Prolactin and somatotropin binding by granulosa cells of cows at different reproductive states

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3-5 to 6-10 mm, specific binding of 125I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data suggest that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1-2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.     

Radioreceptor and autoradiographic analysis of FSH, hCG and prolactin binding sites in primary to antral hamster follicles during the periovulatory period

As measured by radioreceptor assays, binding sites for FSH and prolactin were present at 09:00 h on the day of pro-oestrus in Stage 1-10 follicles (primary to antral) with prolactin receptors 3-6 times higher than FSH sites in Stages 1-3 (3 layers of granulosa cells). Specific binding sites for hCG were present in Stage 1 and 2 follicles (2 layers of granulosa cells) but thereafter their distribution was erratic and they were not consistently detectable until Stage 5, when thecal cells first appeared. Using topical autoradiography, specific binding for FSH was evident in Stage 1-4 follicles (4 layers granulosa cells) whereas specific hCG-binding was not. After the preovulatory gonadotrophin surges, by 21:00 h on pro-oestrus, FSH receptors declined in Stages 5-10, prolactin receptors fell in Stages 8 and 10 (small and large antral follicles) and hCG receptors were reduced in Stages 7 (start of antral cavity) to 10. On the morning of oestrus, for follicles from Stage 4 onwards, receptor numbers usually returned to levels found at 09:00 h on pro-oestrus. At oestrus, the few remaining Stage 10 follicles were all atretic and contained significantly reduced FSH and prolactin receptors but numbers of hCG binding sites comparable to those at 09:00 h of pro-oestrus. These results provide evidence of gonadotrophin receptors in small primary and secondary follicles which is consistent with increased DNA synthesis in small hamster follicles on the afternoon of pro-oestrus and on the morning and afternoon of oestrus. Periovulatory changes in gonadotrophin concentrations may therefore affect early stages of folliculogenesis.     

Prolactin and Somatotropin Binding by Granulosa Cells of Cows at Their Different Reproductive States

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3–5 to 6–10 mm, specific binding of ¹²⁵I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data indicate that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1–2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.     

Insulin-like growth factor (IGF) 2 stimulates steroidogenesis and mitosis of bovine granulosa cells through the IGF1 receptor: role of follicle-stimulating hormone and IGF2 receptor

Little is known regarding the role of insulin-like growth factor 2 (IGF2) and the regulation of the IGF2 receptor (IGF2R) during follicular development. Granulosa cells were collected from small (1-5 mm) and large (8-22 mm) bovine follicles and were treated with IGF2 for 1-2 days in serum-free medium, and steroid production, cell proliferation, specific (125)I-IGF2 binding, and gene expression were quantified. IGF2 increased both estradiol and progesterone production by granulosa cells, and cells from large follicles were more responsive to the effects of IGF2 than those from small follicles. Abundance of aromatase (CYP19A1) mRNA was stimulated by IGF2 and IGF1. The effective dose (ED(50)) of IGF2 stimulating 50% of the maximal estradiol production was 63 ng/ml for small follicles and 12 ng/ml for large follicles, and these values were not affected by FSH. The ED(50) of IGF2 for progesterone production was 20 ng/ml for both small and large follicles. IGF2 also increased proliferation of granulosa cells by 2- to 3-fold, as determined by increased cell numbers and (3)H-thymidine incorporation into DNA. Treatment with IGF1R antibodies reduced the stimulatory effect of IGF2 and IGF1 on estradiol production and cell proliferation. Specific receptors for (125)I-IGF2 existed in granulosa cells, and 2-day treatment with estradiol, FSH, or cortisol had no significant effect on specific (125)I-IGF2 binding. Also, FSH treatment of small- and large-follicle granulosa cells had no effect on IGF2R mRNA levels, whereas IGF1 decreased IGF2R mRNA and specific (125)I-IGF2 binding. Granulosa cell IGF2R mRNA abundance was 3-fold greater in small than in large follicles. These findings support the hypothesis that both IGF2 and its receptor may play a role in granulosa cell function during follicular development. In particular, increased free IGF1 in developing follicles may decrease synthesis of IGF2R, thereby allowing for more IGF2 to be bioavailable (free) for induction of steroidogenesis and mitogenesis via the IGF1R.     

