Fibroblast growth factor 18 regulates steroidogenesis in fetal bovine ovarian tissue in vitro.

In cattle and other species, the fetal ovary is steroidogenically active before follicular development commences, and there is evidence that estradiol and progesterone inhibit follicle formation and activation. Estradiol levels decline sharply around the time of follicle formation. In the present study, we hypothesized that FGF10 and FGF18, which inhibit estradiol secretion from granulosa cells of antral follicles, also regulate fetal ovarian steroid production. Fetuses were collected at local abattoirs, and age determined by crown-rump length measurements. Real-time polymerase chain reaction assays with RNA extracted from whole ovaries revealed that the abundance of CYP19A1 messenger RNA (mRNA) decreased from 60 to 90 days of gestation, which is consistent with the decline in estradiol secretion previously observed. Immunohistochemistry revealed the presence of FGF18 in ovigerous cords in early gestation and in oocytes later in fetal age (≥150 days). The abundance of FGF18 mRNA increased after Day 90 gestation. Addition of recombinant FGF18 to fetal ovarian pieces inhibited estradiol and progesterone secretion in vitro, whereas FGF10 was without effect. Consistent with these results, FGF18 decreased levels of mRNA for CYP19A1 and CYP11A1 in ovarian pieces in vitro. These data suggest that FGF18 may be an intraovarian factor that regulates steroidogenesis in fetal ovaries.

Evidence that fibroblast growth factor 10 plays a role in follicle selection in cattle.

There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection.

Fibroblast growth factor-2 regulation of Sprouty and NR4A genes in bovine ovarian granulosa cells.

Fibroblast growth factors (FGFs) alter ovarian function, at least in part by inhibiting steroid hormone secretion and affecting survival of granulosa cells. The mechanism of action of FGFs in ovarian follicle cells is largely unknown; in the present study we identified the major pathways used by FGF2 in non-luteinizing granulosa cells cultured under serum-free conditions. FGF2 increased abundance of mRNA encoding SPRY1, 2, and 4, but not SPRY3. Common pathways employed by FGF2 in the regulation of SPRY1, 2, and 4, as demonstrated by immunoblot and inhibitor studies, included ERK1/2 and Akt signaling. In contrast, PKC activation was necessary for FGF2-stimulated expression of SPRY1 and 4, but not for SPRY2. Intracellular calcium flux is critical and sufficient for SPRY2 expression, but not for SPRY1 and 4. We also identified the orphan nuclear receptor NR4A1 as a potential early response gene in FGF2 signaling, whose expression, like that of SPRY2, is critically dependent on calcium signaling. Together, these data identify FGF2-target genes in follicular granulosa cells, and demonstrate alternative pathway use for the differential control of SPRY genes.

Regulation and action of fibroblast growth factor 17 in bovine follicles.

Fibroblast growth factor 17 (FGF17) is a member of the FGF8 subfamily that appears to be relevant to folliculogenesis and oogenesis, as the prototype member FGF8 is an oocyte-derived protein that signals to cumulus cells. FGF8 has structural and receptor-binding similarities to FGF17, whose expression in the ovary has not been reported. In this study, we demonstrate localization of FGF17 protein to the oocyte of preantral follicles, and to the oocyte and granulosa cells of antral follicles. Real-time PCR demonstrated the presence of mRNA in oocytes and, to a lesser extent, in granulosa and theca cells. FGF17 mRNA abundance was low in granulosa and theca cells from healthy follicles and increased significantly in atretic follicles. Addition of FSH or IGF-I to granulosa cells in vitro decreased FGF17 mRNA abundance, and treatment with FGF17 inhibited estradiol and progesterone secretion from granulosa cells in relation to control cultures without these additives. We conclude that FGF17 is a potential mediator of granulosa cell differentiation.

Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B, in bovine follicles.

Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro.

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.

Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles.

Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.

Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro.

The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence.

Role of FSH, numbers of FSH receptors and testosterone in the regulation of inhibin secretion during the seasonal testicular cycle of adult rams.

