveness of both SLCs and LLCs treated with progesterone to that observed in vehicle (no progesterone)-treated controls. In summary, intraluteal concentrations of progesterone inhibit the ability of oxytocin to increase intracellular concentrations of calcium in SLCs and the ability of PGF(2 alpha) to increase intracellular concentrations of calcium in LLCs. The highest concentration of progesterone appears to act by influencing cholesterol content of the luteal cell membranes.

Vardenafil citrate is a potent vasodilator used in the treatment of patients with erectile dysfunction. Its mechanism of action is based on the selective inhibition of phosphodiesterase-5 (PDE5), specific to guanosine 3',5'-cyclic monophosphate (cGMP). Recently, chronic treatment with Vardenafil has been successfully used in cases of pulmonary hypertension and, despite being used in high doses for long periods, little is known about its effects on other systems. In the present study, female mice were treated daily with 5 mg/kg Vardenafil for 4 weeks, after which the ovaries were collected for morphological analyses and sera were collected for biochemical assays. This study found that treatment with Vardenafil decreased HDL serum levels and the number of antral follicles as well as induced lesser lipid content in luteal cells, suggesting that high levels of cGMP may affect follicle development.

B-mode sonography is a well-established diagnostic tool for determination of cycle stage in gynaecology. The aim of this study was to determine whether computer-assisted texture analysis of B- mode sonographic images of bovine luteal glands provides further information about the animal's plasma progesterone concentration and cycle stage. Four Simmenthal cows were examined during two consecutive estrous cycles with an ultrasound device equipped with a 7.5MHz microconvex probe. During each examination three B-mode images of the corpus luteum (CL) were digitized and analyzed off-line using a computer-assisted texture analysis program. Size, echogeneity, and echotexture of the CL were characterized by the following texture parameters: area of cross-sectional planes of the CL (A), mean gray level (MGL), correlation (CORR), run percentage (RPERC), and long-run emphasis (LREM). Plasma progesterone levels (P4) were also determined. All parameters showed characteristic changes during the estrous cycle (P<0.05). Variance component estimates for the effect of Day of estrous cycle on A, MGL, CORR, RPERC, and LREM were 56.6%, 64.6%, 77.6%, 89.9%, and 86.0%, respectively, and 20.6%, 24.5%, 7.2%, 0.0%, and 14.0% for the influence of the individual cow. The factor estrous cycle within cows was responsible for 22.8%, 10.9%, 15.2%, 10.1%, and 0.0% of the variability of A, MGL, CORR, RPERC and LREM values, respectively. Cyclic changes were similar in A and P4. In contrast to P4, which decreased already between Days -5 and -3 (Day 0=ovulation), A stayed at constant high values until Day -3. Mean MGL values were higher (P<0.05) on Days 7, 9, and 13 compared to Days 3 and -3. Mean CORR values were constantly high (P>0.05) during the first days after ovulation and decreased continuously (P<0.05) between Days 5 and 13. Thereafter, mean CORR values remained low (P<0.05) until the next ovulation, except on Day -3 (P<0.05). Mean RPERC rose between Days 1 and 9 from low to high values (P<0.0001) remained at these high values (P>0.05) between Days 9 and 15, and decreased (P<0.05) afterwards to baseline values on Day -1. Mean LREM inclined steeply (P<0.0001) from minimum to maximum between Days 1 and 5. From Days 7 to -3, mean LREM remained (P>0.05) at a constant level close below the maximum value, and decreased to baseline values on Day -1. The results of this study show that statistical pattern recognition techniques provide new information about the luteal glands, thus facilitating a more accurate differentiation between different cycle stages in cows.

Pituitary glands were removed from sexually mature female turtles (Chrysemys picta) and they were injected intraperitoneally (i.p.) into other mature females of the same species (experimental). In addition mature females of the same species received saline injection only (controls). Initially all the turtles used in this study were steroidogenically inactive with corpora lutea already undergoing luteolysis (degeneration) as these turtles had ovioposited their eggs approximately 2 weeks earlier. Forty-eight hour post injection the corpora lutea were removed from the control and experimental turtles. In the experimental turtles, the lutein granulosa cells developed ultrastructural features such as tubular and cisternal smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae associated with lipid droplets. However, the controls maintained degenerative corpora lutea without steroidogenic ultrastructural features. The circulating progesterone (Pro) levels in the experimental turtles were significantly higher than the controls (P<0.049). Although the 48h development of steroidogenic ultrastructural features in the lutein granulosa cells was only partial in development, the effect of the pituitary taken from the inactive donor triggered an activating process within a short period, clear evidence of gonadotropic effect on the inactive corpora lutea. The present data offer interesting information on the short-term effect of gonadotropins during the non-reproductive period. This information may have useful implication under natural conditions particularly during the onset of a new reproductive cycle where the ovary is still inactive.

The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

Examination of the rat corpus luteum (CL) provided quantitative data supporting adaptation of the developing vasculature to maximise efficient acceptance of steroids secreted from the luteal cells. Numbers of endothelial cells (ECs) significantly increased during the initial formation of the CL, followed by a further significant proliferation from day 10 to day 16 when there was maximal growth of the CL. As a consequence, there was significant growth of the vascular compartment during this time interval. The final phase of expanding endothelium (days 10 to 16) was a result of increased ECs volume with elongation of the EC in the direction of growth. Continued increase in capillary surface area and a corresponding marked reduction in diffusion distance between LC and ECs evidenced adaptation of the developing microvasculature to enable efficient endocrine function by day 16, when steroid secretion is maximal. Furthermore, from day 1 to day 3 there was close apposition of pericytes to the endothelium, suggesting the important role of pericytes in the initiation of angiogenesis. However, this degree of association was reduced from day 10 to day 16 and was a consequence of expansion of the EC cytoplasm to provide a greater surface area for transport of steroids.

