Luteinizing Hormone Regulation of Inter-Organelle Communication and Fate of the Corpus Luteum.

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.

An objective volumetric method for assessment of ovarian follicular and luteal vascular flow using colour Doppler ultrasonography.

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 ±â€¯0.002 vs 0.030 ±â€¯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 ±â€¯0.008, P < 0.01; three-dimensional: 0.016 ±â€¯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.

Effect of fish oil on lateral mobility of prostaglandin F2α (FP) receptors and spatial distribution of lipid microdomains in bovine luteal cell plasma membrane in vitro.

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) F2α (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 PGF2α 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 PGF2α 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 PGF2α.

Differential cellular localization of galectin-1 and galectin-3 in the regressing corpus luteum of mice and their possible contribution to luteal cell elimination.

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.

Progesterone inhibits oxytocin- and prostaglandin F2alpha-stimulated increases in intracellular calcium concentrations in small and large ovine luteal cells.

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.

Follicle development and luteal cell morphology altered by phosphodiesterase-5 inhibitor.

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.

Examination of cyclic changes in bovine luteal echotexture using computer-assisted statistical pattern recognition techniques.

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.

Partial development of the steroidogenic ultrastructural features in degenerative corpora lutea after a single injection of pituitary extract in the Western painted turtle (Chrysemys picta).

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.

Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

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.

A quantitative study of blood capillary formation (angiogenesis) concomitant with parenchymal tissue differentiation.

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.

Granulosa cell expression of basal lamina matrices: Call-Exner bodies and focimatrix.

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.

Ovarian and placental production of progesterone and oestradiol during pregnancy in reindeer.

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.

Alpha B-crystallin in corpora lutea of pseudopregnant rat.

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.

Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries.

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.

Clinostat rotation induces apoptosis in luteal cells of the pregnant rat.

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.

Effects of second messengers on gap junctional intercellular communication of ovine luteal cells throughout the estrous cycle.

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.

The fate of corpora lutea in the cyclic golden hamster.

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.

Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat.

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.

Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.

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.

Co-localization of Rab3B and oxytocin to electron dense granules of the sheep corpus luteum during the estrous cycle.

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.