The regulation of inhibin secretion has not been elucidated fully in male ruminants. The aim of this study was to determine the relative importance of FSH and testosterone concentrations, and FSH receptors, in the control of secretion of immunoactive inhibin in rams. In Expt 1, temporal changes in hormone concentrations and testicular FSH binding were determined for two groups of rams (n = 4) kept under opposite, alternating 4 month periods of long (16 h light:8 h dark) and short (8 h light:16 h dark) days. Testicular biopsies (1-2 g) were collected when the testes were regressed, redeveloping, redeveloped and regressing. In Expt 2, separate groups of rams (n = 4) kept under natural photoperiod (latitude 45 degrees 48 minutes N) were designated as controls or passively immunized (for 3 weeks) with sufficient oestradiol antiserum to increase testosterone secretion without altering LH and FSH; this was done when the testes were regressed (non-breeding season) and redeveloped (breeding season). In both groups of rams (Expt 1), 'seasonal' increases in FSH concentrations began a few weeks earlier than did increases in inhibin concentrations. FSH reached maximum concentrations during testicular recrudescence, whereas numbers of FSH receptors in the testis and circulatory inhibin concentrations did not reach peak values until the testes were fully developed. Numbers of FSH receptors per testis, but not FSH concentration, were positively correlated (r = 0.65) with inhibin concentrations across the four stages of the testicular cycle. Near the end of testicular recrudescence early in the breeding season (Expt 2), relatively high FSH concentration was associated with increased abundance of FSH receptor mRNA (90%) and number of receptors (45%) in the testis and increased inhibin concentrations (50%), compared with when the testes were regressed. Moderate, physiological increases in testosterone secretion in immunized rams did not affect inhibin in either season. These results indicate that: (i) FSH stimulation of immunoactive inhibin secretion by Sertoli cells as testes recrudesce is via increases in secretion (early) and cognate receptors (late); (ii) FSH upregulates the synthesis of its own receptor late in recrudescence; and (iii) the positive correlation (r = 0.70) observed between circulatory testosterone and immunoactive inhibin does not reflect a causal relationship.

Insulin alters the effects of follicle stimulating hormone on aromatase in bovine granulosa cells in vitro.

It is known that follicle-stimulating hormone (FSH) and insulin stimulate estradiol secretion from cultured non-luteinizing granulosa cells. The interaction between these hormones is less well understood. Granulosa cells from small (2-4 mm) bovine follicles were cultured in serum-free medium to determine if cytochrome P450 aromatase activity is regulated by FSH in the presence of different concentrations of insulin. Insulin significantly stimulated aromatase activity in the absence of FSH. There was a significant interaction between insulin and FSH on aromatase activity, such that FSH stimulated activity at low (0.5, 1 and 10 ng/ml) doses of insulin, whereas at higher (100 ng/ml) doses of insulin FSH failed to stimulate aromatase activity. To determine if the lack of a response to FSH with higher doses of insulin is related to gene expression, the effect of FSH on P450 aromatase mRNA levels was measured. An 'uncoupling' of mRNA and enzyme activity was observed for cells cultured with 100 ng/ml insulin, as FSH significantly increased P450 aromatase mRNA abundance without affecting estradiol secretion or aromatase activity. We conclude that in the presence of high doses of insulin, FSH decreases aromatase activity, and an uncoupling of P450 aromatase mRNA and aromatase activity occurs. This may have implications for infertility treatments when there is a risk of hyperinsulinemia.

Decreased LH pulsatility during initiation of gonadotropin superovulation treatment in the cow: evidence for negative feedback other than estradiol and progesterone.

LH pulse secretion is suppressed during superovulation of cattle. The objective of this study was to determine how soon after initiation of superovulation treatments this suppressive effect occurs, and to test the hypothesis that decreased LH pulsatility is not related to changes in circulating estradiol or progesterone. Heifers (n = 7/group) were injected with eCG (FOLLIGON: a single injection of 2,500 IU) or twice daily injections of decreasing doses of FOLLTROPIN-V (total equivalent of 280 mg of NIH-FSH-P1) or F.S.H.-P (total equivalent of 28 mg of Armour standard) or saline (time controls), starting on Day 10 (Day 0 = estrus). Blood samples were taken every 10 min for 12 h intervals on the day prior to first injection, at 8 to 20 h and 32 to 44 h after initiation of gonadotropin treatment, and also during prostaglandin (PG)-induced luteolysis. A simple method based on robust statistics and on graphical representations of time series was developed to characterize LH pulses. There was a significant interaction between time and treatment for mean LH, estradiol and progesterone when control and treated groups were analyzed together, and no interaction when only the gonadotropin groups were analyzed together. When compared to pretreatment values, pulse frequency of LH was significantly reduced (P<0.05) in each treatment group, 8 to 20 h and 32 to 44 h following initiation of gonadotropin treatment. Mean LH concentrations were also reduced 32 to 44 h following initiation of treatments (P<0.05). Mean estradiol concentrations increased two to threefold at 8 to 20 h following initiation of superovulation treatments (P<0.05). Progesterone concentrations also increased by 20 or 44 h. There was no significant correlation between estradiol or progesterone and LH pulse frequency, amplitude and mean concentrations at any time in control or superovulated animals. This study demonstrates that superovulation treatment in the cow causes a rapid decrease in pulsatile release of LH and suggests that this effect is not mediated through the negative feedback actions of estradiol and progesterone.

