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.

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.

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.

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.

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.

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.

Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model.

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.

Microtubular dynamics in granulosa cells of periovulatory follicles and granulosa-derived (large) lutein cells of sheep: relationships to the steroidogenic folliculo-luteal shift and functional luteolysis.

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.

Luteinization in primates is accompanied by loss of a 43-kilodalton adenosine 3',5'-monophosphate response element-binding protein isoform.

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.

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-alpha and interferon-gamma.

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 comparison of 17 beta-hydroxysteroid oxidoreductase type 1 and type 2 activity of cytosol and microsomes from human term placenta, ovarian stroma and granulosa-luteal 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.

Increased cellular cholesterol upregulates high density lipoprotein binding to rat luteal cells.

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.

[Effects of GTW and T4 from Tripterygium wilfordii Hook f. on LH cells in male rat pituitary glands].

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."

Ultrastructural study on experimentally induced ovarian tumors in the rat.

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.