Uptake and utilization of lipoprotein cholesteryl esters by rat granulosa cells

Earlier studies have shown that rat granulosa cells grown in serum-free medium are exquisitely responsive to exogenously provided lipoprotein cholesterol. In this study we compare the amount of cholesterol (cholesteryl ester) actually delivered from various homologous and heterologous cholesterol-rich lipoproteins and examine the intracellular pathways used in the delivery system. Granulosa cells were incubated for 5 or 24 h with 125I-labeled human (h) HDL3, rat (r) HDL or hLDL equipped with non-releasable apoprotein and cholesteryl ether tags which accumulate within cells, even after degradation. We show that all the tested lipoproteins were similarly efficient in cholesteryl ester delivery; i.e., based on cholesterol: protein ratios of the starting ligands, each delivered approximately the same cholesteryl ester mass and evoked a similar progestin response. However, each lipoprotein was processed quite differently by the granulosa cells: hHDL3-cholesteryl ester was taken up almost exclusively by an non-endocytic pathway, hLDL-cholesteryl ester almost exclusively by an endocytic pathway and rHDL-cholesteryl ester by both pathways. In general, there was no correlation between the total amount of lipoprotein bound or apoprotein internalized and/or degraded by the cells with the amount of cholesteryl ester received or the level of the progestin response. Hormone stimulation upregulated the preferred pathway for each lipoprotein.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Summary Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B. This investigation was supported by PHS Grant CA32444, awarded by the National Cancer Institute. A. R. L. G. is a recipient of a USPHS fellowship, GM09226-01, and S. M. T. was supported by NIH training Grant AMO 7282.     

Androgen aromatization by luteinized bovine granulosa cells in tissue culture

Luteinized bovine granulosa cells in tissue culture contained an active 19-hydroxylase aromatase enzyme system which converted exogenous androstenedione and testosterone to oestradiol-17beta; no oestrone was detected. In the absence of exogenous androgens, the cells failed to synthesize oestrogens due to a limited capacity to synthesize androgen precursor. Theca-lutein cells, present in those CL which synthesize oestrogens, may provide androgen precursor for aromatization by the granulosa-lutein cells.     

Androgens amplify beta-adrenergic and FSH stimulation of granulosa cells

The effects of androgens on granulosa cell stimulation by isoproterenol and follicle-stimulating hormone (FSH) were determined. Two functional parameters of granulosa cell stimulation were monitored: (a) activity of a transfected proopiomelanocortin (POMC) promoter and (b) production of progesterone. Treatment with the beta-adrenergic agonist, isoproterenol, stimulated steroidogenesis, and both isoproterenol and FSH appeared to enhance POMC promoter activity. The non-aromatizable androgen, 5 alpha-dihydrotestosterone (DHT), produced no effect on either parameter, but it potentiated the steroidogenic response to isoproterenol. Preliminary data also indicated a potentiation by DHT of the FSH-mediated increase in POMC promoter activity; results with a combination of DHT and isoproterenol were suggestive of potentiation. A possible role for androgen amplification of adrenergic stimulation in polycystic ovarian syndrome is discussed.     

Prolactin, LH, and estradiol-17 beta in utilization of lipoprotein substrate by porcine granulosa cells in vitro

Porcine granulosa cells cultured under serum free conditions responded by increased progesterone secretion to the addition of the leuteotropic hormones, LH, prolactin, and estradiol. Provision of extracellular substrate for steroidogenesis in the form of porcine high density lipoprotein or low density lipoprotein enhanced progesterone accumulation by granulosa cell cultures. Estradiol, LH, and prolactin all greatly increased progesterone accumulation in the presence of either high or low density lipoproteins. Increases in progesterone accumulation following addition of prolactin or LH in combination with estradiol suggested the presence of a synergistic interaction among leuteotropins. Pre-exposure of granulosa cell cultures to estradiol increased the subsequent stimulatory effect of prolactin on lipoprotein utilization. It is concluded that all three leuteotropins function to enhance and may interact in the utilization of extracellular lipoprotein substrate for progesterone synthesis.     