Effect of growth factors and co-culture with ovarian medulla on the activation of primordial follicles in explants of bovine ovarian cortex.

It has been proposed that the ovarian medulla exerts an intra-ovarian inhibitory effect on primordial follicle activation in cattle. We tested this hypothesis using cortical ovarian explants and determined whether growth factors could alter follicle activation or primary follicle health. Ovaries were obtained from bovine fetuses, and cortical slices were cultured on Millicell culture inserts for up to 8 days. Within 2 d of culture, the proportion of primordial follicles decreased from 70.1 +/- 3.5 to 6.4 +/- 3.4% (P<0.05), and the proportion of primary follicles increased from 23.8 +/- 3.3 to 79.7 +/- 5.5% (P<0.05). The proportion of secondary follicles was relatively stable (6 to 13%). Morphological examination indicated that 91.9 +/- 3.7, 76.7 +/- 8.8, and 71.8 +/- 10.4% of primordial, primary, and secondary follicles, respectively, were considered to be healthy in slices of fresh tissue; these proportions were not altered by up to 8 d of culture (P>0.05). The proportion of all classes of follicles and their morphological health were not affected by the addition of medullary slices to the culture well, nor by the culture of corticomedullary slices (P>0.05). The addition of FSH, insulin-like growth factor-I, epidermal growth factor, basic fibroblast growth factor, or transforming growth factor-beta did not alter primordial follicle activation or the morphological health of primary or secondary follicles. The addition of transforming growth factor-alpha (TGFalpha) decreased the proportion of primary follicles that were healthy from 67.6 +/- 5.1 to 36.8 +/- 4.7% (P<0.05). In conclusion, these data do not support the existence of a medullary inhibitor of primary follicle activation but suggest a role for TGFalpha in the regulation of primary follicle development.

Influence of season and low-level oestradiol immunoneutralization on episodic LH and testosterone secretion and testicular steroidogenic enzymes and steroidogenic acute regulatory protein in the adult ram.

The regulation of LH-dependent and -independent increases in testosterone secretion by key proteins in the testes of adult rams was investigated. Serial blood samples were collected from groups of four control and passively immunized (oestradiol antiserum for 3 weeks) rams and the animals were gonadectomized in either the non-breeding season (April) or the breeding season (September). LH pulse frequency and basal (interpulse) concentrations were several times greater (P < 0.01) in the breeding season than in the non-breeding season. Neither of these parameters nor LH pulse amplitude were affected by oestradiol immunization. Parameters of testosterone episodic secretion and response to an injection (i.v.) of 15 micrograms NIH-LH-S25 were also greater (P < 0.05) in the breeding season and, with the exception of pulse frequency, in immunized rams versus controls. Substrate utilization established that testosterone biosynthesis was predominantly via the 5-ene pathway. Increases in blood testosterone concentration in the breeding season were associated with a fivefold higher (P < 0.01) activity of cytochrome P450 17alpha-hydroxylase/C-17,20 lyase (P450(17alpha)) and a 65% higher (P < 0.05) relative amount of mRNA for cytochrome P450 cholesterol side-chain cleavage enzyme complex (P450scc) in the testis. Of the steroidogenic enzyme activities examined, only that for 17beta-hydroxysteroid dehydrogenase (17beta-HSD) tended to be increased by oestradiol immunization. Blood concentrations of cholesterol lipoproteins and expression of the testicular low density lipoprotein receptor were not affected by season or immunization. The amount of steroidogenic acute regulatory protein (StAR) mRNA was 65% higher (P < 0.01) in the breeding season and 20% higher (P < 0.01) in immunized rams versus controls. These results indicate that greater LH stimulation may increase testosterone biosynthesis in the breeding season by increasing StAR mRNA (and presumably delivery of cholesterol to P450scc) and the activity of P450(17alpha), and possibly that of P450scc (activity not measured). More moderate increases in StAR mRNA and 17beta-HSD activity may explain, in part, the increases in testosterone secretion with oestradiol immunization.

