DETECTION OF MESSENGER-RNA FOR INHIBIN ALPHA-SUBUNITS AND BETA(A)-SUBUNITS IN BOVINE OVARIAN TISSUES AND THE EFFECT OF IN-VIVO ADMINISTRATION OF GNRH

The aims of these studies were to determine which types of bovine ovar ian tissue contain mRNA for inhibin/activin subunits and whether admin istration of GnRH influences concentration of these mRNAs. In experime nt (exp.) one, cows in the luteal phase of the estrous cycle were give n prostaglandin F2alpha (PGF2alpha) to induce luteal regression and in jected after 40 hr with saline (n=5) or 100 mug GnRH (n=6). Ovaries we re removed 6 hr later. In exp. two, unilaterally ovariectomized (OVX) heifers (n=33) in the luteal phase of their estrous cycle were given P GF2alpha to induce luteal regression. Twelve heifers were OVX without injection of GnRH at 24 (n=6) or 40 hr (n=6) after PGF2alpha. The rema ining heifers (n=21) were given 100mug GnRH at 40 hr after PGF2alpha i njection and OVX 8 (n=4), 16 (n=5), 24 (n=6) or 48 (n=6) hr after GnRH injection. Total cellular RNA was isolated from large follicles (exp. one and two), small-medium follicles and stromal tissue (SMS) and cor pora lutea (CL; exp. one) tissues and analyzed by dot blot and Norther n blot techniques by hybridizing with cDNA probes for human inhibin/ac tivin alpha- and beta(A)-subunits. Large follicles were classified as steroidogenically active (EA) if follicular fluid (FF) concentration o f estradiol-17beta (E2) was greater than progesterone (P4), or if P4 a nd E2 concentrations in FF were greater than 100ng/ml, and estrogen in active (EI) if FF concentration of E2 and P4 did not satisfy these cri teria. In exp. one, mRNA for the alpha-subunit was primarily expressed in EA follicles, and detectable in EI follicles, SMS, and CL while be ta(A)-subunit mRNA was detected only in large EA follicles and a few S MS samples. The mRNA (x +/- SEM fmoles/mg DNA) for both subunits of in hibin/activin was higher (P<.05) in EA follicles from GnRH-treated cow s (alpha = 210.2 +/- 38.6; beta(A) = 376.9 +/- 41.0) than in EA follic les from control cows (alpha = 102.5 +/- 28.6; beta(A) = 170.8 +/- 57. 6). Concentration of mRNA for the alpha-subunit of inhibin in other ov arian tissues was not different (P>.10) between saline and GnRH treatm ents. In exp. two, the mRNA (fmol/mg DNA +/- SEM) for the alpha-subuni t decreased slightly 24 to 40 hr after PGF2a injection from 112 +/- 41 to 84 +/- 15, increased (P<.05) transientally to 158 +/- 24 at 8 hr a fter GnRH, and declined (P<.01) to 55 +/- 16, 40 +/- 4 and 42 +/- 14 a t 16, 24 or 48 hr after GnRH, respectively. Similarly, the beta(A)-sub unit decreased slightly from 242 +/- 52 to 189 +/-48 between 24 to 40 hr after PGF2alpha. Following injection of GnRH mean concentrations de creased (P<.10) further from 15 5 +/- 41 to 53 +/- 24, 64 +/- 33 or 10 +/- 7 at 8, 16, 24 or 48 hr, respectively. In summary, mRNA for the a -subunit of inhibin shows a transient rise in response to the preovula tory gonadotropin surges, but declines rapidly (3 fold decrease in 8 h r) following the transient rise and remains at this decreased level th rough ovulation. The mRNA for the beta(A)-subunit of inhibin/activin r ises (exp. one) or remains at a steady state (exp. two) early after th e preovulatory gonadotropin surges and then declines by 16 hr post GnR H injection and remains low through ovulation.