Luteinization of bovine granulosa cells and corpus luteum formation associated with loss of androgen-aromatizing ability.

The relative aromatizing ability of bovine luteinizing granulosa cells and dispersed luteal cells in tissue culture was studied. Luteinization of granulosa cells, as indicated by steadily increasing progesterone production (from 50 to 300 ng/10(5) cells/day over 4--5 days), was accompanied by a dramatic reduction in their capacity to aromatize exogenous androgen; oestradiol-17 beta production falling from 200 to less than 10 ng/10(5) cells/day over 4--5 days. Luteal cells also had only a very limited capacity to aromatize exogenous androgen, maximum oestradiol-17 beta production being less than 600 pg/10(5) cells/day. The loss in aromatizing capacity of granulosa cells during luteinization was also reflected in the relative endogenous steroid content of non-luteinized granulosa cells and luteal tissue, the former containing high levels of oestradiol-17 beta, less than or equal to 28 ng/mg protein, while the latter, although containing substantial amounts of testosterone, less than or equal to 5.7 ng/g tissue, contained very little oestradiol-17 beta, less than or equal to 0.35 ngG TISSUE. These findings suggest that luteinization of bovine granulosa cells and subsequent corpus luteum formation is associated with a loss in androgen aromatase activity.     

Inhibin production in vitro by granulosa cells from Booroola ewes which were either homozygous or non-carriers of a fecundity gene influencing their ovulation rate

The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10-13 of the oestrous cycle, from Booroola x Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3-4.5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P less than 0.05) than that by cells from non-atretic 3-4.5 mm diameter ++ follicles, but similar to that of cells from non-atretic greater than or equal to 5 mm diameter ++ follicles. The production of oestradiol-17 beta by cells cultured in the presence of testosterone (1 microgram/ml) followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17 beta production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17 beta synthesis, testosterone generally had a slight, stimulatory effect on inhibin production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oxytocin,vasopressin and vasotocin and their agonists on steroid secretion by bovine granulosa cells

The effects of oxytocin and vasopressin and their agonists on the secretion of progesterone and oestradiol-17β by bovine luteinised granulosa cells cultured in a serum-supplemented medium were analysed. The effects of oxytocin (OT), its long-acting agonist 2-0-methyl-tyrosin (deamino-karba)-oxytocin (DK-OT), arginine-8-vasopressin (AVP), 1-desamino-arginine-8-vasopressin (D-AVP, a vasopressin analogue with high antidiuretic and without vasopressor properties) and arginine-8-vasotocin (AVT) were investigated. It was found that OT and DK-OT had a stimulatory effect on progesterone release, while AVP, D-AVP and AVT had an inhibitory effect. All peptide hormones investigated significantly increased oestradiol-17β secretion. The results suggest the involvement of nonapeptide hormones of both oxytocin and vasopressin groups in the regulation of steroidogenesis by granulosa cells from bovine ovarian follicles.     

Functional anatomy of the ovaries of pregnant and lactating Cape porcupines, Hystrix africaeaustralis

The constituent cell types of the ovary of the porcupine were similar to those of New World hystricomorph rodents and accessory corpora lutea and luteal bodies were formed through the luteinization of the membrana granulosa or theca interna of antral follicles. All luteal bodies were histologically similar. The total volume of luteal tissue per female was not affected by fetal age and was unrelated to circulating concentrations of maternal plasma progesterone. Maternal plasma progesterone concentrations were correlated with fetal age. Follicular activity occurred throughout pregnancy but was not affected by fetal age or related to circulating values of oestradiol-17 beta. The formation of accessory corpora lutea during pregnancy is regarded as important in supplementing progesterone during pregnancy.     

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley-Liss, Inc.     

Influence of follicular health on the steroidogenic and morphological characteristics of bovine granulosa cells in vitro

In 24-h cultures, steroid production by cells from non-atretic follicles increased with increasing follicular diameter. Cells from atretic follicles, of all sizes, produced low amounts of oestradiol-17 beta, but very high amounts of progesterone, relative to cells from non-atretic follicles. Increasing the culture period to 72 h caused little change in daily progesterone and oestradiol-17 beta production by granulosa cells from atretic follicles. In contrast, in cells from non-atretic follicles, daily progesterone production increased and daily oestradiol-17 beta production decreased to the levels observed with cells from atretic follicles. Dibutyryl cyclic AMP (1.0 mM) significantly stimulated progesterone production by cells from atretic, but not from non-atretic, follicles. Testosterone (1 microgram/ml) had no effect on progesterone production by cells from atretic follicles, while oestradiol-17 beta, oestrone, testosterone, androstenedione and 5 alpha-dihydro-testosterone (0-1000 ng/ml) each significantly suppressed progesterone production by cells from non-atretic follicles in a dose-dependent manner. Morphometric analysis revealed few subcellular differences between cells from non-atretic and atretic follicles. Mean cell volume was significantly higher for cells from atretic compared to non-atretic follicles, but the mean volumes of the major subcellular components were not influenced by follicle health. The mean surface area of the plasma and nuclear membrane, and granular endoplasmic reticulum was also significantly higher in cells from atretic compared to non-atretic follicles.     