Androgen inhibition of FSH-stimulated progesterone production by granulosa cells of prepubertal pigs

Androgens have been reported to stimulate progesterone production by granulosa cells of several species, and to act synergistically with FSH in stimulation of progesterone accumulation by rat granulosa cells. Studies were undertaken to examine the effect of androgens on FSH-stimulated progesterone production in culture by granulosa cells derived from prepubertal pig ovaries. When included in 24-h culture with FSH, both androstenedione and testosterone caused a reduction in progesterone accumulation, but dihydrotestosterone and androsterone did not. Granulosa cells were cultured for 24 h with FSH and [14C]progesterone with or without testosterone; testosterone did not affect the rate of overall metabolism of [14C]progesterone and it was therefore concluded that testosterone inhibited progesterone synthesis, rather than enhancing its catabolism. 17 beta-Estradiol also inhibited FSH-stimulated progesterone accumulation. To determine whether the action of testosterone was mediated by conversion to estradiol, granulosa cells were cultured with FSH and testosterone with or without an aromatase inhibitor (4-acetoxy-androstenedione). The aromatase inhibitor failed to prevent the testosterone-induced reduction in progesterone accumulation, although it markedly inhibited estradiol accumulation. These results indicate that theca-derived androgens can inhibit FSH-stimulated progesterone production by granulosa cells in the prepubertal pig, independently of estradiol.     

Hormone-induced desensitization of cultured rat granulosa cells to FSH

Monolayers of granulosa cells (GC) derived from immature hypophysectomized diethylstilbestrol-treated rats became refractory in terms of FSH-stimulable cyclic AMP production following exposure to the homologous hormone. In the presence of ovine FSH (5 μg/ml), maximal refractoriness was attained after 4 h of incubation. Upon removal of the FSH from the medium, the cells regained their full responsiveness within 24 h. The extent of desensitization was dependent upon the dose of FSH, and could not be overcome by increasing the dose of the hormone during the challenge period. Exposure of GC monolayers for 2–4 h to the protein synthesis inhibitors actinomycin D (8 μg/ml) and cycloheximide (5 μg/ml) on their own enhanced FSH-stimulable cyclic AMP production. When added together with FSH, the inhibitors did not prevent the process of desensitization to the hormone. The results suggest that the initial phases of FSH-induced desensitization do not require de novo protein synthesis.     

Identification and expression analyses of BAMBI mediated by FSH in swine luteinizing granulosa cells

Transforming growth factor-β and related growth factors are essential regulators for the development of follicles. Bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI) was reported as a key factor participating in the transforming growth factor-β signal pathway. To investigate the role of BAMBI in porcine granulosa cells, the full length of the BAMBI was cloned from porcine ovarian cDNA. The results of bioinformatics analyses showed that the signaling peptide was located in between positions 20 and 21. The results of online prediction on phosphorylation sites indicate that the sites of Ser, Thr, and Tyr are 9, 1, and 1, respectively. In addition, BAMBI was highly homologous in rodent and livestock. Real-time quantitative polymerase chain reaction (qPCR) indicated that BAMBI was widely expressed in porcine tissues. Immunofluorescence showed that BAMBI was located in both nucleus and cytoplasm. Stimulating the granulosa cells with FSH in vitro could alter BAMBI expression level in a time-dependent manner. Moreover, the expression level declined after treatment with FSH. These results indicated that BAMBI is an FSH-repressed gene in porcine luteinizing granulosa cells and it may be involved in the regulation of ovarian follicle development and oocyte maturation.     

Androgen actions and the ovary

Although androgens and the androgen receptor (AR) have defining roles in male reproductive development and function, previously no role in female reproductive physiology beyond testosterone (T) as the precursor in estradiol (E(2)) biosynthesis was firmly established. Understanding the role and specific mechanisms of androgen action via the AR in the ovary has been limited by confusion on how to interpret results from pharmacological studies, because many androgens can be metabolized in vivo and in vitro to steroids that can also exert actions via the estrogen receptor (ESR). Recent genetic studies using mouse models with specific disruption of the Ar gene have highlighted the role that AR-mediated actions play in maintaining female fertility through key roles in the regulation of follicle health, development, and ovulation. Furthermore, these genetic studies have revealed that AR-mediated effects influence age-related female fertility, possibly via mechanisms acting predominantly at the hypothalamic-pituitary axis in a dose-dependent manner. This review focuses on combining the findings from pharmacological studies and novel genetic mouse models to unravel the roles of ovarian androgen actions in relation to female fertility and ovarian aging, as well as creating new insights into the role of androgens in androgen-associated reproductive disorders such as polycystic ovarian syndrome.     