Effect of FSH and cell localization on dimeric inhibin-A secretion from bovine granulosa cells in culture.

We tested the hypotheses that the secretion of dimeric inhibin-A from cultured bovine granulosa cells is stimulated by FSH, and that antral cells secrete more inhibin-A than do mural cells. Cells from the antral or mural compartment of follicles were cultured in defined medium in two culture systems, and dimeric inhibin-A was measured by two-site ELISA or by Western immunoblotting. In the first culture system, dimeric inhibin-A secretion declined with time in culture, but was significantly (P<0.05) higher from antral than from mural cells (as was total inhibin-alpha measured by RIA). The secretion of dimeric inhibin-A and inhibin-alpha from antral but not mural cells was responsive to FSH. In the second culture system, dimeric inhibin-A secretion increased with time in culture, and was significantly stimulated by FSH, but FSH responsiveness was dependent on the concentrations of insulin in the culture medium. The major forms of inhibin-A secreted had molecular masses of approximately 58, 62, 103-116 and >116 kDa; the 32 kDa form was barely detectable. These different forms were all stimulated by FSH, but the >116 and 62 kDa forms were most responsive to FSH. We conclude that (i) FSH stimulates dimeric inhibin-A secretion from bovine granulosa cells, (ii) the 62 kDa form of inhibin-A may be more responsive to FSH than the 58 kDa form, and (iii) the spatial differentiation of granulosa cell function within the follicle previously observed for oestradiol secretion was also observed for inhibin-alpha and dimeric inhibin-A secretion.

Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro.

We determined 1) whether the previously observed induction of estradiol secretion in bovine granulosa cells cultured in serum-free conditions is associated with an increase in cytochrome P450 aromatase (P450(arom)) mRNA abundance and 2) whether P450(arom) mRNA levels are responsive to FSH in vitro. Granulosa cells from small (2-4-mm) follicles were cultured in serum-free medium. Estradiol secretion increased with time in culture and was correlated with increased P450(arom) mRNA abundance. Progesterone secretion also increased with time in culture, but P450 cholesterol side-chain cleavage (P450(scc)) mRNA abundance did not. FSH stimulated estradiol secretion and P450(arom) mRNA abundance; the effect was quadratic for both estradiol and P450(arom) mRNA. Estradiol secretion and P450(arom) mRNA levels were correlated. FSH stimulated progesterone secretion and P450(scc) mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450(arom) mRNA levels but not progesterone or P450(scc) mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450(arom) mRNA abundance responsiveness to FSH and insulin, whereas P450(scc) mRNA abundance and progesterone secretion were responsive to FSH but not insulin.

Effects of superovulation on endogenous LH secretion in cattle, and consequences for embryo production.

Stimulation of follicular growth during superovulation is achieved by the injection of FSH or compounds with high FSH-bioactivities. However, some LH-activity is required for follicle maturation. It is of relevance to evaluate, therefore, the effect of superovulatory treatments on endogenous LH secretion. Luteinizing hormone is secreted in discrete pulses, and the pattern of pulsatile LH secretion during superovulation is reviewed. Four of five published studies have shown that LH pulse frequency is significantly reduced by injection of eCG or FSH preparations. This suppression appears within 8 h of treatment Effects of superovulation on LH pulse amplitude are less consistent. The reasons for the decrease in pulse frequency have been investigated, and although the answer is not definitive, it would seem that increased follicular estradiol, acting perhaps in synergism with progesterone, may play a role. Changes in plasma progesterone concentrations are not related to changes in LH pulse frequency. What is the significance of decreased LH pulse frequency? We attempted to investigate this by inducing LH pulses during superovulation, but the result was a major reduction in ovulation rate. More research is required to determine if modification of endogenous LH secretion can improve superovulatory responses.