Hormonal control of uteroglobin secretion in rabbit uterus. Inhibition of uteroglobin synthesis and messenger ribonucleic acid accumulation by oestrogen and anti-oestrogen administration

Investigations were conducted to quantify activity of uteroglobin mRNA and secretion of uteroglobin in rabbit uterus after administration of progesterone and 5alpha-dihydrotestosterone, either alone or concomitantly with oestradiol-17beta and tamoxifen, a non-steroidal anti-oestrogen. Poly(A)-containing mRNA was isolated from the uterine tissue by extraction with phenol/chloroform, precipitation with ethanol and chromatography on oligo(dT)-cellulose. Cell-free translation in vitro of the poly(A)-containing mRNA was carried out in a wheat-germ lysate, and the product isolated by specific immuno-precipitation with anti-uteroglobin antiserum purified by affinity chromatography. Radioimmunoassay was utilized to determine uteroglobin content in the uterine flushings and tissue preparations. When given for 5 days, both progesterone (1mg/kg per day) and 5alpha-dihydrotestosterone (25mg/kg per day) elicited a marked induction of uteroglobin secretion, which was accompanied with accumulation of uteroglobin mRNA in the tissue. Concomitant administration of oestradiol-17beta (50mug/kg per day) or tamoxifen (12.5mg/kg per day) significantly decreased both progesterone- and 5alpha-dihydrotestosterone-induced uteroglobin secretion, with a parallel decrease in the uteroglobin-mRNA activity. The decline in the uteroglobin content of the uterine flushes brought about by oestradiol-17beta or tamoxifen administration was not due to inhibition of secretion of this protein by the endometrial cells, since a simultaneous decrease occurred in the tissue uteroglobin content. After a 5-day pretreatment with progesterone (1mg/kg per day), administration of oestradiol-17beta (50mug/kg per day) during the ensuing 4 days greatly accelerated the decay of the uteroglobin content in the uterine fluid.     

Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation

To determine whether inhibin and its related peptides might act locally to control granulosa cell function and differentiation, the dose- and time-dependent effects of bovine inhibin, the homo-dimer of the beta-chain of bovine inhibin (Activin-A) and porcine TGF beta on rat granulosa cell aromatase activity and progesterone synthesis were investigated in vitro. TGF beta enhanced FSH-induced aromatase activity and progesterone synthesis, and accelerated the peak response for progesterone synthesis. Activin-A on the other hand, augmented FSH-induced aromatase activity while arresting progesterone synthesis, and anti-luteinization effect. By contrast, exogenous inhibin had no detectable effect on the steroidogenic potential of these cells. Thus TGF beta and Activin, unlike their similar effects on the release of FSH by the pituitary, appear to affect ovarian granulosa cell function in different fashion, under conditions where inhibin itself has no effect.     

Inhibitory effect of progesterone on cell death of mouse uterine epithelium

The protective effect of progesterone against cell death of mouse uterine epithelium was evaluated by examining the retention of 5'-[125I]iodo-2'-deoxyuridine [( 125I]IdUrd) incorporated into the whole uterus and the apoptotic index (percentage of apoptotic cells in total cells), which is a good index of physiological cell death. Castrated adult female mice were given a daily injection of oestradiol-17 beta for 3 days, and then an injection of [125I]IdUrd. They were then divided into 4 groups, which received a daily injection of vehicle only, oestradiol-17 beta (E), progesterone (P), or both oestradiol-17 beta and progesterone (EP), and were killed at intervals during these treatments for determination of 125I radioactivity retained in the whole uterus. On treatment with vehicle only, the 125I radioactivity retained in the uterus decreased rapidly, but treatment with E, P or EP reduced the loss of 125I radioactivity significantly. Progesterone did not antagonize the effect of oestradiol-17 beta on the 125I radioactivity retained in the uterus. The apoptotic index of uterine cells was examined by a similar experimental protocol, but without injection of [125I]IdUrd. In the group treated with vehicle only, the apoptotic indices of both luminal and glandular epithelia increased markedly, but the injection of E, P or EP suppressed these increases significantly. Progesterone did not antagonize the effect of oestradiol-17 beta on the apoptotic index. The apoptotic index of stroma was not affected by the injection of E, P or EP. On the other hand, progesterone completely inhibited the increase in the mitotic index of uterine epithelia induced by oestradiol-17 beta. These results show that progesterone alone or in combination with oestrogen reduced cell death in mouse uterine epithelium and that the effects of oestrogen and progesterone on uterine cell death were independent of their actions on cell division.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Structure-function relationships during differentiation of normal and oncogene-transformed granulosa cells.

