W. F. Pope

Attenuation of Estrogenic Effects by Dihydrotestosterone in the Pig Uterus Is Associated with Downregulation of the Estrogen Receptors

Abstract Androgens are known to attenuate some effects of estradiol-17β (E) in the uterus. The objectives of the present experiment were to determine effects of 5α-dihydrotestosterone (DHT) on estrogenic actions in the pig uterus and its associations with changes in expression of the estrogen receptor (ER) α and ERβ. Postpubertal gilts (120–130 kg of body weight; n = 16) were ovariectomized, and 3–4 weeks later received once-a-day injections (i.m.) of one of the following treatments during four consecutive days: 1) vehicle (corn oil), 2) E (250 μg), 3) E (250 μg) plus 1 mg DHT, or 4) E (250 μg) plus 10 mg DHT. Uterine tissues were collected 24 h after the last treatment. Gilts receiving E or E plus 1 mg DHT had greater uterine wet weight, uterine horn diameter, luminal epithelium thickness, and endometrial gland diameter compared with gilts treated with vehicle or E plus 10 mg DHT. Gilts receiving E or E plus 1 mg DHT were not different in these characteristics. Relative amounts of mRNAs in the endometrium for the cell proliferation marker histone H2a and the E-inducible protein complement component C3 increased in gilts treated with E compared with gilts treated with vehicle. E-induced increases in histone H2a and C3 mRNAs were not altered by cotreatment with E plus 1 mg DHT but were inhibited by E plus 10 mg DHT. Androgen receptor (AR) mRNA in the endometrium increased by treatment with E. Cotreatment of gilts with E and DHT did not alter the E-induced AR mRNA increase. Gilts treated with E plus 10 mg DHT had lesser amounts of immunoreactive ERα in cell nuclei of the myometrium and endometrial stroma and a tendency for a decrease in luminal epithelium compared with gilts treated with E. Amounts of immunoreactive ERα in glandular epithelium were not influenced by the treatments. Relative amounts of ERα and ERβ mRNAs decreased in the endometrium of gilts treated with E plus 10 mg DHT compared with gilts treated with E. Downregulation of the ERs, particularly ERα in the myometrium and endometrial stroma, might be a relevant mechanism in the antagonism of estrogenic effects by DHT in the pig uterus.

Survival of small and large littermate blastocysts in swine after synchronous and asynchronous transfer procedures.

Thirty-two crossbred sows were assigned to synchronous and asynchronous embryo transfer procedures to determine if, within a litter, small blastocysts were as viable as large blastocysts. Synchronous embryo transfers were established when donors and recipients displayed the onset of estrus (Day 0) within 6 h of each other. Asynchronous transfers were established when recipients displayed the onset of estrus 18 to 24 h after that of donors. An equal number (four or five) of the smallest and largest diameter blastocysts, from a Day 7 donor, were transferred to separate uterine horns of a Day 7 (synchronous) or a Day 6 (asynchronous) recipient. Each recipients uterine horns were ligated at the external bifurcation to prevent transuterine embryonic migration. The percentage of blastocysts surviving was determined 300 h (12.5 d) after donors exhibited estrus. Small as well as large Day 7 blastocysts survived following asynchronos transfer to a Day 6 recipient. However, fewer (P<0.01) small blastocysts survived synchronous transfer than large blastocysts. These data suggested that small blastocysts were lost due to asynchrony with the uterine environment; however, when transferred to a less advanced environment, small blastocysts were equally viable as large blastocysts.

Estrogen Receptor β in the Sheep Ovary During the Estrous Cycle and Early Pregnancy