We obtained uterine and peripheral venous plasma, and samples of luteal and placental tissues from 2- to 7-year-old, Eurasian mountain reindeer (Rangifer tarandus tarandus) from a free-living, semi-domesticated herd in northern Norway in November 1995, and February and March 1996. In November, ovarian venous blood was also collected from four animals. Plasma samples were assayed for progesterone and oestradiol. The tissue samples were examined by light and electron microscopy, steroid dehydrogenase histochemistry, and northern blot analysis for RNAs for 3beta-hydroxy-steroid dehydrogenase (3beta-HSD) and P450 (side chain cleavage (scc)). Peripheral blood was taken from non-pregnant females in the same herd on the same dates. Peripheral progesterone concentrations in pregnant reindeer (3.4 +/- 0.5 ng/ml, n = 8) clearly exceeded those in non-pregnant animals (0.40 +/- 0.14 ng/ml; P < 0.0004 , n = 10) but oestradiol levels were only marginally higher in pregnant (6.0 +/- 0.7 pg/ml) than in non-pregnant (4.8 +/- 0.5 pg/ml; P = 0.35) reindeer at the stages examined. In pregnant animals, peripheral progesterone and oestradiol concentrations rose slightly between November and March but the differences did not reach significance (progesterone, P = 0.083; oestradiol, P = 0.061). In November, progesterone concentrations in the ovarian vein (79 +/- 15 ng/ml) greatly exceeded (P < 0.03) those in the uterine vein ( 10 +/- 4 ng/ml) which in turn exceeded the levels in the peripheral blood (2.8 +/- 0.4 ng/ml; P < 0.29). Oestradiol concentrations were slightly but significantly (P < 0.05) higher in the ovarian (20 +/- 3 pg/ml) than the uterine vein (13 +/- 1 pg/ml) and, in turn, greater (P < 0.03) than in peripheral blood (4.6 +/- 0.4 pg/ml). All samples of luteal tissue consisted exclusively of normal fully-differentiated cells and stained intensely for 3beta-HSD. Isolated groups of placental cells also stained strongly for 3beta-HSD. RNA for P450 (scc) and 3beta-HSD was abundant in all corpora lutea and lower concentrations of P450 (scc) were present in the placenta. 3beta-HSD RNA in the placenta was below the limit of detection. We conclude that the corpus luteum remains an important source of progesterone throughout pregnancy in reindeer but that the placenta is also steroidogenic.

A Call-Exner body appears as a rosette of granulosa cells radially disposed (polarized) around an aggregate of extracellular matrix. The function of these bodies is unknown. A newly described matrix, focimatrix, also develops as aggregates of basal lamina-like material between granulosa cells. It appears much later in follicular development, and in a far greater amount than Call-Exner bodies. Its possible role is to reduce the polarity of epithelial granulosa cells allowing them to develop into non-polarized mesenchymal luteal cells upon ovulation of the follicle. Call-Exner bodies may arise from early and isolated expression of focimatrix.

During luteolysis, luteal cell apoptosis is induced by the Fas ligand (FasL)/Fas system. In murine luteal bodies, we demonstrated the expression of mRNA of soluble form of Fas (FasB), which binds to FasL and prevents apoptosis induction. By in situ hybridization, strong expression of FasB mRNA was observed in normal luteal bodies, in which no apoptotic cells were detected, but negative/trace expression in regressing luteal bodies, in which many apoptotic cells were observed. Immunohistochemical staining revealed that Fas and TNF-alpha were localized in both normal and regressing luteal bodies, but IFN-gamma was localized only in regressing luteal bodies. Apoptosis was induced in primary cultured luteal cells, when they were pretreated with TNF-alpha and IFN-gamma and then incubated with TNF-alpha, IFN-gamma, and mouse recombinant FasL (rFasL). However, no apoptosis was detected in the cells, when they were treated with rFasL alone, TNF-alpha alone, IFN-gamma alone, TNF-alpha and rFasL, IFN-gamma and rFasL, or TNF-alpha and IFN-gamma. Fas mRNA expression in cultured luteal cells was up-regulated by the treatment of TNF-alpha, IFN-gamma, or TNF-alpha and IFN-gamma. The expression of FasB mRNA was down-regulated, when the cells were treated with TNF-alpha and IFN-gamma, but its expression was not changed by the treatment of TNF-alpha alone or IFN-gamma alone. We conclude that FasB inhibits the apoptosis induction in luteal cells of normal luteal bodies, and that decreased FasB production induced by TNF-alpha and IFN-gamma made possible the apoptosis induction in the luteal cells of regressing luteal bodies., (Copyright 2003 Wiley-Liss, Inc.)

Localization sites and labeling intensity of alphaB-crystallin in corpus luteum (CL) of pseudopregnant rats has been studied using postembedding light and electron microscopical immunohistochemistry. At days 2 and 18 alphaB-crystallin labeling was found to be significantly higher compared with the luteal maintenance period (days 6 and 10). alphaB-crystallin localized both to luteal and interstitial cells of CL. At light microscopical level alphaB-crystallin labeling decreased during CL life span in the central area of luteal cells cytoplasm and increased in the peri-plasmalemmal area. Interstitial cells labeling was found to increase at day 6, followed by almost complete disappearance during functional luteolysis (days 15 and 18). Our results at electron microscopical level showed alphaB-crystallin to localize in cytoplasm with close relationship with endoplasmic reticulum, Golgi apparatus, mitochondria and also in nuclei of luteal and interstitial cells. At day 2 labeling of luteal cells was abundant in cytoplasm but weak in nuclei. During the luteal maintenance period and functional luteolysis labeling in luteal cells was relocalized to the peri-plasmalemmal and perinuclear areas. Labeling in nuclei of luteal cells was weak. At the same time (day 6) interstitial cells including nuclei showed strong labeling, which was significantly decreased during luteolysis. Immunohistochemically detectable tubulin decreased in CL tissue during CL life span allowing to suggest that alphaB-crystallin acts as chaperone, one possible role of which is to stabilize the cytoskeleton in different CL cell types during CL formation and active functioning.

Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzymatically dispersed, and a portion of the cells were elutriated to obtain fractions enriched with small or large luteal cells. Mixed, small, and large luteal cell fractions were incubated with no treatment or with agonists or antagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7), or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependent gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P(4)) radioimmunoassay, and luteal cells cultured with no treatment were fixed for immunocytochemistry or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P < 0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P < 0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P < 0.01), but H-7 did not affect, GJIC for all cell types across the estrous cycle. The A23187 decreased (P < 0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A23187 + EGTA decreased (P < 0.05) GJIC for all cell types across the estrous cycle. For the mixed and large luteal cell fractions, dbcAMP increased (P < 0.05), but TPA and A23187 + EGTA decreased (P < 0.05), P(4) secretion. The A23187 alone decreased (P < 0.05) P(4) secretion by large, but not by mixed, luteal cells. For all days and cell types, the rate of GJIC and P(4) secretion were correlated (r = 0.113-0.249; P < 0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thus, intracellular regulators like cAMP, PKC, or calcium appear to regulate GJIC, which probably is an important mechanism for coordinating function of the ovine CL.

Structural luteolysis shows striking interspecies differences. Morphological changes in the corpus luteum (CL) of the cyclic hamster have been studied alongside the potential involvement of known luteolytic hormones. Ovaries from intact Syrian golden hamsters killed at 1100 h on days 1 and 2 and at 1100 and 1700 h on days 3 and 4 of the estrous cycle were dissected for histological study. The day of ovulation, the day of estrus, was arbitrarily designated day 1 of the estrous cycle. Steroidogenic cells in the CL were scarcely luteinized on day 1 and reached full luteinization on day 2. On the morning of day 3, initial regressive changes (accumulation of lipid droplets, invasion by neutrophils, and accumulation of phagocytic cells) were observed. These regressive changes increased progressively and apoptotic cells as well as phagocytic cells containing phagocytized apoptotic cells were abundant on the evening of day 3. On the morning of day 4, apoptotic cells/bodies and phagocytic cells containing phagocytized material were extremely abundant throughout the CL. However, steroidogenic cells with intact nuclei and well-preserved blood vessels were also found. Surviving cells in the CL showed progressive morphological changes. These cells showed morphological features intermediate between luteal and interstitial cells in the evening of day 4 and were virtually indistinguishable from interstitial cells on day 1 of the following cycle. Additional animals were injected at 1100 h on day 2 with: (a) the dopaminergic agonist CB154 (0.4 mg) to block prolactin secretion, (b) the anti-estrogen LY117018 (1.6 mg) or the anti-androgen Flutamide (3 mg) to block estrogen or androgen receptors, respectively, and (c) progesterone (2 mg) to prevent the fall in serum progesterone concentrations. Ovaries from these animals were collected at 1700 h on day 3 and at 1000 h on day 4. The luteolytic process was not affected by any treatment. These data indicate that, in contrast to its close relatives (e.g., the rat), structural luteolysis in the hamster is independent of the apoptotic inducing luteolytic hormones. In addition, differences in the cellular mechanisms responsible for CL elimination were also present. In the hamster, part of the luteal cells do not undergo apoptosis and seemed to progress through another developmental path giving rise to interstitial-like cells., (Copyright 2001 Academic Press.)

Objective: To localize (by the light and electron microscopy) and partially characterize the antigen recognized by the Mab4E6 in rat ovaries., Methods: Monoclonal antibody (4E6) against a rat ovarian granulosa cell antigen was prepared and identified the 40kDa protein specific for rat steroid producing cells. The localization of this antigen was studied by light and electron microscopic immunocytochemistry., Results: The immunocytochemical observation suggested that the recognized antigen was localized in granulosa and thecal cells in all stages of follicular development. The intensity of immunostaining was found to depend on the developmental stage. In granulosa and thecal stage (health follicle) Mab 4E6 binding molecule was localized on the membranes of rough endoplasmic reticulum (RER) and on the surface of lipid droplets in close association with the RER. In atretic follicles we established that the final destination of the visualized antigen is in structures which we refer as the autophagic vacuoles in close contact with the steroidogenic organelles. In addition, we observed Mab 4E6 binding molecule in the cytoplasm of luteal cells. Leydig cells and adrenocortical cells., Conclusions: The results indicate that the 40kDa antigen may be common to all of rat steroidogenic organs. Our results suggest that the 40kDa protein may be associated with the processes governing steroidogenesis and/or follicular development.

The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.

Oxytocin and its carrier protein, neurophysin, are both associated with luteal secretory granules which migrate from the paranuclear region to the cell membrane where exocytosis takes place. Rab3 proteins are thought to be associated with membrane vesicles or granules undergoing exocytotic fusion with the plasma membrane. The objective of this study was to determine whether Rab3B is co-localized with oxytocin within the same secretory granules of large luteal cells obtained from corpora lutea of 16 Merino cross ewes at day 3, 7, 12 or 15 of the estrous cycle using immunocytochemistry. The mean granule density (granules/microm3) was not significantly different (P > 0.05) between the days examined. Electron microscopic immunocytochemistry showed that oxytocin and Rab3B were co-localized to the secretory granules on all days evaluated. Rab3B immunostaining was primarily located within secretory granules scattered throughout the cytoplasm. The mean intensity of labelling (number of gold particles) for oxytocin per microm2 cytoplasmic luteal tissue was significantly decreased on day 15 compared to those observed on days 3, 7 and 12 of estrous cycle. No significant changes were observed in the mean intensity of the Rab3B label at the different times of the cycle. The present study provides evidence that a member of the subfamily of Rab proteins, Rab3B, is present and co-localized with oxytocin in the same secretory granules of the ovine corpus luteum. These results implicate Rab3B protein directly or indirectly in the hormone secretory pathway of ovarian tissue.