Prolactin-binding activity of bovine granulosa cells on luteinization and exposure to somatotropin in vitro

The influence of luteinization and bovine somatotropin (ST, 5-50 ng/ml) during cultivation of bovine granulosa cells on their ability to bind [125I]-labeled bovine prolactin (PRL) was studied. On the second day of cultivation in serumfree medium, granulosa cells from immature antral follicles underwent spontaneous luteinization, in both the absence and presence of ST. The level of [125I]-PRL specific binding to cells increased after two days of cultivation, with a negative correlation being revealed between estradiol production by the cells and their PRL-binding activity. At the same time, the addition of ST to the culture medium had no effect on the level of [125I]-PRL specific binding to native and luteinizing granulosa cells. The findings suggest a stimulatory influence of the luteal differentiation process on the PRL-binding activity of bovine granulosa cells, this influence is independent of the action of ST.     

Porcine follicular fluid containing water-soluble LH/hCG receptor

Porcine follicular fluid has been shown to have a specific water-soluble receptor for human chorionic gonadotropin (hCG). The binding of [125I] hCG to follicular fluid is inhibited by unlabelled hCG, LH but not FSH, ACTH and GH. The binding of hormone to the receptor in follicular fluid is a saturable phenomenon and Scatchard analysis suggested that the receptor has high affinity to hCG with no changes as the follicle enlarges. In contrast, follicular fluid from large follicles (6-12 mm) has higher binding capacity (2.04 +/- 0.12 fmol/mg protein) than follicular fluid isolated from medium (3-5 mm) and small (1-2 mm) follicles (0.60 +/- 0.05 and 0.44 +/- 0.04 fmol/mg protein, respectively). With the aid of affinity chromatography on hCG-CNBr-Sepharose 6-B a homogeneous fraction with Mr about 65,000 as estimated by SDS-PAGE was isolated. Treatment of follicular fluid with several protein-modifying reagents changed interactions of [125I] hCG with both soluble receptor and that bound to granulosa cell membrane in the similar manner. The [125I] hCG binding capacity of follicular fluid represents about 9.5% of the total binding capacity of granulosa cells. Finally, soluble LH/hCG receptor is probably secreted actively by follicular cells into follicular fluid. Dead granulosa cells do not release receptor into follicular fluid or incubation medium.     

Receptors for insulin-like growth factor-I and tumor necrosis factor-alpha are hormonally regulated in bovine granulosa and thecal cells.

Mastitis induces release of tumor necrosis factor-alpha (TNFalpha) and has been linked with reduced reproductive performance. To further elucidate the role and mechanism of action of TNFalpha on ovarian cells, the effect of TNFalpha on insulin-like growth factor-I (IGF-I)-induced steroidogenesis and IGF-I binding sites in granulosa and thecal cells as well as the hormonal regulation of TNFalpha receptors were evaluated. Granulosa and thecal cells were obtained from small (1-5mm) and large (> or =8mm) bovine ovarian follicles, respectively, and cultured for 3-4 days. During the last 2 days of culture, cells were treated with various hormones and steroid production and specific binding of 125I-IGF-I and 125I-TNFalpha was determined. Two-day treatment with 30 ng/ml of TNFalpha decreased (P<0.05) IGF-I-induced estradiol production by granulosa cells and IGF-I-induced androstenedione production by thecal cells. Two-day treatment with 10 and 30ng/ml of TNFalpha decreased (P<0.05) specific binding of 125I-IGF-I to thecal cells, but had no effect on specific binding of 125I-IGF-I to granulosa cells, or on specific binding of 125I-IGF-II to thecal cells. TNFalpha did not compete for 125I-IGF-I binding to granulosa or thecal cells whereas unlabeled IGF-I suppressed 125I-IGF-I binding. Insulin inhibited (P<0.10) whereas FSH had no effect on the number of specific 125I-TNFalpha binding sites in granulosa cells. In contrast, LH increased (P<0.10) whereas insulin had no effect on specific 125I-TNFalpha binding sites in thecal cells. These results suggest that IGF-I and TNFalpha receptors in granulosa and thecal cells are regulated by hormones differentially.     

Ontogeny and cellular localization of 125I-labeled basic fibroblast growth factor and 125I-labeled epidermal growth factor binding sites in ovaries from bovine fetuses and neonatal calves.