Abstract Objectives were to sequence and examine the expression of the estrogen receptor β (ERβ) in the sheep ovary. The sequence of the ovine ERβ (oERβ) was determined using reverse-transcription polymerase chain reaction (RT-PCR) and cloning techniques. The reading frame of oERβ contained 527 amino acids and exhibited high overall homology with cow (98%), rat (88%), and human (88%) ERβ. In addition, an oERβ isoform having a 139-base pair deletion (oERβ1) was identified. The predicted amino acid sequence of this isoform is lacking the ligand-binding and carboxyl-terminal transactivation domains. The oERβ protein and mRNA were determined in ovaries obtained from ewes on Days 0 (first day of estrus), 2, 6, and 10 of the estrous cycle and Day 30 of gestation. Immunohistochemistry showed that oERβ protein was located in granulosa cells, the ovarian surface epithelium, endothelium, and Day 2 corpus luteum (CL). Weak immunostaining for ERβ was detected in the theca interna. Relative steady-state amounts of oERβ mRNA in the CL were determined using semiquantitative RT-PCR. Amounts of oERβ mRNA were greater (P < 0.05) during CL formation (Day 2) than at later stages. The oERβ to oERβ1 mRNA ratio was lower (P < 0.05) on Day 2 than on Day 10 or Day 30 due to a decrease in amounts of oERβ1. Results indicate that the oERβ is a 527-amino acid protein expressed in specific cells of the ovary. Changes in relative amounts of full-length oERB and a deletion isoform in CL occurred during the estrous cycle, suggesting that these two types of ERβ might regulate estrogen actions during early CL development in sheep.

Intracellular adenosine triphosphate and glutathione concentrations in oocytes from first estrous, multi-estrous, and testosterone-treated gilts

Cytoplasmic maturation refers to a variety of cellular changes that must occur in the oocyte in order to progress through subsequent fertilization and embryonic development. Intracellular concentrations of ATP (ATPi) or glutathione (GSHi), indicative of metabolic activity or the ability of the oocyte to form a male pronucleus and cope with cellular stress, respectively, have been used as markers of cytoplasmic maturation in vitro. In the current study, our objective was to determine if concentrations of ATPi and GSHi in oocytes recovered from three groups of gilts were associated with known differences in developmental competence within these populations. In vivo matured oocytes were surgically recovered 36-38 h after the onset of estrus from first estrous gilts, multi-estrous gilts, and multi-estrous gilts receiving testosterone (1mg/2 ml per day; day 13 to estrus, day 0=day of estrus). Concentrations of ATPi and GSHi were determined using a bioluminescent somatic cell assay kit (luciferin-luciferase reaction) and the dithiobisnitrobenzoic acid (DTNB)-glutathione reductase recycling reaction, respectively. There were no differences (P>0.05) between ATPi concentrations in oocytes from the three groups (1.52 +/- 0.10, 1.51 +/- 0.11, 1.56 +/- 0.11pmol per oocyte). In contrast, oocytes from multi-estrous gilts had higher (P<0.05) concentrations of GSHi (31.53 +/- 1.66 to 33.67 +/- 2.30 pmol per oocyte) than oocytes from first estrous gilts (25.07 +/- 0.82). Administration of testosterone did not affect (P>0.05) GSHi concentrations in oocytes from multi-estrous gilts. Differences in developmental potential between the three groups of gilts were apparently not due to different concentrations of ATPi. However, GSHi concentrations were higher in oocytes from multi-estrous gilts, suggesting that reduced developmental potential of oocytes from first-estrus gilts may be related to insufficient amounts of GSHi. The beneficial effect of exogenous testosterone on the percentage of embryos surviving early gestation does not appear to be due to increased GSHi. Of the numerous potential markers of developmental potential, two were examined in the current study, and GSHi appeared to be useful for assessing porcine oocytes.

Dose-response relationships of exogenous progesterone shortly after ovulation on estrous cycle length, blastocyst development and fertility in sheep

Maternal recognition of pregnancy is a period of competing signals; a luteolytic signal from the endometrium and an antiluteolytic signal from the blastocyst (s). Exogenous progesterone indirectly enhances these signals, causing premature release of prostaglandin and stimulating growth of blastocysts. In Experiment 1, 78 ewes were injected with vehicle or varying dosages of progesterone on Days 2–4 (Day 0 represents onset of estrus) for the purpose of comparing the minimal dose that shortened the estrous cycle, in non-pregnant ewes, to the minimal dose that enhanced growth of blastocysts to Day 13, in pregnant ewes. In Experiment 2, a field trial was conducted using Targhee and Polypay ewes to evaluate their fertility after treatment with vehicle or 6 mg of progesterone (n = 55). Analysis of plasma samples indicated that none of the dosages of exogenous progesterone altered concentrations of progesterone by Days 5 or 10, suggesting that treatment with exogenous progesterone failed to alter luteal function to Day 10. The minimal dose of progesterone that shortened (P<0.05) the estrous cycle was 6 mg and was the same dose that began to stimulate (P<0.05) blastocyst growth. Lambing rates of Targhee ewes were not different following treatment with exogenous progesterone. However, the lambing rate of Polypay ewes increased (P<0.05) from 200 to 256%. These data suggest that treatment of sheep, predisposed to an ovulation rate greater than two, with progesterone improved embryonic survival by a still unknown mechanism(s) that might have also advanced luteolytic and antiluteolytic signals.