This study addresses the question of whether the level of expression of SR-BI (an HDL receptor) is linked to the expression of selective lipoprotein-cholesteryl ester delivery in a steroidogenic cell model. Rat ovarian granulosa cells are physiologically normal cells which show no selective uptake of HDL-cholesteryl esters and no progestin production until luteinized by trophic hormones or adenylate cyclase stimulators, after which expression of the selective cholesterol pathway and production of steroid hormone is dramatically up-regulated. The current study demonstrates that at every cell stage studied, the protein content and level of expression of SR-BI mRNA are linked to changes that occur in HDL-cholesteryl ester uptake; i.e., SR-BI is not present in basal (non-luteinized) cells, develops slowly (from 6-9 h) after hormone treatment, increases robustly from 9-48 h after stimulation, and remains high after incubation with HDL. In contrast, another structural protein, caveolin, did not follow this pattern; caveolin expression showed an inverse relationship to selective cholesteryl ester uptake, and was most prominent in basal cells and least prominent in luteinized, HDL-incubated cells. Morphologically, SR-BI appears to be associated with cell surface sites showing high levels of cholesteryl ester uptake (after luteinization and/or incubation with HDL labeled with fluorescent cholesteryl esters), and at the electron microscope level, SR-BI is most clearly associated with microvillar regions on the cell surface which also bind HDL-labeled with colloidal gold. Thus, induction of the SR-BI receptor system and induction of the HDL-selective cholesterol uptake pathway in rat granulosa cells appear to be linked morphologically, biochemically, and functionally.

Electron-microscope immunocytochemistry was used to determine the subcellular distribution and presence of immunoreactive relaxin throughout pregnancy and early lactation in the corpus luteum of a marsupial, the tammar wallaby. Membrane-bound, electron-dense granules were a prominent feature of the luteal cell cytoplasm. The highest numbers of granules were observed between days 20 and 24 of the 26-day gestation, with a rapid clearance immediately after birth. Relaxin immunogold particles were present only in small, electron-dense granules (200-350 nm in diameter), with no particles observed in larger granules (>400 nm diameter), nuclei or mitochondria. Relaxin immunoreactivity was low throughout early and mid pregnancy but increased markedly between days 21 and 22 and remained high over the last 4 days of pregnancy. The number of granules containing relaxin immunogold particles and the density of immunostaining were both reduced on the day of expected births (day 26). Our data demonstrate that electron-dense granules in the luteal cell cytoplasm of a pregnant marsupial contain relaxin. The peptide is produced in greatest amounts at the end of pregnancy, consistent with a role in parturition.

Indirect immunofluorescence light microscopy was used to monitor temporal perturbations in the microtubular (tubulin) system of granulosa/lutein cells in paraffin-embedded sections of periovulatory follicles and corpora lutea of sheep. Estrogen-active granulosa cells of preovulatory follicles not yet exposed to the gonadotropin surge immunostained intensely for tubulin. Immunostaining of the microtubular matrix diminished after the onset of the surge and coincident with an abrupt fall in follicular estradiol production. A transient period of microtubular retraction was characterized by low-level steroid hormone output. Microtubules reappeared with the approach of ovulation and increase in follicular progesterone biosynthesis (luteinization). Treatment of animals during the preovulatory period with colchicine, a drug that binds specifically with tubulin and interferes with microtubular assembly, obstructed the follicular shift toward progesterone. Microtubular dynamics (polymerization<->depolymerization) evidently underpin fundamental mechanisms of follicular steroidogenesis. Finally, corpora lutea were isolated from ewes on Day 10 of the estrous cycle before (0 h) and after administration of prostaglandin (PG) F2 alpha. There was a small augmentation in luteal concentrations of progesterone at 2 h, followed by a sharp decrease from 4 to 16 h. Luteal weights were reduced (structural regression) at 24 h. Sections of large (PG-sensitive) steroidogenic cells of control corpora lutea typically displayed a radiating microtubular network. After administration of PGF2 alpha, tubular matrices of large cells were scant; mitochondrial clustering was evident in transmission electron micrographs. Affixed disassembly of the cytoskeleton of large luteal cells may be a heretofore unrecognized event in the biomechanics of functional luteolysis--perhaps uncoupling cholesterol translocation to mitochondrial cytochrome P450 side-chain cleavage.

Although granulosa cell differentiation and corpus luteum function are both regulated by cAMP, there are development-dependent differences, particularly at the level of gene expression and cell proliferation, between the responses of follicular granulosa cells and luteal cells to trophic hormone stimulation. In this study, we sought to determine whether these differences could be due to changes in the cellular expression of cAMP response element (CRE)-binding protein (CREB). Immunocytochemical analysis of macaque ovaries revealed a development-related alteration in the subcellular distribution of CREB-immunoreactive material. Immunoreactive CREB was present in nuclei of follicular granulosa cells from maturing follicles, whereas after ovulation and luteinization, no CREB-immunoreactive proteins were visualized in luteal cell nuclei. Anti-CREB immunoblotting of granulosa cell extracts from macaque preovulatory follicles as well as extracts of granulosa cells from luteinizing human follicles revealed a 43-kilodalton (kDa) protein, a size typical of native CREB. In contrast, whole cell extracts of monkey corpora lutea collected during the early, mid-, and late luteal phases completely lacked a 43-kDa CREB signal. The absence of 43-kDa CREB isoforms in corpora lutea was confirmed using three different antisera directed against different regions of CREB. Using a human collagenase gene CRE to probe Southwestern blots, a 43-kDa CREB was observed in follicular cell extracts, whereas no CRE-binding activity was found in corpora lutea extracts using this probe. We also sought to determine whether the loss of expression of the 43-kDa CREB isoform may be functionally correlated with the cessation of cellular proliferation that accompanies luteinization. Expression of proliferating cell nuclear antigen (PCNA), an obligatory component of DNA polymerase delta, is essential for proliferation and has been shown by others to be CRE dependent. Immunoblotting of follicle cell and luteal cell extracts with an anti-PCNA monoclonal antibody revealed PCNA expression in granulosa cells and no detectable PCNA expression in corpora lutea. These findings indicate that as follicular granulosa cells progress from the proliferative state to terminally differentiated luteal cells, there is a cessation of expression of a 43-kDa member of the CREB family of transcription factors, and there may be an association between the loss of CREB isoforms and cessation of PCNA expression.

