Effects of long-term treatment with LH on induction of cyclic ovarian activity in seasonally anoestrous ewes.

The aim of the present study was to establish whether cyclic ovarian activity could be induced and then maintained in anoestrous Romney ewes by the long-term administration of regular intravenous pulses of LH (10 micrograms ovine LH i.v. once every 1 or 2 h for 29-91 days). The LH pulse regimen was designed to generate plasma profiles of LH that were comparable to those experienced during the luteal and follicular phases of the oestrous cycle. The results showed that the LH treatments were capable of inducing cyclic ovarian activity, as assessed from the concentrations of progesterone in plasma, but that the treatments were inadequate for sustaining cyclic activity beyond two consecutive progestational phases. After 35-56 days of treatment, the plasma concentrations of FSH declined significantly (P less than 0.05) relative to those in the untreated animals. These data suggest that FSH supplementation as well as LH might be required for the long-term maintenance of cyclic ovarian activity in seasonally anoestrous ewes.

Accumulation of luteinizing hormone, oestradiol and androstenedione by sheep ovarian follicles in vivo.

The temporal relationship between the levels of LH in peripheral plasma and in follicular fluid of ovarian follicles in anaesthetized sheep were investigated for a 10-h period after a single i.m. injection of LH releasing hormone (LH-RH; 100 microgram). The ovarian secretion rates of oestradiol and androstenedione and the levels of these steroids accumulating in different sized follicles at varying time-intervals after the LH-RH injection were also compared. The data show that the rates at which pituitary LH enters and leaves the intrafollicular fluid-filled spaces are substantially slower than those of peripheral blood. Two hours after LH-RH injection the levels of LH in plasma had increased from 1 to 200 ng/ml, whereas in the follicle the levels remained at approximately 2ng/ml. Ten hours after the LH-RH injection, the levels of LH in plasma had returned to basal values (approximately 1.4 ng/ml) but in both small and large follicles the levels of LH (approximately 20 ng/ml) were comparable to those present in similar sized follicles 4h earlier. The data also indicate that more than 90% of the oestradiol produced by a large antral follicle (greater than or equal to 5 mm diameter) probably enters the bloodstream without first accumulating within the follicular antrum. Finally it is concluded that the clearance of the small amount of oestradiol which does accumulate in the follicular antrum is negligible compared with the clearance of this hormone from peripheral plasma.

Influence of follicular atresia on LH-induced cAMP and steroid synthesis by bovine thecae interna.

The aim of the present study was to examine the interrelationships between the luteinizing hormone (LH) receptor, the LH-induced changes in adenosine cyclic 3', 5'-monophosphate (cAMP) and steroid synthesis in theca interna tissue of large antral follicles (greater than or equal to 8 mm diameter) from oestrous cycling cows. Three distinct types of theca interna were identified (types I, II and III), all of which contained an LH receptor: type I was capable of secreting increased amounts of cAMP dehydroepiandrosterone, androstenedione and testosterone when exposed to LH; type II was capable of secreting increased amounts of cAMP and progesterone but not the androgens when exposed to LH; type III was incapable of cAMP or steroid synthesis when exposed to LH. Follicles with type I thecae contained: a full complement of granulosa cells; high intrafollicular concentrations of oestradiol; and granulosa cells with a high capacity to metabolise testosterone to oestradiol. These follicles were considered to be non-atretic structures. Follicles with types III thecae contained: fewer granulosa cells; low intrafollicular concentrations of oestradiol; and granulosa cells with a low capacity to metabolise testosterone to oestradiol. Moreover, follicles with type III thecae contained the highest concentrations of progesterone and the lowest concentrations of androstenedione and testosterone. These follicles were considered to be severely atretic structures. Follicles with type II thecae contained granulosa cell populations and progesterone, and androgen concentrations which were intermediate between those with thecae of types I and III. These follicles were considered to be at an intermediate stage of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)

[125I]hCG binding to bovine thecal tissue from healthy and atretic antral follicles.