We have recently reported that although specific 125I-FSH receptors are present in granulosa cells from primary and secondary follicles, gonadotropin responsiveness is very low in ovaries from bovine fetuses, which consist mainly of preantral follicles with few early antral follicles. It is well established that a number of polypeptide growth factors show pronounced mitogenic effects on follicular cells. Therefore, we have compared autoradiographically the ontogeny and cellular localization of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) binding activities to assess their possible involvement in the regulation of early follicular growth in fetuses and neonatal calves. Follicular growth was initiated around Day 180 of gestation in fetuses. 125I-bFGF binding values were high in granulosa cells from preantral follicles (mean +/- SEM, 7.8 +/- 1.1-9.8 +/- 0.7 grains/cell, 0.05-0.15-mm diam.) but decreased in early antral follicles (0.16-3.0 mm) to a constant level (5.7 +/- 1.2 grains/cell). Specific 125I-EGF binding values were low in preantral follicles but showed a 2.5- and 5.0-fold increase in both granulosa cells and the theca interna from antral I (0.16-0.5 mm) and antral II follicles (0.6-3.0 mm), respectively. In atretic follicles, 125I-bFGF specific binding values were high (10.4 +/- 0.8 grains/cell), whereas 125I-EGF binding levels were significantly reduced or absent. None of the radioligands tested bound significantly to primordial follicles. There was no age-related difference in any ligand binding to follicles of comparable size. These results provide novel evidence that bFGF, a potent mitogen, is involved in the regulation of granulosa cell function as early as the preantral stage in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Epidermal growth factor binding sites in porcine granulosa cells and their regulation by follicle-stimulating hormone.

Epidermal growth factor (EGF) modulates ovarian function, including folliculogenesis and steroidogenesis. We investigated the localization of EGF binding sites in the porcine ovary, and the effect of FSH on EGF binding to cultured granulosa cells. Autoradiographic study demonstrated that the binding sites for 125I-labeled mouse EGF in the porcine ovary were present in the granulosa and luteal cells, but not in the thecal cells. Porcine granulosa cells were collected by the needle aspiration method from small (1-2 mm) and medium-sized (3-5 mm) follicles. Scatchard analysis showed that a single class of the specific binding sites for EGF was present in the granulosa cells. The number of binding sites and the apparent dissociation constant were 5,540 binding sites/cell and 0.23 nM (medium-sized follicle), respectively. No significant difference was observed between small and medium-sized follicles. Granulosa cells were cultured for 48 h at 37 degrees C in medium alone or with increasing doses of ovine FSH (1-100 ng/ml). FSH treatment significantly increased EGF binding in a dose-dependent manner. In conclusion, it is suggested that the specific high affinity, low capacity binding sites for EGF are present in porcine granulosa cells, and that they are up-regulated by FSH.     

Ontogeny and cellular localization of 125I-labeled insulin-like growth factor-I, 125I-labeled follicle-stimulating hormone, and 125I-labeled human chorionic gonadotropin binding sites in ovaries from bovine fetuses and neonatal calves.

In a previous study we reported that ovaries from bovine fetuses, which consist mainly of preantral follicles with few antral follicles, are weakly responsive to gonadotropins (FSH and LH). Insulin-like growth factor-I (IGF-I) is known to enhance gonadotropin responsiveness in vitro, but there is a lack of consistent data on the involvement of IGF-I, FSH, and LH during early stages of folliculogenesis in cattle. In the study reported here, we assessed autoradiographically the ontogeny of 125I-gonadotropin and 125I-IGF-I binding activities during preantral and early antral stages in cattle. Follicular growth was initiated around Day 180 of gestation in fetuses. The density of 125I-FSH binding was high in granulosa cells from primary (mean +/- SEM 10.5 +/- 0.7 grains/cell, 0.05-mm diam.) and secondary follicles (10.8 +/- 0.8 to 13.6 +/- 1.2 grains/cell, 0.06-0.15 mm) but increased significantly (p < 0.05) in early antral follicles (18.2 +/- 1.1 grains/cell, 0.16-3.0 mm). Specific 125I-IGF-I binding levels were low in granulosa cells from preantral follicles, averaging 2.5 +/- 0.6-3.1 +/- 0.9 grains/cell. However, after antrum formation, the density of 125I-IGF-I binding increased significantly (p < 0.05) with follicular diameter in granulosa cells and was 5.7 +/- 0.7 and 9.1 +/- 0.6 grains/cell for antral I (0.16-0.5 mm) and antral II (0.6-3.0 mm) follicles, respectively. 125I-FSH and 125I-IGF-I binding densities were low in theca cells from preantral and early antral follicles as well as in the interstitial tissue and granulosa cells from atretic follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reciprocal modulation of regulatory action of prolactin and somatotropin on DNA synthesis in granulosa cells of cows