In the present investigation, we have studied peroxisomes and sterol carrier protein-2 (SCP2) in control and luteinizing hormone stimulated rat luteal cells. Superovulated immature rats in mid-luteal phase (8 days after ovulation) were divided into two groups (n = 4/group) and treated with vehicle (0.2 ml saline), or luteinizing hormone (LH, 20 micrograms/rat). In this animal model, LH acutely stimulates steroidogenesis. Thirty minutes later, corpora lutea were fixed by whole body perfusion and processed for (1) electron microscopic immunocytochemistry to localize SCP2 via the protein A gold immunolabeling technique, and for (2) electron microscopic histochemistry to stain peroxisomal catalase via the alkaline 3,3'-diaminobenzidine tetrahydrochloride method. In the steroidogenic, mid-phase luteal cells of vehicle treated rats (controls), SCP2 was highly concentrated in peroxisomes and sparsely scattered on mitochondria, but no labeling was observed in lipid droplets. In the luteal cells of rats acutely stimulated with LH, peroxisomes immunolabeled for SCP2 were observed within the luteal cell lipid droplets and mitochondria, and in union with lipid droplets and mitochondria. Moreover, in contrast to control luteal cells, significant immunolabeling for SCP2 was detected within the lipid droplets and mitochondria in luteal cells of LH-treated rats. As SCP2 binds cholesterol to 1:1 molar ratio and is known to be involved in the intracellular movement of cholesterol, these findings suggest that peroxisomes and SCP2 may possibly be involved in delivering cholesterol from lipid droplets to the mitochondria when luteal cell steroidogenesis is acutely stimulated by LH.

Background: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), we investigated the effects of these two cytokines on death of luteal cells in vitro., Methods: Mouse luteal cells were cultured in serum-free medium with TNF-alpha at 0, 500, 1,000, 3,000, or 5,000 U/ml in the presence or absence of IFN-gamma at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3' end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy., Results: On day 3 of culture, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500-5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-gamma (1,000 U/ml) and TNF-alpha (5,000 U/ml) did. On day 6, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-alpha alone at 5,000 U/ml did, and combinations of IFN-gamma and TNF-alpha at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-alpha. On days 3-6 of culture, combinations of IFN-gamma and TNF-alpha that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis., Conclusions: The presence of IFN-gamma modulates the ability of TNF-alpha to induce a reduction in the number of viable cells, although TNF-alpha alone at high concentrations can induce a reduction in the number of viable cells.

A large body of evidence suggests multiple forms of 17 beta-hydroxysteroid oxidoreductase (17-HOR) regulate estrogen and androgen levels within gonadal and peripheral tissues. Two kinetically-differing 17-HOR activities have been detected in placental homogenates. 17-HOR type 1, found mainly in the cytosol, is highly reactive with estradiol-17 beta (E2) and estrone (E1) but not testosterone (T) (high E2/T activity ratio). Microsomal 17-HOR type 2 is reactive with both E2 and T (low E2/T activity ratio). In this study, 17-HOR activity of cytosol and microsomes from term placenta, ovarian stroma and granulosa-luteal cells was assayed under conditions which specifically differentiate between the two forms of the enzyme. Placenta had the highest activity with either E2 or T in both cytosol and microsomes and stroma the lowest. The highest specific activity with E2 and E1 was cytosolic in all samples. The highest specific activity with T was microsomal in placenta and ovarian stroma. E2/E1 activity ratios were comparable for cytosol and microsomes while E2/T activity ratios were comparable for placenta and stroma, but markedly elevated in granulosa-luteal (G-L) cell cytosol and microsomes. The results indicate trophoblast and ovarian stroma have more 17-HOR type 2 relative to type 1. G-L cells, in contrast, are relatively enriched in 17-HOR type 1 and thus have a greater capacity for net conversion of E1 to E2 under physiologic conditions. These differences may contribute to increasing serum and follicular fluid E2/E1 ratios during development of the dominant follicle.

Corpora lutea from Sprague-Dawley rats that were orally administered 0.0 (control), 1.0, 10.0, and 100.0 mg/kg hexachlorobenzene (HCB) for 21 days were analyzed by electron microscopy. Granulosa lutein cells (GLC) from animals of the 10.0 mg group showed differences from the cells of animals that served as the controls. Golgi complexes and smooth endoplasmic reticulum appeared more conspicuous, possibly due to dilation resulting from hyperactivity. Free polysomes seemed more prominent in the cells of the 10.0 mg group. The GLC architecture from animals of the 1.0 and 100.0 mg groups was similar to that of the corresponding cells in the control group. Since smooth endoplasmic reticulum is involved in the synthesis of steroid hormones, and that free polysomes are engaged in synthesis of cytoplasmic proteins, it is suggested that HCB at a dose of 10.0 mg/kg given for 21 days may alter the synthetic activity of the GLC of the rat.

An ultrastructural study to the corpus luteum (CL) from 263 non pregnant female cattle slaughtered in slaughter-houses of León (Spain) was done. The luteal cells are classified in five types, noted with roman numbers. We made a comparison between these cells and the cells described in the literature. We believe that luteal cells have a development from type I to V, the first type is the principal inside the developing CL. The types II and III are in the mature CL, and the two last types are in the degenerating CL. It will be the same cell in different stages of development. We did not find characteristic structures to differentiate luteal cells originating from the granulosa or the theca.

Rat luteal cells preferentially utilize cholesterol derived from high density lipoproteins (HDL) as a substrate for steroid hormone synthesis. The uptake of cholesterol from HDL by these cells is in contrast to nonsteroidogenic cells, which export cholesterol to HDL. A previous study demonstrated that HDL binding to luteal cell membranes was increased in conjunction with in vivo cholesterol depletion or cholesterol loading of the ovary induced by pharmacological agents. These results suggest a biphasic regulation of the HDL receptor in luteinized rat ovaries. In the present studies, the in vitro regulation of HDL binding in rat luteal cells by increased intracellular cholesterol was examined. Cultured luteal cells were incubated with increasing doses of low density lipoproteins (LDL) for 2 days after which the cellular sterol content and the effects on progesterone production and HDL binding were measured. As expected, the LDL treatment increased total cellular sterol content in a dose-dependent manner, resulting in a 2.1-fold increase over control at a dose of 1 mg LDL/ml. Increased cellular cholesterol was accompanied by a comparable increase in progesterone secretion. These results suggest that exogenous cholesterol was utilized by these cells. The LDL treatment also increased the binding of HDL to the cells in a dose-dependent manner to a maximum of 2.2-fold over control. The effect of increased cellular sterol on HDL binding was also examined using a more polar cholesterol derivative, 25-hydroxycholesterol. Cells were cultured for 2 days in media containing 0.3-40 micrograms/ml 25-hydroxycholesterol in the presence of 100 micrograms/ml aminoglutethimide, an inhibitor of cholesterol metabolism. The HDL binding to luteal cells exhibited dose-dependent up-regulation by 25-hydroxycholesterol with a 5.8-fold increase in binding at the maximum dose tested. Equilibrium binding studies using cells treated with 10 micrograms/ml 25-hydroxycholesterol revealed a 2.1-fold increase in the number of HDL binding sites on the luteal cells without affecting the binding affinity. From the results of this study, it is concluded that HDL binding in rat luteal cells is up-regulated by an increase in the intracellular cholesterol level.

