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.     

Differences in gonadotrophin-stimulated cyclic AMP production by granulosa cells from booroola x merino ewes which were homozygous, heterozygous or non-carriers of a fecundity gene influencing their ovulation rate

Granulosa cells from follicles of different sizes from Booroola x Merino ewes which were homozygous (FF), heterozygous (F+) or non-carriers(++) of a fecundity gene were obtained 0-48 h after cloprostenol injection on Day 10 of the oestrous cycle. The highest mean amounts of cAMP produced by the cells did not differ between the genotypes. However, in the ++ ewes it was attained by cells from follicles greater than or equal to 5 mm in diameter, whereas in F+ and FF ewes it was attained by cells from follicles 3-4.5 mm in diameter. Cells from 1-2.5-mm diameter follicles of FF ewes were more sensitive to FSH and LH than were corresponding cells from F+ or ++ ewes. Granulosa cells from greater than or equal to 5 mm diameter follicles of ++ ewes 12-24 h after injection of cloprostenol had a lower mean response to FSH and LH than did cells obtained 0-6 or 36-48 h after cloprostenol. No such effect of time was evident for cells from any size of follicles obtained from F+ or FF ewes. In 1-2.5-mm diameter follicles, the mean aromatase activity of granulosa cells from ++ and F+ ewes was similar, but significantly lower than that of cells from FF ewes. In 3-4.5 mm diameter follicles, the mean aromatase activity of cells from F+ and FF ewes was similar, and significantly higher than that of cells from ++ ewes. For all 3 genotypes, there was a significant positive relationship between FSH or LH stimulation of granulosa cell cAMP production and cellular 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 follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.     

Gonadotrophins, fecundity genes and ovarian follicular function

The Booroola Merino is a sheep breed having a major gene(s) (F) influencing its ovulation-rate. Homozygous (FF), heterozygous (F+) and non-carriers (++) of the gene have ovulation-rates of greater than or equal to 5, 3 or 4 and 1 or 2 respectively with the durations of each oestrous cycle and oestrous behaviour being similar in all genotypes. Although the principal site(s) of gene expression are obscure, FF genotypes have mean plasma concentrations of FSH and LH which are higher than in the F+ ewes, which in turn are higher than in the ++ animals. Thus, the FF and F+ animals provide a unique system in which to examine ovarian function under continual exposure to elevated gonadotrophin concentrations. At the ovarian level, F gene-specific differences in follicular development and function were noted. In small follicles (0.1-1.0 mm dia.), the basal levels of cAMP and the in vitro synthesis of cAMP, progesterone, androstenedione and oestradiol-17 beta in response to LH and FSH were significantly influenced by genotype (FF greater than F+ greater than ++; P less than 0.05). In larger follicles (1-4.5 mm dia.) the granulosa cells from FF and F+ ewes were more responsive to FSH and/or LH than in ++ ewes with respect to cAMP synthesis and they also had higher levels of aromatase activity. In vivo, the ovarian secretion-rates of oestradiol from greater than or equal to 5 ("oestrogenic") follicles in FF ewes, 3-4 such follicles in F+ ewes, and 1-2 such follicles in ++ animals during the follicular phase were similar. In FF and F+ ewes, the preovulatory follicles ovulated at a smaller diameter (i.e. 3-5 mm) than in ++ ewes (greater than 5 mm diam.) and also produced smaller corpora lutea. Thus, after continual exposure to elevated levels of gonadotrophins, follicles may synthesize steroid and mature at smaller diameters compared to those exposed to normal levels of FSH and LH.     

Effects of oestradiol-17 beta, progesterone or bovine follicular fluid on the plasma concentrations of FSH and LH in ovariectomized Booroola ewes which were homozygous carriers or non-carriers of a fecundity gene

The plasma concentrations of FSH and LH were measured in ovariectomized Booroola FF and ++ ewes before and after treatment with subcutaneous implants of oestradiol-17 beta (0, 2 or 8 cm Silastic capsules; 5 ewes/genotype per dose) or progesterone (0, 1 or 3 Silastic envelopes; 5 ewes/genotype per dose) or subcutaneous injections of steroid-free bovine follicular fluid (bFF; 0, 0.5, 1.0, 2.5 or 5 ml; 4 ewes/genotype per dose). During the first 50 h after implantation of oestradiol or progesterone, or the first 24 h after bFF treatment, the FSH and LH concentrations in plasma were not different between the genotypes although there were significant effects of the steriods and bFF with respect to dose (P less than 0.05). At 6 days after steroid implantation, no gene-specific effects were noted for the plasma concentrations of FSH although significant effects of dose of oestradiol (P less than 0.01) but not progesterone were noted. Also at 6 days after steroid implantation, no gene-specific differences in the pulsatile patterns (i.e. peak frequency or amplitude) of plasma LH concentrations were noted although there were significant effects of steriod dose (P less than 0.05) on frequency and/or amplitude. It is concluded that the higher ovulation-rate in FF than ++ Booroola ewes is unlikely to be due to gene-specific differences in the sensitivity of the hypothalamic-pituitary axis to ovarian hormones.     

