A luteolytic interaction between estradiol benzoate and prostaglandin F2 alpha in cattle.

A possible luteolytic interaction between prostaglandin F2 alpha (PGF2 alpha) and estradiol benzoate (E2B) in cattle was investigated by randomly assigning 20 heifers to one of four groups of a 2 x 2 factorially designed experiment. The treatments for the groups consisted of im administration of: 1) 200 micrograms of E2B, given twice daily on d 10, 11 and 12 of the estrous cycle plus 7 mg PGF2 alpha, given at a separate site, but concurrently with the last injection of E2B; 2) the vehicles for E2B (sesame oil) and PGF2 alpha (saline); 3) E2B and saline and 4) sesame oil and PGF2 alpha. Administration of both E2B and 7 mg PGF2 alpha resulted in luteolysis as evidenced by a shorter mean length of the estrous cycle (P less than .05) when compared with the vehicle-treated control group, and a decline in systemic concentrations of progesterone to less than 1 ng/ml in four of five animals. Administration of PGF2 alpha with sesame oil was luteolytic in only one of five animals and E2B plus saline had no effect on the mean length of the estrous cycle or systemic concentrations of plasma progesterone. These observations suggested a luteolytic interaction between E2B and PGF2 alpha. This luteolytic interaction was examined further in a second experiment. Corpora lutea was removed 12 h after treatment with 200 micrograms E2B or .5 ml sesame oil, administered twice on d 10, 11 and 12 of the cycle, and incubated with luteinizing hormone (LH) and PGF2 alpha in vitro. The effect of both LH and PGF2 alpha was to increase progesterone synthesis (P less than .01), and this effect was observed irrespective of E2B pretreatment. These findings were not consistent with the hypothesis that the luteolytic site of action for either E2B or PGF2 alpha was inhibition of the steroidogenic effects of LH as assessed under in vitro conditions.

Progesterone secretion by the bovine fetoplacental unit and responsiveness of corpora lutea to steroidogenic stimuli at two stages of gestation.

To evaluate the response of luteal cells to in vitro stimulation with luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (dbcAMP) and determine the secretion of progesterone by the fetoplacental unit, the corpora lutea were removed surgically in 10 cows (luteectomy) at 250 days (5 cows) or 270 days (5 cows) of gestation. During surgery, but before luteectomy, catheters were placed in the middle uterine artery and vein, carotid artery, and jugular vein. Blood samples were collected from all catheters just before luteectomy and at 8-hour intervals after luteectomy for 72 hours or until calving, whichever occurred first. Luteal tissue was prepared as a dispersed cell preparation and incubated with 0, 0.1, 1.0, 10, or 100 ng of LH/ml of medium or was incubated with 0, 0.5, or 2 mM dbcAMP. Synthesis of progesterone in response to LH by dispersed cells prepared from corpora lutea at 270 days was less (P less than 0.01; analysis of variance) than that by similar preparations at 250 days because a dose-response relationship was not observed for incubations of luteal tissue with LH at 270 days of gestation. Progesterone synthesis in response to the addition of dbcAMP also was less (P less than 0.01) at 270 than at 250 days of gestation. This difference in responsiveness to LH and dbcAMP between the 2 stages of gestation was not reflected by a significant difference between stages of gestation in systemic concentrations of progesterone before luteectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of estrone infusion on secretion of F series prostaglandins and on function of the corpus luteum during late gestation in cattle.

