Graduate Thesis Or Dissertation
 

In vitro estradiol-induced protein synthesis in the endometrium of cyclic and steroid-treated ovariectomized ewes

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  • The ability of ovine endometrium incubated in vitro with estradiol -17(3 to synthesize a specific induced protein (IP) was studied. Endometrium from cycling, pregnant and ovariectomized ewes was used. Twelve ewes were allotted in equal numbers to be sacrificed on days 0 (estrus), 3, 6, or 10 of the estrous cycle. In addition uteri from nonpregnant ewes (day 14 or 15, N = 3) were compared with day 15 pregnant ewes (N = 3). Experiment 2 involved 16 ovariectomized ewes assigned randomly to one of four treatment regimes. The ewes were treated as follows: a) controls, no exogenous steroids, b) estradiol -17β, c) progesterone and d) progesterone plus estradiol. Estradiol was packed into silastic capsules and implanted subcutaneously on day 1 of treatment. Progesterone (15 mg 2x daily) or vehicle (oil) were injected for 5 consecutive days beginning 48 hr (day 3) after implantation of estradiol. All ewes were sacrificed on the morning of day 8. Blood samples were taken at sacrifice and estradiol and progesterone levels were measured. The ewes treated with estradiol tended to have increased estradiol levels and ewes receiving progesterone had significantly higher serum progesterone levels. Upon sacrifice of the ewes, the uterus was removed and transported to the laboratory. Three-50 mg endometrial segments were incubated for 1 hr with either 2.5 x 10⁻⁸ M estradio1-17β (treated) or ethanol (control), followed by a 2 or 4 hr incubation with labelled amino acids. Treated and control tissues were homogenized together and the resulting supernatant was stored at -20 C until electro phoresed. Following disc gel electrophoresis, the gels were placed into a dye for staining or were fixed in acetic acid. The gels fixed with acetic acid were sliced into sections and then counted in a scintillation spectrometer. Experiments utilizing immature rat uteri were conducted to verify that IP synthesis in the rat uterus could be detected in our laboratory. Induced protein was detected following estradiol stimulation in vivo and in vitro. Synthesis of an estradiol-induced endometrial protein was not detected during the ovine estrous cycle, on day 15 of pregnancy or in steroid-treated ovariectomized ewes. However, increased incorporation of both isotopes, running in front of the tracker dye, was detected in two of the four ewes in both the control and estradioltreated ovariectomized ewes. This increased isotope incorporation was detected in all four ewes receiving progesterone injections and was not present in any of the ewes receiving both estradiol and progesterone. Problems with excess estradiol during the in vitro incubation probably prevented any expression of IP during the estrous cycle experiment (experiment 1). No substantial change in the prealbumin, non-specific protein patterns were observed. Protein profiles of uteri varied from 1 to 3 bands during the estrous cycle and day 15 of pregnancy. The number of uterine prealbumin bands observed in the ovariectomized ewes receiving exogenous steroids varied from 0 to 4 within and among the treatment groups.
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