Using anti-beta-LH monoclonal antibodies, studies were undertaken to assess the effects of the glycosides (GTW) and T4 from Tripterygium wilfordii Hook f. on LH cells in male rat pituitary glands using immunohistochemical methods and ultrastructural observation. The results showed that there were more vacuoles in the cytoplasm of LH cells and the color density of immunohistochemical staining was much stronger in the treated groups than that in the control group. Electron microscopic results showed that the nulei of LH cells were shrunken and the Golgi complexes and the rough endoplasmic reticula were largely expanded in the treated groups. The mechanism of these changes is similar to that in "castration cells."

Ovarian tumors induced by intrasplenic ovarian grafting were studied ultrastructurally to obtain the details with particular references to cytoplasmic organelles associated with steroid synthesis. The grafted cells were transformed ultrastructurally at around 7 months following intrasplenic ovarian grafting, in fact, intermediate type of cells were also seen in the grafts at this period. The grafted cells before the transformation showed fine structural evidence of steroid hormone secretions, as indicated by the presence of abundant smooth endoplasmic reticulum, lipid droplets and mitochondria with tubular or vesicular criste. After transformation, the cells, however, had no fine structural features associated with the production of steroid hormones.

Large steroidogenic cells of the bovine corpora lutea were evaluated for morphological changes on Days 3, 7, 11, 14, 17, and 19 of the estrous cycle. Large cells were readily identified by size (25-50 microns diameter), numerous mitochondria, and the presence of dense secretory granules (150-300 nm in diameter). These granules were found in a discrete cluster and were not dispersed throughout the cytoplasm. Only 3% of the large cells contained a cluster of granules on Day 3. The percentage was highest during midcycle (Day 7, 84%; Day 11, 64%), dropped on Day 14 (26%), and was lowest on Days 17 (16%) and 19 (8%). Electron microscopic immunocytochemistry showed that oxytocin and neurophysin were co-localized in these granules on all days evaluated. As early as Day 14, large cells were observed with characteristics typical of regressing corpora lutea, i.e., a reduction in cells with secretory granules, large cytoplasmic lipid droplets, and swollen mitochondria with dense inclusions. However, since this was a time of the cycle when plasma concentrations of progesterone were very high, this corpus luteum is referred to as involutive rather than regressive. Our results may be summarized as follows: 1) from Day 7 to Day 14 there was a 69% decline in the number of large cells containing oxytocin-laden secretory granules. This occurred prior to the rise in uterine oxytocin receptors and the large luteolytic pulses of prostaglandin that reportedly occur after Day 14. The role of this apparent early release of oxytocin is not known. 2) Large steroidogenic luteal cells of the estrous cycle have morphological characteristics similar to those of large luteal cells during pregnancy. However, large luteal cells of the estrous cycle contain oxytocin whereas those of pregnancy are devoid of oxytocin.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.

Luteal cells (10(6)/chamber) from pregnant pigs (Day 18, n = 5) were incubated in a perfusion system with a flow rate of 0.11 ml/min and samples collected every 10 min. This medium 199 had been supplemented with 1% BSA, 0.1% ascorbic acid and 10 mM Hepes buffer. The effects in vitro of adrenergic agonists (adrenaline, noradrenaline and isoproterenol) at concentrations of 10(-5) M on progesterone (P4) production by pig luteal cells were studied. The alpha or beta receptors were blocked by pretreatment of the luteal cells with phentolamine or propranolol for 30 min and then the cells were treated with adrenergic agonists for 60 min followed by a further 80 min with medium alone. The steroidogenic response to noradrenaline in terms of increased P4 production was greater than that for adrenaline and isoproterenol (P less than 0.05). Response to both natural and synthetic catecholamines was suppressed by pretreatment of luteal cells with propranolol but after pretreatment with phentolamine all the agonists significantly (P less than 0.05) elevated P4 production. The results demonstrate that catecholamines can affect steroidogenesis in luteal cells from the early pregnant pig and their action is mediated by beta adrenergic receptors.

Three enriched populations of cells [C alpha, non-steroidogenic cells less than or equal to 15 microns in diameter; R1, small (less than or equal to 15 microns) steroidogenic cells; and R3, large (greater than 20 microns) steroidogenic cells] have been isolated from the macque corpus luteum using flow cytometry based on light scatter properties. To determine whether the cell populations differ in their ability to bind and internalize low-density lipoprotein (LDL), collagenase-dispersed cells were prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. Cells were incubated in Hams F-10 medium containing fluorescent-tagged LDL (DiI-LDL). Optimal labeling occurred at 10 micrograms DiI-LDL/10(6) cells.ml incubated for 20 min at 37 degrees C. Labeled cells were analyzed and sorted by flow cytometry based on light scatter and fluorescence. Only 8.2 +/- 1.0% (n = 10) of C alpha cells exhibited fluorescence intensity greater than the autofluorescence of unlabeled cells. In contrast, 52.3 +/- 3.4% of R1 cells and 83.9 +/- 2.3% of R3 cells were fluorescent. Uptake of DiI-LDL was competitively inhibited when cells were conincubated with either unlabeled monkey or human LDL, but human high-density lipoprotein and very low-density lipoprotein were less effective. Progesterone (P) production by fluorescent [DiI-LDL(+)] R1 luteal cells was increased (P less than 0.001) in the presence of pregnenolone, but not human (h) CG, consistent with earlier results for the R1 population. Surprisingly, basal P production by the nonfluorescent [DiI-LDL(-)] R1 cells was similar to that by fluorescent cells and was stimulated by both hCG (P less than 0.01) and pregnenolone (P less than 0.001). Basal P production by DiI-LDL(+) R3 cells was nearly 10-fold greater than that by DiI-LDL(-) R3 cells. P secretion by both DiI-LDL(+) and (-) R3 cells was stimulated by hCG (P less than 0.01) and pregnenolone (P less than 0.001). DiI-LDL(-) C alpha cells produced barely detectable levels of P, but DiI-LDL(+) C alpha cells secreted P at levels similar to R1 cells. We conclude that: 1) multiparameter (light scatter and fluorescence) cell sorting is a useful method for separating enriched populations of cells from the corpus luteum; and 2) small and large luteal cell populations from the macaque corpus luteum consist of subtypes of steroidogenic cells that differ in lipoprotein uptake and/or gonadotropin sensitivity.