A comparative analysis of the individual and joint influence of bovine prolactin (BPRL) and bovine somatotropin (BST) on DNA synthesis in cultured bovine granulosa cells from follicles of 3-5 mm in diameter was conducted, and a possibility for regulation of BPRL binding to receptors on the cells by BST was examined. Despite a unidirectional growth-promoting action of BPRL and BST on granulosa cells, their joint mitogenic effect on the cells was not additive at low concentrations of BPRL. The addition of BST to the culture medium did not alter the biphasic character of dependence of DNA synthesis in the cells on BPRL concentration, but increased the maximum effective concentration of the latter. When culturing granulosa cells with BST, the absence of its influence on the level of BPRL specific binding to the cells was shown. This fact suggests that BST modulating action on the mitogenic effect of BPRL is not a result of a change in the number of free receptors for PRL on granulosa cells. The results obtained are discussed in relation to the notion of similarity of receptor functional properties and intracellular signal ways for PRL and ST.     

Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell

A cellular receptor for erythroid differentiation factor (EDF) was demonstrated by incubation of 125I-labeled EDF with rat follicular granulosa cell cultures. The specific binding of labeled EDF to the cells showed saturation; Scatchard analysis of binding data indicated a single class of receptors having Kd = 3.4 x 10(-10) M. A large excess of unlabeled EDF reduced labeled EDF binding almost completely, whereas similar doses of inhibin and transforming growth factor type beta, which are quite similar to EDF in protein structure and subunit organization, had no effect; EDF did not share receptors with those factors. EDF receptor (activin A receptor) expression was enhanced in granulosa cells cultured in the presence of follicle-stimulating hormone; follicle-stimulating hormone raised the number of EDF binding sites/cell from 13,000 to 96,000 without altering the binding affinity.     

The effect of periovulatory imbalance of prolactin on the expression of prolactin receptors in rat ovary cells

Using the technique of immunohistochemistry in combination with cytophotometry, we have studied the effect of periovulatory hyper- and hypoprolactinemia on the expression of prolactin receptors in various cell types of rat ovaries during early estrus. It has been shown that intense specific staining of oocytes is positively controlled by prolactin. The maximal intensity of specific staining was found in cells of the cumulus and the inner layer of granulosa cells in mature follicles; staining intensity gradually diminished towards the outer boundary cell layer. Postovulatory follicles are distinct from those mature follicles in which there was no ovulation in their more intense manifestation of prolactin receptors in cells of the inner layer and cumulus, as well as in increased positive staining (after prolactin administration) only in the granulosa layer cells closest to theca. In follicles which did not ovulate by the time of the early estrus, prolactin administration leads to a proportional growth of specific immunoreactivity in all cell layers of the granulosa. The administration of bromocryptin, an inhibitor of prolactin secretion, leading to a 10-fold decrease in the prolactin level in the blood, results in a twofold decrease in the intensity of specific staining of all cell layers of the granulosa in either type of follicle. Corpora lutea of the previous cycle have irregularly positioned luteocytes with weak and strong specific staining, the intensity of which is not changed in response to prolactin and diminishes slightly after the administration of bromocryptin. We conclude that the most intense changes in the content of prolactin receptors under the conditions of imbalance of this hormone during the periovulatory period are observed in those follicles where the oocyte did not ovulate by the time of early estrus.     

125I-labelled hCG binding characteristics in theca interna and other tissues from Romney ewes and from Booroola x Romney ewes with and without a major gene influencing their ovulation rate