Follicle-stimulating hormone stimulates circulating gonadotropin surge-attenuating/inhibiting factor bioactivity in cows.

This study aimed to determine whether superovulation in cattle stimulates gonadotropin surge-attenuating/inhibiting factor (GnSAF/IF) bioactivity, as it does in humans. Blood samples were collected from cows (n = 7 per treatment) at -4, 8, 20, 32, 44, 56, and 68 h after injections of saline, eCG, or FSH. Equal volumes of plasma at each treatment and time point were pooled, and GnSAF/IF and inhibin bioactivities were measured using an established rat pituitary cell culture bioassay. Plasma from saline- and eCG-treated cows had little effect on GnRH-induced LH secretion (116.3 +/- 8.3%-81.6 +/- 6.0% of control), while plasma from FSH-treated cows produced a time-dependent suppression of GnRH-induced LH secretion, falling to 64.6 +/- 4.0% of the control value at 56 h after first FSH injection (p < 0.001). The GnSAF/IF bioactivity from the 56-h plasma eluted at pH 5.73 by pseudochromatofocusing-similar to the GnSAF/IF isoelectric point value of 5.81 determined using serum from superovulated women. Plasma from FSH-treated cows reduced basal FSH secretion more than plasma from eCG-treated cows (to 55.5 +/- 5.7% and 63.2 +/- 6.6% of the control value, respectively, p < 0.01) although immunoreactive inhibin concentrations were similar between the two groups. We conclude that FSH, but not eCG, treatment causes a time-dependent production of circulating GnSAF/IF bioactivity in cattle.

Ovarian follicular steroidogenic acute regulatory protein, low-density lipoprotein receptor, and cytochrome P450 side-chain cleavage messenger ribonucleic acids in cattle undergoing superovulation.

Ovarian hyperstimulation with eCG in cattle results in increased plasma estradiol and progesterone concentrations, whereas hyperstimulation with FSH increases only estradiol concentrations. This study tested the hypothesis that eCG, compared to FSH, increases mRNA abundance for steroidogenic acute regulatory protein (StAR), low-density lipoprotein receptor (LDL-R), and/or cytochrome P450 cholesterol side-chain cleavage (P450scc), the main elements of the progesterone biosynthetic pathway. Heifers were stimulated with eCG (n = 10) or commercial FSH (n = 10), and ovaries were removed by colpotomy the day before and at 12 and 24 h after luteolysis was induced with prostaglandin (PG) F2alpha. RNA was extracted from individual follicles, and relative abundance of StAR, LDL-R, and P450scc mRNA was assessed by slot blots. In ovaries of abattoir origin, StAR mRNA was detected in all follicles and was present in the theca but not the granulosa cell layer, as shown by Northern blotting. Levels of StAR mRNA increased 2-fold (p < 0.05) after PGF2alpha injection in small (< 6 mm) follicles from eCG-treated but not from FSH-treated animals. After PGF2alpha, injection, StAR mRNA levels were 2- to 3-fold higher (p < 0.01) in large (> 9 mm) and medium (6-9 mm) follicles in eCG- compared with FSH-treated heifers. In contrast, P450scc and LDL-R mRNA levels did not consistently differ according to treatments. We show that StAR is expressed in the theca cells of bovine follicles and that stimulation with eCG increases follicular accumulation of StAR mRNA in comparison to stimulation with FSH.

Follicular 3 beta-hydroxysteroid dehydrogenase and cytochromes P450 17 alpha-hydroxylase and aromatase messenger ribonucleic acids in cattle undergoing superovulation.