Changes in follicular endocrinology during final maturation of porcine oocytes

Thirty-one gilts were ovariectomized between 21 and 34 hr after the onset of estrus to compare changes in follicular endocrinology with stages of oocyte maturation. Oocytes were recovered from 6 to 8 mm follicles and classified by stage of meiosis. Remaining follicular fluid was assayed for steroids and dermatan sulfate. Amounts of prostaglandin F2 alpha (PGF2 alpha) and E2 (PGE2) were measured in intramural tissues. Coincident with germinal vesicle breakdown, the follicular content of all steroids except testosterone decreased (P less than .05). As oocytes approached metaphase II, the amount of progesterone within follicles increased (P less than .05), and estradiol continued to decrease (P less than .05). The pattern of dermatan sulfate content was biphasic and peaked at germinal vesicle breakdown and anaphase stages. Amounts of PGF2 alpha and PGE2 within intramural tissues increased (P less than .05) throughout oocyte maturation. Follicular atresia was evident during estrus; however, more (P less than .05) atretic follicles were recovered at germinal vesicle than metaphase II stages (20 vs 3%, respectively). Follicular development, within a gilt, was skewed (P less than .05) and classification of follicles by hormone content demonstrated that a majority were more mature than a minority of less mature follicles. These data suggest that follicular maturation and oocyte development are highly correlated in swine. Furthermore, partitioning the follicular variability by hour and stage of oocyte maturation allowed for more precise assessment of follicular endocrinology than previously reported.

Effect of the rate of progesterone decline at luteolysis on the ovulatory follicles and subsequent estrous cycle length in ewes

Follicular and interestrous characteristics were examined in 34 ewes after experiencing either a rapid decline in plasma concentrations of progesterone at luteolysis [prostaglandin F2 alpha (PGF2 alpha)-induced] or a slow rate of decline, lasting over 72 h. All ewes were given PGF2 alpha on day 10 (day 0 = estrus). A slow rate of decline was established in 17 ewes by the intravenous infusion of progesterone initially at 72 ml h-1, delivering 4.5 mg progesterone h-1, then decreasing the infusion rate by 1 ml h-1 for the next three days. Seventeen additional ewes, predestined to experience a rapid decline in progesterone, were infused with vehicle. In Experiment 1, after infusion, ewes (6 ewes/group) were necropsied at the onset of estrus and follicle diameter was determined, follicular fluid was aspirated and the remaining follicular wall was microscopically examined to determine the number of granulosa cell layers. In Experiment 2, the interestrous interval, after infusion, was observed in both groups of ewes (11 ewes/group). Ewes experiencing a rapid rate of progesterone decline at luteolysis had no differences in follicle diameter nor follicular concentration of progesterone or estradiol but their ovulatory follicles contained fewer (P < 0.01) granulosa cell layers and the resulting estrous cycle was longer (P < 0.05) than ewes experiencing a slow rate of progesterone decline.

Influence of estradiol on duration of anestrus and incidence of short estrous cycles in postpartum cows.