Granulosa cells, which nurse the oocyte, luteinize in response to the preovulatory surge of gonadotropins and become a major producer of progesterone during the normal estrous cycle and pregnancy. This process is characterized by a dramatic change in inter- and intracellular organization and modulation of gene expression that leads to enhanced steroidogenesis. Culturing of human, porcine, and rat cells has permitted detailed studies concerning the microenvironment (hormones, growth factors, and extracellular matrices) that control steroidogenesis in a coordinated fashion both in vivo and in vitro. Reorganization of the cytoskeleton--mainly characterized by down-regulation of the actin network, which is associated with changes in cell contacts and intercellular communication--seems to be a prerequisite for up-regulation of the steroidogenic enzymes. These processes can be investigated now in more detail due to the transfection of rat granulosa cells with specific oncogenes, which leads to their immortalization, concomitantly with the preservation of their capacity for inducible steroidogenesis. The effect of oncogene expression on granulosa cell differentiation suggests that different members of the ras oncogene superfamily may be involved in controlling development and luteinization of both normal and transformed granulosa cells. The identification of the specific forms of the ras protooncogene that may be involved in the differentiation of primary granulosa cells remains a challenging objective.     

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.     

Secretion of 17 alpha-hydroxyprogesterone, androstenedione, and estrogens by porcine granulosa and theca interna cells in culture

The steroid secreting activities of dispersed granulosa and theca interna cells from preovulatory follicles of prepubertal gilts 72 h after pregnant mare's serum gonadotropin treatment (750 IU) were compared. The cells were cultured for 24 h with or without steroid substrate (10(-8) to 10(-5) M progesterone, 17 alpha-hydroxyprogesterone, or androstenedione), FSH (100 ng/mL), LH (100 ng/mL), and cyanoketone (0.25 microM, an inhibitor of 3 beta-hydroxysteroid dehydrogenase). Granulosa cells cultured alone secreted mainly progesterone. Theca interna cells secreted mainly 17 alpha-hydroxyprogesterone and androstenedione, with secretion being markedly enhanced by LH. In the presence of cyanoketone, which inhibited endogenous progesterone production, theca interna but not granulosa cells were able to convert exogenous progesterone to 17 alpha-hydroxyprogesterone and androstenedione, and exogenous 17 alpha-hydroxyprogesterone to androstenedione and estradiol-17 beta in high yield. The secretion of the latter steroids from exogenous substrates was unaffected by LH. Theca interna cells secreted more estradiol-17 beta than did granulosa cells in the absence of aromatizable substrate, but estradiol-17 beta secretion by the latter was markedly increased after the addition of androstenedione. These apparent differences in steroid secreting activity between the cell types suggest that the enzymes responsible for conversion of C21 to C19 steroids, i.e., 17 alpha-hydroxylase and C17,20-lyase, reside principally in the theca interna cells. However, aromatase activity appears to be much higher in granulosa cells.     

The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells

To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor beta1 (TGFbeta1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFbeta1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFbeta1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFbeta1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFbeta1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFbeta1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.     

Inhibin production by bovine ovarian tissues in vitro and its regulation by androgens

No detectable amounts of inhibin were produced by cultured ovarian stroma or luteal tissue. Follicular tissue produced inhibin in vitro and removal of the granulosa cells from the follicle wall caused inhibin production to fall by 80%. Granulosa cells alone had the greatest ability of any ovarian cell type to produce inhibin in vitro, and are probably the major site of follicular inhibin production. Cyproterone acetate at concentrations of 35 and 350 microM inhibited basal and testosterone (3.5 microM)-stimulated inhibin production by cultured intact follicle wall and granulosa cells. In addition, each concentration of cyproterone acetate inhibited progesterone but not oestradiol-17 beta production by the follicle wall and granulosa cell cultures. The synthetic, non-aromatizable androgens, methylestrenolone and mesterolone, at concentrations of 5 and 25 microM, mimicked the effect of testosterone and stimulated granulosa cell inhibin production, methylestrenolone being the more potent. These findings provide further evidence that androgens regulate follicular inhibin and progesterone production and that these may be receptor-mediated processes, and suggest that inhibin production may be a general property of androgenic compounds. Preliminary examination of the physicochemical characteristics of inhibin indicated that the inhibin activity of bovine granulosa cell culture medium was (a) retained by an Amicon XM100A filter with a nominal molecular weight cut-off point of 100 000; and (b) destroyed by heating to 80 degrees C for 30 min.     

