Inhibin/activin beta-subunit monomer: isolation and characterization.

Using an activin RIA that showed limited cross-reaction with inhibin, activin immunoactivity was monitored throughout the isolation of activin from bovine follicular fluid and side-fractions during the isolation of human recombinant inhibin. Two peaks of activin immunoactivity were identified in both materials and isolated to homogeneity by dye affinity chromatography, hydrophobic interaction and gel permeation chromatography, and reverse phase HPLC. The purified proteins in all four peaks had terminal amino acid sequences identical to those of the inhibin/activin beta-subunit. The molecular masses determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing (and reducing) conditions were 25 and 15 and 15 and 15 kilodaltons (kDa) for each pair of proteins from both sources. Based on these criteria, the bovine and human recombinant 25-kDa proteins correspond to the inhibin/activin beta A-subunit dimer (activin-A), while the 15-kDa proteins correspond to the inhibin/activin beta A-subunit monomer. The activity of the monomer was 17% of the activity of the dimer in the activin RIA. Based on this level of cross-reaction and the proportion of monomer to dimer immunoactivity found after reverse phase HPLC of bovine follicular fluid, it is estimated that the levels of monomer in bovine follicular fluid are 25-60% those of the dimer. The biological activities of the human recombinant activin monomer and dimer were investigated in two different cell culture systems. In a rat pituitary cell system the activity of the activin monomer was 19% of the activity of the dimer in stimulating FSH release, while in rat thymocyte cultures the activity of the monomer was 45% the activity of the dimer in suppressing lectin-stimulated [3H]thymidine uptake. It is concluded that the beta A-subunit monomer is found in bovine follicular fluid at a level 25-60% that of the beta A-subunit dimer (activin-A). The monomer displays in vitro responses similar to those of the dimer, although the monomer is less active (18-45%) than the dimer. It is unclear if dimerization of the monomer is a necessary prerequisite for biological activity.

Serum FSH-suppressing activity of human recombinant inhibin A in male and female rats.

After a single i.v. injection of purified human recombinant inhibin A (hr-inhibin) or bovine follicular fluid (bFF) to 3-day castrated 35-day-old male rats, serum FSH concentrations fell (P less than 0.05) between 4 and 8 h, returning to control concentrations by 16-24 h. Administration of graded doses of hr-inhibin (0.625-10 micrograms/100 g body wt) and bFF (31.3-250 microliters/100 g body wt) resulted in a parallel dose-related suppression of serum FSH with a maximum suppression 50% of controls. Similar experiments in 2-day ovariectomized 85-day-old female rats also showed a dose-related suppression with a maximum suppression approximately 30% of controls. Serum LH concentrations remained unchanged in all studies with male or female rats. The biological activity of hr-inhibin in vivo was determined for male and female rats in terms of a standard bFF preparation defined by an in-vitro bioassay based on the suppression of FSH content in rat pituitary cells in culture. In males hr-inhibin exhibited a biopotency of 407 (159:1050; fiducial limits) U/micrograms protein and in females the biopotency was 358 (226:565) U/micrograms protein. These potencies are lower than that measured in the in-vitro bioassay (1120 (1040:1210) U/micrograms protein) and differences between in-vivo and in-vitro systems were attributed to the use of bFF rather than a purified human inhibin preparation as standard. These results indicate that hr-inhibin behaves similarly in vivo to bFF. Furthermore, based on the large working range and relatively good precision, the female rat system provides a good basis for an inhibin in-vivo bioassay method.

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.

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

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

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.