GnRH-induced gonadotrophin secretion in ovariectomized Booroola ewes with hypothalamic-pituitary disconnection

To test whether the F gene-specific differences in the plasma concentrations of FSH and LH are due to differences in the pituitary responsiveness to exogenous GnRH, ovariectomized Booroola ewes with hypothalamic-pituitary disconnection (HPD-ovx) were treated with GnRH (250 ng i.v.) once every 2 h for up to 5 weeks. In Exp. 1, jugular venous blood was collected once weekly from 13 FF and 14 ++ HPD-ovx ewes for 6 weeks before GnRH treatment and every 2nd, 3rd or 6th day for 5 weeks during treatment. In Exp. 2, jugular venous blood was collected from another 8 FF and 7 ++ HPD-ovx ewes at 5- or 10-min intervals over 4 GnRH pulses (250 ng i.v. once every 2 h) on 3 separate occasions after the animals had been subjected to the GnRH pulse regimen for approximately 7 days beforehand. Also in Exp. 2, the animals were extensively sampled around a larger (10 micrograms) i.v. injection of GnRH and the pituitary FSH and LH contents assessed after the animals had been re-exposed to the once every 2 h GnRH (250 ng i.v.) pulse regimen for several days following the larger GnRH bolus. In Exp. 3 the distributions of mean plasma concentrations of FSH and LH in individual GnRH-treated HPD-ovx ewes were compared with those in ovariectomized and ovary-intact FF and ++ ewes. During the 6 weeks before GnRH treatment (Exp. 1), the plasma concentrations of FSH (approximately 1 ng/ml) and LH (less than or equal to 0.8 ng/ml) were not different between the genotypes. After GnRH treatment both the mean FSH and LH concentrations increased significantly (P less than 0.01) above basal values after 2 days with F gene-specific differences being noted for FSH but not LH (FSH; FF greater than ++; P less than 0.05). Thereafter, the mean FSH but not LH concentrations increased at a faster rate in FF than in ++ ewes with the overall mean FSH concentrations between the genotypes being significantly different (P less than 0.05). In Exp. 2 considerable between-animal variation in the pulsatile pattern of FSH but not LH concentrations was seen in ewes of both genotypes during GnRH treatment. The overall mean FSH concentrations were higher in FF than in ++ ewes (P less than 0.05) and the mean FSH response to each GnRH pulse was significantly higher in FF than in ++ ewes (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of long-term hypophysectomy on ovarian follicle populations and gonadotrophin-induced adenosine cyclic 3',5'-monophosphate output by follicles from Booroola ewes with or without the F gene

Long-term (i.e. approximately 70 days) hypophysectomy led to a significant (P less than 0.05) reduction in ovarian weight but no reduction in the total number of antral follicles (greater than 0.1 mm in diameter). In hypophysectomized ++ Booroola ewes (N = 8) follicles were always less than or equal to 3 mm and in hypophysectomized FF Booroola ewes (N = 6) follicles were always less than or equal to 2 mm in diameter; in ewes of both genotypes follicles reached diameters which were approximately 40% of their predicted final size at ovulation. Under in-vitro conditions, follicles from the FF and ++ hypophysectomized ewes produced significant increases in cAMP within 1 h of exposure to gonadotrophins (P less than 0.05) although no genotypic differences in cAMP production were noted. We conclude that ovarian follicles in FF and ++ ewes have absolute requirements for pituitary hormone on reaching diameters of 2 mm and 3 mm respectively and that appreciable numbers of antral follicles in ewes of both genotypes remain responsive to pituitary gonadotrophins despite prolonged deprivation of these hormones.     