Catheters were surgically implanted in the carotid artery, jugular vein, and middle uterine vein of 8 cows at 245 days of gestation. Four cows were given 4 mg of estrone/hour via continuous jugular infusion from 0800 hours on day 246 of gestation through 0800 hours on day 250 of gestation; the remaining 4 cows (controls) were given the vehicle for estrone at 10 ml/hour for the same period. Blood samples were collected from the carotid artery every 3 hours during the infusion. Samples were collected hourly from the middle uterine vein from 0 through 8, 54 through 66, and 112 through 120 hours of the infusion periods. After completion of the infusion, corpora lutea were enucleated and blood samples were collected from the carotid artery and uterine vein at hourly intervals for an additional 8 hours. Dispersed cell preparations of the corpora lutea were incubated with and without luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Circulating concentrations of unconjugated and conjugated estrone and estradiol-17 beta were higher (P less than 0.05) in the group infused with estrone than in the vehicle-infused group. Mean and base-line concentrations of F series prostaglandins (PGF) for each blood collection period tended to increase (P less than 0.10) during infusion with estrone, but not during infusion with vehicle. After luteectomy, mean and base-line concentrations of PGF also tended (P less than 0.10) to be greater in the estrone-infused cows than in the control cows, but a surge in PGF concentrations due to removal of the ovarian source of progesterone did not develop.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of equine growth hormone, insulin-like growth factor-I and its interaction with gonadotropins on in vitro maturation and cytoskeleton morphology in equine oocytes.

In horses, successful in vitro fertilization procedures are limited by our inability to consistently mature equine oocytes by in vitro methods. Growth hormone (GH) is an important regulator of female reproduction in mammals, playing an important role in ovarian function, follicular growth and steroidogenesis. The objectives of this research were to investigate: the effects of equine growth hormone (eGH) and insulin-like growth factor-I (IGF-I) on the in vitro maturation (IVM) of equine oocytes, and the effects of eGH in addition to estradiol (E2), gonadotropins (FSH and LH) and fetal calf serum (FCS) on IVM. We also evaluated the cytoskeleton organization of equine oocytes after IVM with eGH. Equine oocytes were aspirated from follicles <30 mm in diameter and matured for 30 h at 38.5°C in air with 5% CO2. In experiment 1, selected cumulus-oocyte complexes (COCs) were randomly allocated as follows: (a) control (no additives); (b) 400 ng/ml eGH; (c) 200 ng/ml IGF-I; (d) eGH + IGF-I; and (e) eGH + IGF-I + 200 ng/ml anti-IGF-I. In addition to these treatment groups, we also added 1 µg/ml E2, 5 IU/ml FSH, 10 IU/ml LH and 10% FCS in vitro (experiment 2). Oocytes were stained with markers for microtubules (anti-α-tubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and chromatin (TO-PRO3-iodide) and assessed via confocal microscopy. No difference was observed when eGH and IGF-I was added into our IVM system. However, following incubation with eGH alone (40%) and eGH, E2, gonadotropins and FCS (36.6%) oocytes were classified as mature v. 17.6% of oocytes in the control group (P < 0.05). Matured equine oocytes showed that a thin network of filaments concentrated within the oocyte cortex and microtubules at the metaphase spindle showed a symmetrical barrel-shaped structure, with chromosomes aligned along its midline. We conclude that the use of E2, gonadotropins and FCS in the presence of eGH increases the number of oocytes reaching oocyte competence.

The involvement of growth hormone in equine oocyte maturation, receptor localization and steroid production by cumulus-oocyte complexes in vitro.

The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5% CO2. The COCs were stained with 10 µg/mL propidium iodide and 10 µg/mL fluorescein isothiocyanate-labeled Lens culinaris agglutinin. Chromatin configuration and distribution of cortical granules were assessed via confocal microscopy. Compared to control, COCs incubated with eGH had: more oocytes that reached metaphase II (35/72, 48.6% vs. 60/89, 67.4%, respectively; P=0.02); greater concentrations of testosterone (0.21 ± 0.04 vs. 0.06 ± 0.01 ng/mL; P=0.01), progesterone (0.05 ± 0.01 vs. 0.02 ± 0.00 ng/mL; P=0.04), and oestradiol (76.80 ± 14.26 vs. 39.58 ± 8.87 pg/mL; P=0.05) in the culture medium, but no significant differences in concentration of androstenedione. Based on Real Time RT-PCR analyses, expression of the eGH-R gene was greater in cumulus cells and COCs at the start than at the end of in vitro maturation. Positive immunostaining for eGH-R was present in cumulus cells, the oocytes and granulosa cells. In conclusion, addition of eGH to maturation medium increased rates of cytoplasmic maturation and had an important role in equine oocyte maturation, perhaps mediated by the presence of eGH-R in ovarian follicles.