The influence of exposure to exogenous estradiol on the interval from parturition to first ovulation, luteinizing hormone (LH) secretion and luteal function was examined in cows. Cows were assigned at parturition to one of three treatments. Cows received either a 3.0 (1-E; n = 30) or .75 cm (1/4-E; n = 28) implant containing 17 beta-estradiol or served as untreated control animals (C; n = 33). Implants were administered within 2 days following parturition and removed on day 40 postpartum (day 0 = day of parturition). Single blood samples were collected twice weekly and analyzed for progesterone to determine length of postpartum anestrus and duration of the initial increase in progesterone. Sequential blood samples were collected on day 35 +/- .1 postpartum (15 min intervals for 18 hrs) from 5 cows in each treatment and analyzed for LH. Concentrations of estradiol were higher (P less than .01) in the 1-E (5.3 +/- .24) than in C (3.9 +/- .23) or 1/4 E (3.9 +/- .25) cows on day 35 postpartum. The interval from parturition to the first estrous cycle of normal duration was similar for cows in the C and 1-E treatment (53 +/- 2.4 and 56 +/- 2.4 days, respectively). Cows in the 1/4-E treatment had a longer (P less than .05) interval (68 +/- 2.5 days). Secretion of LH was similar among treatments on day 35 postpartum. The first normal luteal phase after parturition was preceded by a transient rise in progesterone in 81, 64 and 85% of the cows in the C, 1-E and 1/4-E treatments, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Dihydrotestosterone influenced numbers of healthy follicles and follicular amounts of LH receptor mRNA during the follicular phase of the estrous cycle in gilts

The present experiments were conducted to determine androgenic effects on numbers, health, and amounts of gonadotropin receptor mRNA in late developing follicles of gilts. Gilts (n=5 per group) received daily injections of one of the following treatments on days 13-16 or days 13-18 of the estrous cycle: corn oil, 5alpha-dihydrotestosterone (DHT, 10 mg), flutamide (1.5 g, an androgen receptor inhibitor), DHT (10 mg) plus flutamide (1.5 g), testosterone (10 mg), and testosterone (10 mg) plus flutamide (1.5 g). Ovarian follicles > or =5 mm in diameter were evaluated on day 17 or 19, 24 h after receiving the last treatment dose. Follicles were classified as healthy (H), moderately atretic (MA), or very atretic (VA). Treatment with DHT increased (P<0.05) the numbers of H follicles relative to control gilts on days 17 and 19. DHT administration from days 13 to 16 diminished (P<0.05) the amounts of LH receptor (LHR) mRNA in H follicles from day 17 (relative amounts: 1.45+/-0.33 and 2.72+/-0.33 for DHT- and vehicle-treated gilts respectively). The effects of DHT on numbers of H follicles and LHR mRNA were not observed in gilts receiving DHT plus flutamide. Androgens did not influence numbers of MA, VA, and total follicles, or follicular estradiol-17beta concentrations and amounts of FSHR mRNA. Treating gilts with DHT during follicular recruitment and selection did not induce changes in the numbers of total follicles > or =5 mm, but rather increased the numbers of healthy follicles in this follicular population in association with decreased amounts of LHR mRNA.

Extension of short cycles in postpartum beef cows by intrauterine treatment with catecholestradiol

Eight multiparous beef cows were used to examine the effects of intrauterine infusion of catecholestradiol (4-hydroxylated estradiol) on development and function of the first corpus luteum after parturition. Calves were weaned on day 1 (day 0 = parturition) to initiate formation of a corpus luteum (CL) by approximately day 10 or 11. Before CL formation, on days 5 to 9, cows received twice daily infusions of catecholestradiol (4 micrograms; n = 4) or vehicle (n = 4) into the uterine horn opposite the previous pregnancy. Plasma progesterone during the first estrous cycle was elevated longer (P less than .001) and reached a higher (P less than .001) concentration in cows treated with catecholestradiol. The decline in progesterone was associated with an increase in plasma 13,14-dihydro, 15-keto-prostaglandin F2 alpha (PGFM) in all cows infused with catecholestradiol. In contrast, a rise in PGFM at the end of the first short cycle was detected in only one of four cows treated with vehicle. Furthermore, PGFM concentrations were linearly related (R2 = .870; P less than .001) to concentrations of progesterone. Estradiol-17 beta concentrations were not different during the infusion period, but after formation of the first CL, estradiol remained elevated (P less than .01) in cows that received vehicle. Results of this experiment suggest that exposure of postpartum beef cows to catecholestradiol extended luteal function in association with enhanced PGFM release.