Secretion and reabsorption of uterine luminal fluid in rats

Treatment of ovariectomized rats with oestradiol-17beta and progesterone demonstrated that oestradiol-17beta causes secretion of sodium, potassium and water into the lumen of the uterine horn and that progesterone causes reabsorption of these substances.     

Secretion of fibronectin by rat granulosa cells occurs primarily during early follicular development

Rats were pretreated with oestradiol-17 beta or PMSG, treatments producing mainly preantral and antral follicles respectively. The granulosa cells from these two treatments, E2- and PMSG-cells respectively, were cultured for 2 successive 24-h periods. Basal progesterone secretion, stimulated 3- to 5-fold by FSH, was almost 8-fold higher by PMSG-cells than by E2-cells at 24 and 48 h. Similarly, PMSG-cells secreted 16-fold more 20 alpha-dihydroprogesterone than did E2-cells, in the absence of FSH, and 6- to 10-fold more of the 20 alpha-reduced metabolite in the presence of FSH. In contrast, fibronectin secretion by PMSG-cells was only about 60% and 30% that by E2-cells at 24 and 48 h, respectively. Fibronectin secretion by E2- and PMSG-cells was reduced in the presence of FSH at 24 and at 48 h of culture. While cells in all treatment groups underwent spreading during culture to become elongated and irregular in outline, elevated fibronectin secretion in vitro was accompanied by enhanced cellular spreading. At 24 and 48 h of culture respectively, the mean area occupied by E2-cells on the culture surface was x 2 and x 1.6 that by PMSG-cells. Coincident with its inhibitory effect on fibronectin secretion, FSH reduced the mean area occupied by E2- and PMSG-cells on the culture surface at both time intervals. These findings suggest that granulosa cell fibronectin secretion is a feature of early follicular development. It may be that the secretion of this adhesive glycoprotein by granulosa cells provides a pool of fibronectin which is used for basement membrane deposition during follicular growth.     

Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH

Granulosa cells from fully differentiated bovine follicles were cultured in serum-free medium for 4 days. At the end of culture, the number of viable cells was low (10-15% of cells plated on day one) and only progesterone secretion responded to FSH. Insulin increased the number of viable cells at the end of culture (ED50 # 70 ng/ml) and stimulated progesterone secretion (ED50 # 50 ng/ml); the secretion of oestradiol-17 beta over basal value was evident only for concentrations of 1000 and 10,000 ng/ml. FSH acted synergistically with insulin to modify steroid secretion. In the presence of 50 ng/ml of insulin, dose-response studies indicated that secretion of progesterone was maximal at 10 ng/ml of FSH and plateaud thereafter, while oestradiol output peaked at 2 ng/ml of FSH, decreasing at higher concentrations. When cells were seeded in wells precoated with fibronectin, a comparison with cells cultured on plastic showed an increase (30-40%) in the number of viable cells at the end of culture and in oestradiol secretion but a decrease in progesterone output. These results indicate that granulosa cells from large bovine follicles, cultured in a serum-free medium containing insulin, maintain their steroidogenic potency for at least 4 days. Moreover, they show that oestradiol and progesterone synthesis are differentially sensitive to FSH concentrations and that fibronectin increases oestradiol secretion in response to FSH.     

In-vitro zona-lytic activity in uterine fluid from ovariectomized mice treated with oestradiol-17 beta and progesterone

Mouse embryos collected before implantation were incubated in vitro for 24 h with fluid rinsed from the uteri of ovariectomized female mice injected with progesterone, oestradiol-17 beta + progesterone, oestradiol-17 beta + progesterone, or oestradiol-17 beta alone. Although none of the zonae was completely dissolved, those incubated in fluid from animals treated with oestradiol + progesterone were subsequently more soluble in sodium thiocyanate (NaSCN) than those incubated similarly in control buffer, indicating a sublytic change during the incubation with uterine washings. Zonae incubated in fluid from animals injected with either hormone alone did not undergo such a change.