Ovarian follicular activity in Booroola lambs with and without a fecundity gene

The ovaries of 3-month-old Booroola lambs which were heterozygous carriers of a major gene (F) influencing the ovulation rate in mature ewes (i.e. F + lambs) were compared to those ofsimilarly-aged Booroola lambs which were non-carriers of the F-gene (i.e. ++ lambs). The ovaries of the F + Booroola lambs were significantly lighter (P less than 0.01) than those of ++ lambs even though the mean +/- s.e.m. number of follicles (greater than or equal to 1 mm diam.) in the F + lambs was greater than that in the ++ lambs (i.e. F + lambs, 30.2 +/- 2.5 follicles; ++ lambs, 18.4 +/- 1.2 follicles; P less than 0.01). In granulosa cells from non-atretic follicles (greater than or equal to 1 mm diam.) from F + and ++ Booroola lambs, FSH (NIAMDD-FSH-S16) doses of 100 and 1000 ng/ml caused significant stepwise increases (P less than 0.05) in cyclic adenosine 3',5'-monophosphate (cAMP) production compared to that achieved at FSH doses of 0 and 1 ng/ml or at any FSH dose in cells from atretic follicles. However, no significant differences in FSH-induced cAMP production were noted with regard to Booroola genotype or follicular diameter. None of the granulosa cell preparations from non-atretic follicles of 1-2.5 mm diameter from F + lambs (N = 13) or from non-atretic follicles of 1-4.5 mm diameter from ++ lambs (N = 16) responded to LH (NIAMDD-LH-S24; 10 or 1000 ng/ml) to produce significantly more cAMP than did the controls. In contrast, the granulosa cell preparations from non-atretic follicles of 3-4.5 mm diameter from F + lambs (N = 4) and from non-atretic follicles of greater than or equal to 5 mm diameter of ++ lambs (N = 4) produced significantly more cAMP (P less than 0.05) in response to LH (1000 and/or 10 ng/ml) relative to that in the controls. The theca interna from follicles of lambs of both genotypes had functional LH receptors as judged by the androstenedione responses to exogenous LH although no genotypic differences were noted. In F + lambs, the follicular fluid concentrations of testosterone but not oestradiol (i.e. in 1-4.5 mm diam. follicles) and granulosa cell aromatase activity (i.e. in 3-3.5 mm diam. follicles) were significantly higher (both P less than 0.05) than in corresponding follicles or cells from ++ lambs. Collectively the results suggest that the Booroola F-gene influences the composition and function of sheep ovaries before puberty.     

Differences in the plasma concentrations of FSH and LH in ovariectomized Booroola FF and ++ ewes

During 12 sampling days before ovariectomy the mean plasma FSH but not LH concentrations in FF ewes were higher (P less than 0.01) than those in ++ ewes (16 ewes/genotype). After ovariectomy increases in the concentrations of FSH and LH were noted for ewes of both genotypes within 3-4 h and the rates of increase of FSH and LH were 0.18 ng ml-1 h-1 and 0.09 ng ml-1 h-1 respectively for the first 15 h. From Days 1 to 12 after ovariectomy, the overall mean +/- s.e.m. concentrations for FSH in the FF and ++ ewes were 8.1 +/- 0.6 and 7.1 +/- 0.4 ng/ml respectively and for LH they were 2.7 +/- 0.3 and 2.1 +/- 0.2 ng/ml: these differences were not statistically significant (P = 0.09 for both FSH and LH; Student's t test). However, when the frequencies of high FSH or LH values after ovariectomy were compared with respect to genotype over time, significant F gene-specific differences were noted (P less than 0.01 for both FSH and LH; median test). In Exp. 2 another 21 ewes/genotype were blood sampled every 2nd day from Days 2 to 60 after ovariectomy and the plasma concentrations of FSH and LH were more frequently higher in FF than in ++ ewes (P less than 0.01 for FSH and LH). The F gene-specific differences in LH concentration, observed at 21-36 days after ovariectomy were due to higher mean LH amplitudes (P less than 0.025) but not LH peak frequency in FF than in ++ ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian activity in Booroola X Romney ewes which have a major gene influencing their ovulation rate

A marked difference in both the function and composition of individual ovarian follicles was noted in Booroola X Romney ewes (6-7 years of age) which had previously been segregated on at least one ovulation rate record of 3-4 (F + ewes, N = 21) or less than 3 (++ ewes, N = 21). Follicles in F + ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In F+ ewes (N = 3), the presumptive preovulatory follicles were 4.4 +/- 0.5 (s.e.m.) mm in diameter and contained 2.1 +/- 0.3 X 10(6) (s.e.m.) granulosa cells, whereas in ++ ewes (N = 3), such follicles were 7.3 +/- 0.3 mm in diameter and contained 6.5 +/- 0.8 X 10(6) cells. During a prostaglandin (PG)-induced follicular phase, the secretion rate of oestradiol from ovaries containing 3 presumptive preovulatory follicles in F + ewes was similar to that from ovaries with only one such follicle in ++ ewes. We suggest that the putative 'gene effect' in F + ewes is manifested during early follicular development and that it may be mediated via an enhanced sensitivity of granulosa cells to pituitary hormones. As a consequence, the development of 3 preovulatory follicles in F + ewes may be necessary to provide a cell mass capable of producing the same quantity of oestradiol as that from one preovulatory follicle in ++ ewes.     