Specific receptors for 125I-labelled hCG in ovarian follicle wall were located in the theca interna. No specific binding of 125I-labelled hCG was found in theca externa and/or stromal tissue. The kinetics of 125I-labelled hCG binding to theca interna followed second order kinetics with calculated association rate constants (ka +/- s.d.) of 1.57 +/- 0.16 X 10(6) and 0.57 +/- 0.02 X 10(6) litres mol-1 sec-1 at 37 degrees C and 22 degrees C respectively. Dissociation of specifically bound 125I-labelled hCG from theca interna was minimal at 37 degrees C and 22 degrees C. The binding of 125I-labelled hCG to theca interna could be displaced with PMSG, FSH-P and sheep LH but other sheep pituitary hormones and LH-releasing hormone showed little or no cross-reaction. The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hCG binding to theca interna did not differ between Romney ewes and Booroola x Romney ewes with and without the fecundity (F) gene on Day 10 of the oestrous cycle, during anoestrus or at 36 h after an injection of cloprostenol on Day 10 of the oestrous cycle. When the data for Day 10 and anoestrus were pooled, the median (range) Bmax and Kd values in non-atretic follicles (greater than or equal to 3 mm diameter) were 12.0 (5.1-23.5) fmol/mg protein and 0.10 (0.05-0.16) nM respectively. At 36 h after cloprostenol injection the respective median (range) Bmax and Kd values in non-atretic follicles (greater than or equal to 3 mm diam.) increased to 46.9 (28.4-70.3) fmol/mg protein and 0.23 (0.13-0.65) nM respectively. In corpora lutea the hCG binding characteristics were similar in all the above breeds/genotypes. On Day 10 of the cycle, the mean Bmax but not the mean Kd value was significantly higher (P less than 0.01) than the corresponding value at 36 h after cloprostenol injection. In granulosa cells, from follicles of greater than or equal to 5 mm diameter of Romney and Booroola x Romney (++) ewes and from follicles of greater than or equal to 3 mm diameter of Booroola x Romney (F+) ewes, the hCG binding characteristics were similar. In granulosa cells from smaller sized follicles from the above breeds/genotypes, no specific hCG binding was noted.     

The effect of a periovulatory prolactin imbalance on prolactin receptor expression in rat ovary cell

Using the technique of immunohistochemistry in combination with cytophotometry, we have studied the effect of periovulatory hyper- and hypoprolactinemia on the expression of prolactin receptors in various cell types of rat ovaries during early estrus. It has been shown that intense specific staining of oocytes is positively controlled by prolactin. The maximal intensity of specific staining was found in cells of the cumulus and the inner layer of granulosa cells in mature follicles; staining intensity gradually diminished towards the outer boundary cell layer. Postovulatory follicles are distinct from those mature follicles in which there was no ovulation in their more intense manifestation of prolactin receptors in cells of the inner layer and cumulus, as well as in increased positive staining (after prolactin administration) only in the granulosa layer cells closest to theca. In follicles which did not ovulate by the time of the early estrus, prolactin administration leads to a proportional growth of specific immunoreactivity in all cell layers of the granulosa. The administration of bromocryptin, an inhibitor of prolactin secretion, leading to a 10-fold decrease in prolactin level in blood, results in a twofold decrease in the intensity of specific staining of all cell layers of the granulosa in either type of follicle. Corpora lutea of the previous cycle have irregularly positioned luteocytes with weak and strong specific staining, the intensity of which is not changed in response to prolactin and diminishes slightly after the administration of bromocryptin. We conclude that the most intense changes in the content of prolactin receptors under the conditions of imbalance of this hormone during the periovulatory period are observed in those follicles where the oocyte did not ovulate by the time of early estrus.     

Relationships between luteinizing hormone, follicle-stimulating hormone and prolactin secretion and ovarian follicular development in the weaned sow

Folliculogenesis was studied by assessing development of the largest 10 follicles obtained from 10 sows 48 h after weaning and by analyzing changes in plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) for 24 h before weaning until 48 h after weaning. Follicular diameter, follicular fluid volume, and concentrations of estradiol and testosterone and granulosa cell numbers were determined in all follicles, and 125I-hCG binding to theca and granulosa and maximal aromatase activity in vitro was determined in five follicles/sow. Overall, a significant rise in LH, but not in FSH, occurred at weaning, although in individual sows an increase in LH was not necessarily related to subsequent estrogenic activity of follicles. In 9/10 sows, PRL fell precipitously after weaning. In lactation, LH was negatively, and after weaning, positively, correlated with FSH and PRL. Marked variability in follicular development existed within and between sows. Overall, most follicular characteristics were positively correlated to follicular diameter; however, in larger follicles the number of granulosa cells was variable and unrelated to estrogenic activity, which--together with theca and granulosa binding of hCG--increased abruptly at particular stages of follicular development. Differences in maturation of similarly sized follicles from different sows were related to estrogenic activity of the dominant follicles but not to consistent differences in LH, FSH or PRL secretion. Both the dynamics and the control of folliculogenesis in the sow, therefore, appear to be complex.     