Synthesis of lipoprotein lipase in cultured avian granulosa cells

Avian granulosa cells cultured as a homogeneous parenchymal population contain lipolytic activity. This activity is stimulated 2--5-fold by serum, inhibited 90% by 1 M NaCl and inhibited 80% by specific anti-lipoprotein lipase immunoglobulins. 85% of the activity binds to heparin-Sepharose 4B, and 70% of bound activity is eluted with 1.5 M NaCl. Thus, the lipolytic activity of cultured granulosa cells is lipoprotein lipase. Granulosa cells were shown to synthesize lipoprotein lipase in culture by incorporating [3H]leucine into the enzyme protein, as measured with an immunoadsorption technique. Finally, colchicine was shown to increase intracellular lipolytic activity, suggesting an inhibition of secretion of this enzyme by cultured granulosa cells.     

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.     

Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy

Oxidative stress-induced granulosa cell (GCs) death represents a common reason for follicular atresia. Follicle-stimulating hormone (FSH) has been shown to prevent GCs from oxidative injury, although the underlying mechanism remains to be elucidated. Here we first report that the suppression of autophagic cell death via some novel signaling effectors is engaged in FSH-mediated GCs protection against oxidative damage. The decline in GCs viability caused by oxidant injury was remarkably reduced following FSH treatment, along with impaired macroautophagic/autophagic flux under conditions of oxidative stress both in vivo and in vitro. Blocking of autophagy displayed similar levels of suppression in oxidant-induced cell death compared with FSH treatment, but FSH did not further improve survival of GCs pretreated with autophagy inhibitors. Further investigations revealed that activation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling pathway was required for FSH-mediated GCs survival from oxidative stress-induced autophagy. Additionally, the FSH-PI3K-AKT axis also downregulated the autophagic response by targeting FOXO1, whereas constitutive activation of FOXO1 in GCs not only abolished the protection from FSH, but also emancipated the autophagic process, from the protein level of MAP1LC3B-II to autophagic gene expression. Furthermore, FSH inhibited the production of acetylated FOXO1 and its interaction with Atg proteins, followed by a decreased level of autophagic cell death upon oxidative stress. Taken together, our findings suggest a new mechanism involving FSH-FOXO1 signaling in defense against oxidative damage to GCs by restraining autophagy, which may be a potential avenue for the clinical treatment of anovulatory disorders.     

Binding of LH and FSH to porcine granulosa cells during follicular maturation.

The uptake of 125I-labelled LH by equal numbers of granulosa cells from small, medium or large follicles was greater by cells from large follicles. In contrast, granulosa cells obtained from small follicles bound much more 125I-labelled FSH per cell than did cells obtained from medium and large follicles. Competition studies with unlabelled hormones indicated that porcine granulosa cells have specific receptors for LH and FSH. The addition of diethylstilboestrol enhanced the binding of 125I-labelled LH and inhibited the binding of 125I-labelled FSH to granulosa cells harvested from small and medium-sized follicles, but had no effect on those from large follicles.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

FSH induced stimulation of catalase activity in goat granulosa cells in vitro

Reactive oxygen species scavenging enzymes like catalase play diverse role in mammals. The presence of catalase in mammalian ovary is now well established. In the present investigation, changes in catalase activity in granulosa cells isolated from follicles at various stages of differentiation in response to FSH were studied. The follicles were dissected out from goat ovaries and classified as small (<3 mm), medium (3–6 mm) or large (>6 mm). Granulosa cells were isolated from categorized follicles. Results showed that there was a three-fold increase in catalase activity in granulosa cells from large follicles as compared to small and medium follicles. The catalase activity was stimulated significantly when granulosa cells were treated with FSH in vitro. The minimum effective dose that could stimulate catalase activity and estradiol secretion in case of granulosa cells from small and medium sized follicles was 100 ng/ml; for larger follicles, this value was 200 ng/ml. Concomitant to the increase in catalase activity, the estradiol secretion was significantly enhanced when cultured goat granulosa cells were treated with FSH. It was concluded that enzyme catalase may have a functional role in goat ovarian follicular development under endocrine regulation.