Differences in ovarian activity between booroola X merino ewes which were homozygous, heterozygous and non-carriers of a major gene influencing their ovulation rate

Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of greater than 5 (FF ewes, N = 15), 3-4 (F+ ewes, N = 18) or less than 3 (++ ewes, N = 18). Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean +/- s.e.m. diameters for the presumptive preovulatory follicles were 3.4 +/- 0.3, 4.1 +/- 0.2 and 6.8 +/- 0.3 mm and in each of these follicles the respective mean +/- s.e.m. numbers of granulosa cells (X 10(6)) were 1.8 +/- 0.3, 2.2 +/- 0.3 and 6.6 +/- 0.3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4.8 +/- 0.4 'oestrogenic' follicles, F+ ewes with 3.2 +/- 0.2 'oestrogenic' follicles and ++ ewes with 1.5 +/- 0.02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5.4 X 10(6) cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0.91, P less than 0.001). Collectively, these findings support the notion that the maturation of greater than or equal to 5 follicles in FF ewes and 3-4 follicles in F+ ewes may each be necessary to provide a follicular-cell mass capable of producing the same quantity of oestradiol as that from 1-2 preovulatory follicles in ++ ewes.     

Differences in gonadotrophin concentrations and pituitary responsiveness to GnRH between Booroola ewes which were homozygous (FF), heterozygous (F+) and non-carriers (++) of a major gene influencing their ovulation rate

The mean plasma concentrations of FSH and LH were significantly higher in FF ewes than in ++ ewes with those F+ animals being consistently in between. These gene-specific differences were found during anoestrus, the luteal phase and during a cloprostenol-induced follicular phase, suggesting that the ovaries of ewes with the F-gene are more often exposed to elevated concentrations of FSH and LH than are the ovaries of ewes without the gene. The gene-specific differences in LH secretion arose because the mean LH amplitudes were 2-3 times greater in FF compared to ++ ewes with the LH amplitudes for F+ ewes being in between. The LH pulse frequencies were similar. In these studies the pulsatile nature of FSH secretion was not defined. The pituitary contents of LH during the luteal phase, were similar in all genotypes whereas for FSH they were significantly higher in the F-gene carriers compared to ++ ewes. The pituitary sensitivity to exogenous GnRH (0.1, 0.5 and 25 micrograms i.v.) was related to genotype. Overall the LH responses to GnRH were lower in FF ewes than in ++ ewes with the results for the F+ ewes being in between. The FSH responses to all GnRH doses in the FF genotype were minimal (i.e. less than 2-fold). In the other genotypes a greater than 2-fold response was noted only at the highest GnRH dose (i.e. 25 micrograms). Treatment of FF and F+ but not ++ ewes with GnRH eventually led to a reduced FSH output, suggesting that the pituitary responses to endogenous GnRH were being down-regulated in the F-gene carriers whereas this was not the case in the non-carriers. Collectively these data confirm that peripheral plasma and the pituitary together with the ovary are compartments in which F-gene differences can be observed. In conclusion, these findings raise the possibility that F-gene-specific differences may also extend to the hypothalamus and/or other regions of the brain.     

Identification of specific FSH binding sites in the testes of adult monkeys of different species

The interaction of 125I-labelled hFSH with primate testicular tissue from 4 species of adult monkeys (Macaca mulatta, M. nemestrina, M. fascicularis and Papio cynocephalus) was investigated. 125I-labelled hFSH binding to a particulate fraction (P1, 40 000 g) of frozen testes was highly specific and saturable. Displacement curves generated using the P1 fraction of testes from the 4 species and 125I-labelled hFSH and unlabelled FSH were very similar. The binding of FSH to the monkey testicular receptor was not species specific because purified FSH from heterologous species such as horse, sheep, pig and rat were very effective in competing with 125I-labelled hFSH for binding. The equine FSH was about 10 times more active than hFSH in this respect. Similarly, 125I-labelled ovine FSH bound as well as labelled hFSH to the testes fractions of all 4 monkey species. In marked contrast to the high binding of 125I-labelled hFSH, binding of 125I-labelled hCG with rhesus monkey testis homogenates and P1 fractions was very low. The FSH receptor in the adult rhesus monkey testis was present in much larger quantity than the LH receptor and was more readily detectable. Our studies show that frozen primate testis can be utilized for investigating testicular-FSH interactions.     