Binding characteristics of 125I-labelled human FSH to granulosa cells from Booroola ewes which were homozygous, heterozygous or non-carriers of a major gene(s) influencing their ovulation rate

At 37 degrees C 125I-labelled human (h) FSH (NIAMDD-hFSH-I-3) bound rapidly to granulosa cells from Booroola and Romney ewes with 50% maximum binding achieved after 3 min and equilibrium being reached within 45 min, irrespective of whether the cells were obtained from the FF, F+ or ++ Booroola genotypes or from Romney ewes. Binding of 125I-labelled FSH followed second order kinetics and there was no effect of follicle diameter (1-2.5 mm vs greater than or equal to 3 mm). Irrespective of breed, genotype or follicle size, the mean (+/- s.e.m.) calculated association rate constant, (ka) was 7.3 (+/- 0.8) x 10(5) litres mol-1 sec-1 (n = 12). Dissociation of receptor bound 125I-labelled hFSH was less than 5% after 30 min and low but variable (i.e. between 0 and 30%) after 2-6 h irrespective of breed, genotype or follicle size. No gene-specific differences were noted in binding specificity between F+ and ++ genotypes: studies were not performed with cells from FF ewes because of insufficient cells. The binding of 125I-labelled hFSH could be displaced with sheep FSH (NIH-FSH-S16; 10% cross-reaction) and FSH-P (2.5% cross-reaction) but other sheep pituitary hormones and hCG showed little or no cross-reaction (less than or equal to 0.1%). The calculated binding capacities (Bmax) and equilibrium dissociation constants (Kd) for 125I-labelled hFSH binding to granulosa cells did not differ between the Booroola genotypes or between Booroola or Romney follicles of different diameter (i.e. 1-2.5 mm; or greater than or equal to 3 mm). The overall mean +/- s.e.m. (n = 24) Bmax and Kd values were 16.7 +/- 0.8 fm/mg protein (i.e. approximately 800 available receptor binding sites/cell) and 1.1 +/- 0.1 nM respectively. Collectively, these findings suggest that the earlier maturation of follicles in FF or F+ ewes compared to ++ ewes is unlikely to be due to gene-specific differences in the FSH binding characteristics of the granulosa cells.     

A granulosa cell differentiation-stage-specific cytotoxic activity in bovine and rat sera

Heat-inactivated serum is cytotoxic to granulosa cells from preantral follicles but not to cells from preovulatory follicles. A dominant feature of the granulosa cells of preovulatory follicles is the presence of luteinizing hormone (LH) receptors on the surface of the cells. In the present study, we have examined the relationship between the process of LH receptor induction and the acquisition of serum tolerance in granulosa cells in vitro. Granulosa cells from the ovaries of immature rats primed with diethylstilbestrol (DES) were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium containing 30 ng of ovine follicle-stimulating hormone (oFSH; NIH-15). At either 0, 24, or 48 h of culture, heat-inactivated fetal bovine serum (FBS) was added (10% by volume) to separate groups of culture tubes. All cells were cultured for a total of 72 h, at which time the cultures were assessed for LH receptor (specific 125I-human chorionic gonadotropin [hCG] binding) and DNA content. LH receptors were induced in all FSH-containing serum-free cultures by 48 h. Receptors were not induced, however, when serum was added after either 0 or 24 h of culture. Furthermore, serum addition at these times resulted in a cell loss (assessed by DNA) of 40-60%. Serum addition at 48 h to FSH-containing cultures resulted in an inability to detect LH receptors at 72 h and with no significant effect on the culture DNA content. Addition of a protein extract of FBS at the initiation of cell culture prevented FSH-stimulated LH receptor induction and was cytotoxic. A lipid extract of FSH did not interfere with receptor induction and was not cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Somatotropin- and Prolactin-Binding Sites on Bovine Granulosa Cells Using Homologous Hormones

Specific binding of bovine somatotropin (BST) and bovine prolactin (BPRL) to cow granulosa cells from antrumcontaining follicles of different diameter was studied. Scatchard analysis of the data revealed a single type of low affinity BST-binding sites on the granulosa cells with dissociation constants similar to those for the BPRL-binding sites. The number of BST-binding sites on the cells decreased with increasing follicle diameter from 3-5 to 6-10 mm. However, the binding capacity to BPRL decreased only in the case of cells from follicles 11-20 mm in diameter. The findings are discussed in relation to the homologous binding phenomenon.