[125I]hCG binding to thecal tissue from healthy bovine follicles was examined and compared to [125I]hCG binding to other bovine ovarian tissues. [125I]hCG bound specifically to theca interna but not to theca externa. Binding to theca interna was a time- and temperature-dependent process, the rate of association obeying second-order kinetics with calculated rate constants of 1.97 +/- 0.13 X 10(5) and 0.85 +/- 0.04 X 10(5) 1 M-1 sec-1 at 37 and 22 degrees C, respectively. The dissociation of [125I]hCG from theca interna was a slow biphasic process with only 40% of specifically bound [125I]hCG being liberated after 8 h at 37 degrees C. Unlabelled hCG and LH, but not FSH, prolactin, GH, TSH or GnRH, inhibited [125I]hCG binding to theca interna. The specific binding of [125I]hCG to theca interna was saturable and equilibrium binding data produced a linear plot when fitted to the Woolf equation. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) calculated from Woolf plots were 0.21 +/- 0.02 nM (mean +/- SEM) and 34 +/- 4 fmoles/mg protein, respectively. Constants for [125I]hCG binding to granulosa cells and luteal tissue, respectively, were 0.29 +/- 0.02 and 0.31 +/- 0.04 nM for the Kd values and 32 +/- 6 and 116 +/- 13 fmoles/mg protein for the Bmax values. [125I]hCG binding constants for small (less than 8 mm dia.) and large (greater than or equal to 8 mm dia.) follicles (healthy or atretic) were not significantly different. In addition, there was no difference in the [125I]hCG binding constants of healthy and atretic follicles (large or small).(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotrophin-stimulated cyclic AMP production by granulosa cells from Booroola x Romney ewes with and without a fecundity gene.

The influence of follicular size and health on FSH and LH stimulation of cAMP production by granulosa cells in vitro was studied in cells from Booroola X Romney ewes, with (F+) and without (++) a fecundity gene. The granulosa cells were obtained 0-48 h after the initiation of luteolysis on Day 10 of the oestrous cycle by cloprostenol. The highest mean amounts of cAMP produced by granulosa cells challenged with FSH or LH were not significantly different between the genotypes. However, they were achieved using granulosa cells from follicles greater than 3-4 mm in diameter in F+ ewes but from follicles greater than 4 mm in diameter in ++ ewes. Follicles may thus attain ovulatory maturity at a smaller diameter in F+ ewes than in ++ ewes. Granulosa cells from most atretic follicles gave a poor cAMP response to FSH or LH, compared to cells from non-atretic follicles. Granulosa cell responsiveness to FSH was independent of the time the cells were recovered after cloprostenol treatment in F+ ewes, but not in ++ ewes. Cellular responsiveness to LH was independent of time for sheep of both genotypes. There was a significant positive relationship for sheep of both genotypes between the level of aromatase activity in granulosa cells and cellular responsiveness to FSH and LH.

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.

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.

Use of bovine follicular fluid to increase ovulation rate or prevent ovulation in sheep.

Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0.1, 0.5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1.0 ml bFF twice daily (1.9 +/- 0.2 ovulations/ewe, mean +/- s.e.m. for N = 34) or 5.0 ml once daily (2.0 +/- 0.2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1.4 +/- 0.1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (greater than or equal to 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17 beta in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17 beta and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1-2.5 mm diam.) or medium (3-4.5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.

FSH influences follicle viability, oestradiol biosynthesis and ovulation rate in Romney ewes.

Injection of steroid-free bovine follicular fluid (bFF; 2 X 5 ml s.c. 12 h apart) into anoestrous ewes lowered plasma FSH concentrations by 70% and after 24 h had significantly (P less than 0.01) reduced the number of non-atretic follicles (greater than or equal to 1 mm diam.) without influencing the total number of follicles (greater than 1 mm diam.) compared to untreated controls. Hourly injections of FSH (10 micrograms i.v. NIH-FSH-S12) for 24 h did not influence the number of non-atretic follicles but did negate the inhibitory effects of bFF on follicular viability. Hourly injections of FSH (50 micrograms i.v., NIH-FSH-S12) + bFF treatment for 24 h significantly increased the total number of non-atretic follicles, and particularly the number of medium to large non-atretic follicles (greater than 3 mm diam.) compared to the untreated controls (both P less than 0.01). The 10 micrograms FSH regimen (without bFF) significantly increased aromatase activity in granulosa cells from large (greater than or equal to 5 mm diam.; P less than 0.01) but not medium (3-4.5 mm diam.) or small (1-2.5 mm diam.) follicles compared to controls. The 10 micrograms FSH + bFF regimen had no effect on granulosa-cell aromatase activity compared to the controls. However, the 50 micrograms FSH plus bFF regimen increased the aromatase activity of granulosa cells from large, medium and small non-atretic follicles 2.6-, 8.3- and greater than or equal to 11-fold respectively compared to that in the control cells. Ewes (N = 11) that ovulated 2 follicles had significantly higher plasma FSH concentrations from 48 to 24 h and 24 to 0 h before the onset of a cloprostenol-induced follicular phase (both P less than 0.01) than in the ewes (N = 12) that subsequently ovulated one follicle. Hourly FSH treatment (1.6 micrograms i.v., NIAMDD-FSH-S15) for 24 h but not for any 6 h intervals between 48 and 24 h or 24 and 0 h before a cloprostenol-induced luteolysis also resulted in significant increases (P less than 0.05) in the number of ewes with 2 ovulations.(ABSTRACT TRUNCATED AT 400 WORDS)