Consequences of the presence of the Booroola F gene on the intraovarian insulin-like growth factor system and terminal follicular maturation in Mérinos d'Arles ewes

In sheep, the presence of the Booroola F gene has several important consequences for ovarian function. This study investigated the consequences of the presence of the F gene for the insulin-like growth factor (IGF) system in the ewe ovary. Studies were undertaken in ovaries from F+ and ++ Mérinos d'Arles ewes to determine 1) the levels of type I IGF receptors and IGF binding proteins (IGFBPs) in follicular cells by quantitative autoradiography of [(125)]-IGF-I binding sites on ovarian sections; 2) the pattern of intrafollicular IGFBPs, by Western-ligand blotting on follicular fluids; and 3) the effects of IGF-I and FSH on proliferation and differentiation of granulosa cells in vitro, assessed by progesterone secretion and cytochrome P450 side-chain cleavage (P450(scc)) expression. The amounts of type I IGF receptors were similar in F+ and ++ follicular cells; however, at the same follicular size, F+ healthy follicles contained lower concentrations of IGFBPs smaller than 40 kDa (particularly IGFBP-2) than ++ healthy follicles. In vitro, in basal conditions as well as in IGF-I- or FSH-stimulated conditions (or both), granulosa cells from F+ follicles had a lower proliferative activity, secreted higher amounts of progesterone, and expressed higher levels of P450(scc) than granulosa cells from ++ follicles of the same size. When F+ and ++ preovulatory follicles were compared at the end of the follicular phase, IGFBPs <40 kDa concentrations were slightly higher, and responsiveness of granulosa cells to FSH in vitro was lower in F+ than in ++ follicles, suggesting that terminal maturation of F+ follicles, although precocious, was less complete than it was in ++ follicles. The early decrease in intrafollicular IGFBPs <40 kDa concentrations observed in F+ antral follicles, which likely leads to an early increase in IGF bioavailability, may at least partly account for the increased ovulation rate that characterizes F-carrier ewes.     

Progesterone pretreatment has a direct effect on GnRH-induced preovulatory follicles to determine their ability to develop into normal corpora lutea in anoestrous ewes

In two experiments carried out during seasonal anoestrus, Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals with and without progesterone pretreatment. In Exp. 1, 8/8 progesterone-primed ewes ovulated and produced functionally normal corpora lutea compared with 2/9 non-primed ewes. Follicles were recovered from similarly treated animals 18 or 28 h after the start of GnRH treatment (at least 14 h before the estimated time of the LH peak) and assessed in terms of diameter, granulosa cell number, oestradiol, testosterone and progesterone concentrations in the follicular fluid, oestradiol production in vitro and binding of 125I-labelled hCG to granulosa and theca. There were no significant differences in any of these measures in 'ovulatory' follicles recovered from the progesterone-pretreated compared to non-pretreated animals. In Exp. 2, follicles were removed from similar treatment groups just before and 2 h after the start of the LH surge. Unlike 'ovulatory' follicles recovered from the non-pretreated ewes, those recovered from progesterone-pretreated ewes responded to the LH surge by significantly increasing oestradiol secretion (P less than 0.01) and binding of 125I-labelled hCG (P less than 0.05) to granulosa cells. Overall there was also more (P less than 0.05) hCG binding to granulosa and theca cells from progesterone-pretreated animals. Non-ovulatory follicles recovered from progesterone-primed ewes had more (P less than 0.05) binding of 125I-labelled hCG to theca and a higher testosterone concentration in follicular fluid (P less than 0.05) than did those from non-primed ewes. These results suggest that inadequate luteal function after repeated injections of GnRH may be due to a poor response to the LH surge indicative of a deficiency in the final maturational stages of the follicle.     