In this study, we tested the hypothesis that there is altered abundance of transcripts of genes coding for the enzymes cytochrome P450 17 alpha-hydroxylase (P450(17 alpha)), cytochrome P450 aromatase (P450arom), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in follicles of cattle hyperstimulated with eCG compared to FSH. Treatments were initiated on Day 10 of the cycle, and all cows received prostaglandin (PG) on Day 12. In experiment 1, blood samples were taken to determine plasma progesterone and estradiol concentrations during ovarian stimulation. In experiment 2, both ovaries were removed from stimulated cows by colpotomy before (n = 4 cows/treatment) and at 12 (n = 3/treatment) and 24 h (n = 3/treatment) after PG injection, and from nonstimulated controls (n = 4) 72 h after PG. The preovulatory follicle from nonstimulated heifers, and all follicles greater than 3 mm in diameter from superovulated heifers, were isolated and classified as small (3-5 mm), medium (6-9 mm), or large (> 9 mm). Steady-state levels of RNA for 3 beta-HSD, P450(17 alpha), and P450arom genes were determined by Northern analysis in the individual follicles. In experiment 1, stimulation with eCG significantly (p < 0.01) increased plasma progesterone concentrations compared to FSH-stimulated and nonstimulated controls, and increased (p < 0.05) plasma estradiol concentrations compared to FSH-stimulated controls. Stimulation with FSH did not alter progesterone concentrations, but significantly increased plasma estradiol concentrations compared to those of controls. In experiment 2, the number of large follicles increased significantly with time (p < 0.01), but there were no differences between eCG and FSH treatments in size distribution of follicles (p > 0.05). Relative abundance of P450(17 alpha) message (per 20 micrograms RNA) was significantly higher in large and small follicles (p < 0.05) in eCG-treated compared to FSH-treated heifers after PG injection. Analysis within this period revealed significant treatment effects at 12 h but not 24 h after PG injection. The bovine P450arom cDNA hybridized to 3 transcripts: a 6.5-kilobase (kb) polyadenylated transcript, and non-polyadenylated messages of 3.4 and 1.8 kilobases (kb), all of which hybridized with an oligonucleotide probe specific for the heme-binding region. In medium and small follicles, the 6.5-kb and 3.4-kb transcripts were present in similar quantities, and the 1.8-kb transcript was 25% less abundant. In large follicles recovered after luteolysis, the 3.4 and 1.8-kb transcripts were 3- to 4-fold more abundant in eCG-treated compared with FSH-treated and nonstimulated animals (p < 0.05). There were no significant differences between eCG and FSH treatments on steady-state 3 beta-HSD mRNA levels. Levels of 3 beta-HSD and P450(17 alpha) mRNA in large follicles in hyperstimulated heifers were not different from those in preovulatory follicles in nonstimulated cows. We conclude that hyperstimulation with eCG results in greater stimulation of follicular P450(17 alpha) message abundance compared to hyperstimulation with FSH, and that this may contribute to increased follicular estradiol secretion.

Oocyte quality in small antral follicles in the presence or absence of a large dominant follicle in cattle.

The aim of these experiments was to determine whether the presence of a dominant follicle affects the developmental competence of oocytes from small antral follicles of cattle. In Expt 1, oocytes or follicular fluid samples were collected from follicles 2-7 mm in diameter before (day 3 of the oestrous cycle; n = 4) or after (days 6 and 7 of the oestrous cycle; n = 6) emergence of the first wave dominant follicle (verified by rectal ultrasonography). Five to ten follicles were aspirated for the determination of individual follicular fluid concentrations of oestradiol, progesterone, testosterone and dimeric inhibin; oocytes from the remaining follicles from each cow were pooled and developmental capacity assessed by in vitro fertilization and maturation. In Expt 2, ovaries containing a young corpus luteum and with or without a large oestrogen-active follicle were collected from the abattoir. Follicular aspirates from small follicles in each pair of ovaries were pooled, and oocyte quality and steroid concentrations were determined. In Expt 1, small follicles obtained before emergence of the dominant follicle contained significantly more oestradiol than they did after emergence (19.5 +/- 1.5 versus 0.7 +/- 1.1 ng ml-1, respectively; P < 0.05), but there were no significant differences in concentrations of progesterone, testosterone or dimeric inhibin. The percentage of blastocysts obtained from oocytes collected before (12.1 +/- 9.0) or after emergence (11.8 +/- 7.0) of the dominant follicle did not differ significantly (P > 0.05). In Expt 2, follicular steroid concentrations did not differ in small follicles taken in the presence versus the absence of a large oestrogen-active follicle, and there were no differences in the developmental capacity of the oocytes. There were significant negative correlations between follicular oestradiol concentration and the percentage of blastocysts formed from two-cell (r = -0.90; P < 0.01) and eight-cell embryos (r = -0.65; P < 0.05). These data suggest that in cattle the developmental competence of oocytes from small antral follicles is not adversely affected by the presence of a dominant follicle.