A comparison was made of the ultrastructural features of the remaining corpora lutea (CL) of five unilaterally ovariectomised and five control pregnant rats. Unilateral ovariectomy (ULO) was carried out on Day 8 of gestation; this treatment has been shown to double the normal rate of progesterone secretion by the remaining ovary within 8 days. On Day 16, the ovaries from both ULO and control rats were examined stereologically. The mass of luteal tissue remaining in ULO rats was only 44% of that in the controls, but their plasma progesterone concentration was 81% and not significantly different from the control value. The CL were 10% heavier in ULO rats and the amount of luteal cell cytoplasm per CL was 21% greater. The percentage of the CL occupied by luteal cells was 15% greater in ULO rats but the interstitial space was 50% less. There was no significant change in the percentage of the luteal cell cytoplasm occupied by cytosol, agranular endoplasmic reticulum (AER), mitochondria and electron-dense granules. The surface area per unit cytoplasmic volume of AER and outer and inner mitochondrial membranes (OMM and IMM, respectively) was not affected. However, expressed per CL, both the AER and IMM membrane surface areas were increased by around 30%. These morphometric changes would only account for about 30% of the reported increase in hormone secretion by the CL of ULO rats, and they suggest that the CL of Day 16 pregnant rats may have reached their optimal structural condition.

Changes in cytosolic free calcium concentrations [( Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) were measured in single rat luteal cells, using the calcium-sensitive fluorescent dye fura-2. A total of 112 cells were studied in 20 experiments. The average resting [Ca2+]i was 113 +/- 6.4 nM. In 59 cells (53%), there was a 4.6 +/- 0.2-fold increase in [Ca2+]i within 29.3 +/- 1.0 sec of PGF2 alpha administration, and the cells recovered by 78.0 +/- 4.5 sec. The magnitude of the increase in [Ca2+]i in response to PGF2 alpha was not changed by an increase in the concentration of PGF2 alpha. Perifusion with low calcium buffer reduced, then eliminated, the [Ca2+]i response to PGF2 alpha. Perifusion of cells with PGF2 alpha resulted in a single transient [Ca2+]i response, similar to that after short term exposure to PGF2 alpha. Many (67%) of the cells that responded to PGF2 alpha also responded to GnRH. No additive increase in [Ca2+]i was seen when PGF2 alpha and GnRH were administered together. The source of the calcium appears to be intracellular stores that are shared by GnRH and PGF2 alpha.

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc) activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 microM) induced morphological changes in cellular organelles.

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.

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.

Corpora lutea (CL) from cows on day 12 of the oestrous cycle were studied by electron microscopy to investigate whether, and how, different subpopulations of luteal cells can be identified in tissue sections. Tissues from 6 CL were examined, and nucleated profiles of luteal cells were classified as large, medium or small on the basis of their areas in electron micrographs. Cut-off points for area categories for large, medium and small-sized cells were based on diameters of greater than 25, 20-25 and less than 20 microns, respectively, if the measured areas were converted to a circular shape after correction for shrinkage. The only qualitative features which distinguished cells of large size from those of small size were the presence of clusters of secretory granules, and of exocytosis of these granules, in large cells only. However, these features were observed in only 59% of large cells, probably primarily due to sampling limitations in single sections. Other qualitative features which have been regarded as diagnostic of large or small luteal cells were observed in cells in all size categories. It was concluded that large and small luteal cells in the cyclic CL of the cow are distinguishable by their ultrastructural features. However, these data do not support the recent suggestion that the mid-cycle CL of the cow contain two subpopulations of large luteal cells in approximately equal numbers.

The compartmentalization of the parenchyma of the corpus luteum in the dog was studied by both 100 and 1000 KV electron microscopy. The organells within the luteal cell are oriented with a high degree of consistency towards the pericapillary space. Characteristically, the avascular pole and the lateral margins of the cell posses predominantly stacked and whorled cisternae of agranular ER. In the central medial portions of the cell, pleomorphic mitochondria with tubulo-vesicular cristae and anastomosing tubules of agranular ER predominate. However, the distribution of organelles in this compartment is graded. Mitochondria predominate in the central medial areas while tubular ER is more dominant peripherally. Microfilaments are ubiquitous in this compartment and run a longitudinal course between and around the subcellular components towards the pericapillary space. The Golgi apparatus is large and prominent and is positioned over the pole of the nucleus that faces the basal lamina. Coated vesicles are abundant in the Golgi regions and along the lateral surface of the cell. Three distinct regional specializations of the cell surface exist. The basal surface contains long pleomorphic cytoplasmic folds that fill the pericapillary space, are interconnected by small gap junctions and contain abundant multivesicular bodies. The lateral cell surface is covered with microvilli and is organized into tortuous intercellular channels and canaliculi. These are interrupted at intervals by cytoplasmic protrusions that extend from one cell well into the cytoplasm of the next. Large, well-developed gap junctions line the margins of the cells furthest removed from the pericapillary space. Finally, the individual cells exhibit heterogeneity with respect to the amount one subcellular organelle or compartment is expressed relative to another. These observations are discussed in relation to the subcellular compartmentalization of progesterone synthesis and release.