Effect of castration on plasma concentrations of luteinizing hormone and follicle-stimulating hormone in adult Merino rams which were homozygous carriers or non-carriers of the Booroola fecundity gene

Before castration, the mean plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not differ between FF and ++ Booroola rams. After castration, mean LH and FSH concentrations increased after 8 h, and for the next 14 days the rate of increase in FSH, but not LH, secretion was significantly faster in FF than in ++ rams (P less than 0.05). Mean FSH concentrations over this period were significantly higher in FF than in ++ rams (P less than 0.05). In both genotypes, the ranked FSH values did not significantly change their order over time, i.e. a significant within-ram effect was noted (P less than 0.05). Repeated-measures analysis of variance indicated a significant effect of genotype on mean FSH secretion (P less than 0.05) and a significant effect of sire in the FF (P less than 0.05), but not the ++ (P = 0.76), genotype. From Day 28 to Day 58 after castration, FSH and LH concentrations were variable and no overall increases in concentrations were observed. The mean concentrations of both hormones over this period were not related to genotype. There were no gene-specific differences in pulsatile LH secretion 14 weeks after castration. However, the mean LH, but not FSH, response to a bolus injection of 25 micrograms of gonadotrophin-releasing hormone (GnRH) was significantly higher in FF than in ++ rams (P less than 0.05) and this was not significantly affected by sire. These studies support the hypothesis that the F gene is expressed in adult rams, in terms of pituitary responsiveness to an injection of GnRH and to the removal of the testes, but it is not clear from this study whether the influence of sire is related to or independent of the apparent gene-specific differences.     

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.     

Adenosine cyclic 3',5'-monophosphate and steroid production by small ovarian follicles from Booroola ewes with and without a fecundity gene

The tissue contents of adenosine cyclic 3',5'-monophosphate (cAMP) in freshly dissected follicles (0.13-1.00 mm diam.) were significantly higher in Booroola ewes containing a major fecundity gene (FF and F+ ewes) compared to those values in Booroolas with no copy of the gene (++ animals; P less than 0.025). After a 1 h incubation with LH + FSH, the respective proportions of follicles with a diameter of 0.13-0.52 mm (n = 288) and 0.53-1.00 mm (n = 271) that had synthesized greater than or equal to 0.6 pmol cAMP and greater than or equal to 1.0 pmol cAMP were significantly influenced by genotype (Booroola ewes homozygous for the F-gene, FF greater than heterozygous, F+ greater than ++; P less than 0.01 for both follicle size ranges). The contents of progesterone, androstenedione, testosterone and oestradiol-17 beta in minced ethanolic extracts of freshly dissected follicles (n = 188) were undetectable regardless of Booroola genotype. However, when follicles of 0.53-1.00 mm but not 0.13-0.52 mm diameter were cultured for 48 h with LH + FSH under 70 kPa of a 50% O2, 45% N2 and 5% CO2 gas mixture, the proportions that synthesized high levels of progesterone (greater than or equal to 4.0 ng), androstenedione (greater than or equal to 3 ng), and oestradiol (greater than or equal to 0.8 ng) were significantly influenced by genotype (FF greater than F+ greater than or equal to ++; P less than 0.05 for each steroid). No significant genotypic differences were noted for testosterone synthesis. Collectively, these results show that the Booroola F-gene has an influence on the maturation of ovarian follicles from an early stage of growth.     

Plasma LH, FSH and testosterone concentrations in adult rams which were homozygous carriers or non-carriers of the Booroola fecundity gene

No gene-specific differences were found with respect to LH or testosterone pulsatile secretion (over 12 h), or in 12 hourly mean FSH concentrations in adult Booroola FF and ++ rams. Also, no differences between genotypes in the LH response to an injection of testosterone propionate, the FSH response to an infusion of bovine follicular fluid, or the testosterone response to injections of PMSG were noted. However, during the phase of seasonal testicular development, mean testosterone pulse amplitude (over 12 h) and the FSH response to 25 micrograms GnRH were higher in FF than in ++ rams (P less than 0.05); there were also significant effects of sire (P less than 0.05 in FF genotype only) and litter size (P less than 0.05) on testosterone pulse amplitude and GnRH-stimulated FSH release, respectively. During the breeding season, mean LH, but not FSH, concentrations were higher in FF than in ++ rams, after an injection of 0.5 micrograms GnRH; LH release was not affected by sire or litter size (P greater than 0.05). Long-term studies revealed that the FF rams were born in significantly larger litters, they weighed significantly less than ++ rams (P less than 0.05), and that bodyweight was significantly correlated (P less than 0.05) with litter size. There were no differences in testis size, and testis size was not significantly correlated with bodyweight. There was a strong tendency (P = 0.056) for overall mean FSH concentrations, measured weekly for 9 months, to be highest more often in FF than in ++ rams.(ABSTRACT TRUNCATED AT 250 WORDS)