Biopsies of human ovaries from functional and nonfunctional polycystic ovary patients 24 to 56 years of age were processed by critical point drying and viewed by scanning electron microscopy. The superficial epithelium was characterized by polyhedral-shaped cells with abundant evenly distributed apical microvilli with some intercellular connection and occasional solitary cilia. The fine structure modifications in the ovarian superficial epithelium (e.g., microvilli, blebs, ruffles, and micropapillae) reflect physiological changes in cell cycle or reproductive cycle. Dramatic changes were noted in the superficial epithelium of polycystic, atrophic, and sclerotic ovaries: distribution of microvilli, proportion of cells of different morphology, abundance of solitary cilia, blebbing, and the size of rufflings. In developing follicles, the granulosa cells were spherical and/or polyhedral in shape and in close contact. In atretic follicles, the granulosa cells were flattened and very irregular in shape without cytoplasmic evaginations. The loss of such evaginations within the atretic follicles may correspond to functional changes, e.g., inability to bind and respond to gonadotropic receptors. Follicles in polycystic ovaries were characterized by the disruption of the granulosa layer. Thus, areas of the follicle were devoid of any granulosa cells and appeared patchy, whereas the granulosa cells were irregular in size and shape.

Incubation of ovaries from pseudopregnant rats have been performed with the gonadotropin LH and with two inhibitors, aminoglutéthimide and cycloheximide for periods comprised between 1 and 6 hours. Then the luteal cells have been studied with electron microscope. Measurements in have been used to express the variations in volume of two organels, mitochondria and lipid droplets. The principal modifications induced by these compounds bear on the mitochondria. Treatment with LH is followed by an increase in the volume of individual mitochondria resulting probably of fusion of organels. Treatments with the inhibitors are followed by reduction in the volume of individual mitochondria and in the total volume of mitochondrial apparatus. Mixed treatments using LH and an inhibitor allow to obtain organels of intermediate size. Volume of lipids is increased by incubation with LH and probably decreased by the inhibitors. Smooth endoplasmic reticulum shows rearrangements with the various treatments. It seems not convenient to extend incubations over 4 hours in order to avoid cytolysis.

The structural changes in the granulosa lutein cells in the pregnant cows between 60 and 245 days, were observed. The polyhedral or pump cells has an acidophilic cytoplasm around the spherical nucleus. The glycogen granules increased in number up to 230 days of pregnancy. The lipid globules increased in size and amount during advanced pregnancy. The mitochondria showed a wide range of variations in shape and size. The matrix of many mitochondria contained dense inclusions which replaced the entire matrix in late pregnancy. The endoplasmic reticulum was chiefly of smooth type but a few stacks of the rough variety could be seen in the early days of pregnancy. Sometimes, whorls of smooth-surfaced endoplasmic reticulum enclosing mitochondria were seen. The Golgi complex was fully developed in the granulosa cells of all cases studied. The dense granules and lipid globules increased in size and number in the older corpora lutea. The cell surface showed numerous infolding of the plasma membrane.

Functionally active or regressing luteal cells were obtained from pseudo-pregnant (psp) rats between days 5-8 of psp or on day 15 of psp, respectively. They were monolayer-cultured (10(6)/dish) in the presence of 0.2 micrograms/ml LH 2.0 micrograms/ml PRL and 10 micrograms/ml pregnenolone for 4 days with or without macrophages, although functionally active luteal cells secreted progesterone dominantly during day 1 of culture (Day 1), the amounts of progesterone and 20 alpha-OH-P secreted were inverted on Day 2, and the dominance of 20 alpha-OH-P continued from Day 2 to Day 4. In the functionally regressing luteal cell culture, more 20 alpha-OH-P than progesterone was secreted throughout the culture period. The addition of peritoneal macrophages (2.5 X 10(6] to the active luteal cell monolayer lengthened the dominance of progesterone secretion for an additional day and the inversion occurred on Day 3. The progestin ratio (progesterone/20 alpha-OH-P) on Day 2 was maintained significantly higher. The daily addition of macrophages maintained the progesterone dominance throughout the culture period. On the other hand, macrophages had no effect on luteal cells already functionally regressing. These results indicate that macrophages are effective in maintaining the progesterone secreting activity of luteal cells in vitro.

Previous studies showed that perfused 125I-labeled low and high density lipoproteins (LDL, HDL) have affinity for specialized microvillar regions of luteal cells in hormone-primed, luteinized rat ovaries. In the current report, we re-examined the interaction of cholesterol-rich lipoproteins with these specialized plasma membrane regions using native lipoproteins visualized as discrete particles by standard electron microscopic techniques. In ovaries perfused with the various lipoproteins, spherical particles (varying in size from 12 to 28 nm depending on the particle used) were found over the surfaces of all luteal cells and filling up extensive "channel" space formed by the apposed plasma membranes of adjacent microvilli or cytoplasmic surfaces. Only 30% of these tissue-associated particles were removable after prolonged washing with perfused media or heparin. Few intact particles were found inside the cells, despite the fact that the lipoproteins induced a substantial hormone response by the ovary. To determine the total protein internalized by cells during the course of the experiments, parallel biochemical experiments were carried out with nonreleasable (14C-sucrose-coupled) human LDL. Of the total bound 14C-sucrose LDL, only 8.5% was degraded (trichloroacetic acid-soluble) and presumed internalized by the cells. Thus, while large numbers of cholesterol-rich lipoprotein particles interact with the luteal cell surface in specialized microvillar channels and elicit a progesterone response, relatively few intact lipoprotein particles appear to enter the cells to be degraded. We speculate that in the luteinized ovary, a large majority of the lipoprotein-cholesterol transfer occurs at the surface of the luteal cells, and that the membranes of the microvillar channels are involved in this process.

The fine structure of luteal tissue from sheep and goats at the mid-stage of the estrous cycle and during pregnancy was examined. At the mid-stage of the estrous cycle, when the corpus luteum of both species is secreting progesterone, the ultrastructure of the luteal cells is similar in that granule secretion is prevalent. In late pregnancy, the corpus luteum of the sheep produces very little progesterone and there is an absence of granule secretion. In the pregnant goat, the production of progesterone from the corpus luteum is high and there is plentiful evidence of granule formation and secretion.