E.A. Van Kirk

Progesterone induces expression of endometrial messenger RNA encoding for cyclooxygenase (sheep).

Exogenous progesterone given early in the ovine estrous cycle results in precocious luteolysis. It has been suggested that under this circumstance regression of the corpus luteum is caused by an advancement in the timing of uterine secretion of prostaglandin F2 alpha. Uterine tissues were obtained from ewes following administration of progesterone (or injection vehicle), fixed in paraformaldehyde, and embedded in paraffin. Tissue sections were hybridized using an 35S-labeled cRNA probe specific for cyclooxygenase mRNA. There was approximately an eight-fold increase in the level of hybridization signal, localized mainly to uterine glands, consequential to treatment with progesterone. Thus, progesterone appears to control expression of the endometrial gene and(or) the stability of the message encoding for a rate-limiting enzyme involved in metabolism of arachidonic acid to its luteolytic product, thereby resolving the length of the nonpregnant cycle.

Localization of stress protein-70 in ovine corpora lutea during prostaglandin-induced luteolysis

Accumulation of heat shock (stress) protein-70 (HSP-70) was assessed in corpora lutea of sheep obtained after in vivo administration of a luteolytic dose of prostaglandin (PG) F2 alpha. A quantitative immunofluorescence technique was used to localize inducible HSP-70 production to specific cell-types. The number and intensity of immunostained large luteal cells increased within 2 h of injection of PGF2 alpha. A drop in luteal progesterone concentrations (functional regression) was not manifested until 4 h post-treatment. A dramatic increase in intensely-stained mononuclear leukocytes was observed in luteal tissues at 16 h, when glandular weights had begun to diminish (structural regression). Stress proteins could mediate intracellular protein processing and cell-surface autoimmune mechanisms underlying luteal regression.

Secretion of platelet-activating factor by periovulatory ovine follicles

Secretion of platelet-activating factor (PAF) in vitro by ovine follicles and ovarian interstitium obtained at various times before, during and after the endogenous preovulatory surge of luteinizing hormone (LH) and ovulation was quantified by radioimmunoassay. Release of PAF by the preovulatory follicle increased within 2 h after initiation of the surge of LH. Capacity for secretion of PAF was greatest at the time of ovulation, then declined thereafter. Production of PAF by ovarian interstitium throughout the periovulatory period was relatively low and did not change with time. It appears that PAF could act as an intrafollicular mediator in the mechanisms of ovulation and(or) luteinization.

Induction of apoptotic or lytic death in an ovarian adenocarcinoma cell line by antibodies generated against a synthetic N-terminal extracellular domain gonadotropin-releasing hormone receptor peptide.

A polyclonal antiserum was generated in ovariectomized sheep against a synthetic peptide corresponding to amino acids 5-17 of the deduced mouse pituitary gonadotropin-releasing hormone (GnRH) receptor. Antipeptide antibodies did not bind native cells, but did react strongly with a human ovarian cancer cell line (OVCAR-3) reportedly sensitive to GnRH. Growth of cultured OVCAR-3 cells was specifically suppressed by antipeptide serum. This was attributed in part to programmed death (chromatin condensation and DNA fragmentation) of cells by antibody-induced apoptosis. Antibodies also exhibited a cytolytic effect (lactate dehydrogenase release) toward OVCAR-3 cells in the presence of the complement. Endometria of passively immunized mice lacked development; thus, antipeptide antibodies evidently recognize Mullerian duct derivatives. Experiments are in progress to determine whether the putative antigen is a variant of the pituitary GnRH receptor or a largely dissimilar protein. Effector-functional antibodies could be useful in the management of ovarian or uterine neoplasia.

Inhibition by tocopherol of prostaglandin-induced apoptosis in ovine corpora lutea.

Accumulation of toxic oxidants within corpora lutea is a prelude of apoptotic cell death. Vitamin E (alpha-tocopherol) is a biological antioxidant that protects cells from the inductive effects of reactive oxygen on DNA damage and nuclear/cytoplasmic condensation that dictate apoptosis. Ewes were challenged with a luteolytic dose of PGF2 alpha on d 10 of the estrous cycle. The acute decline in circulatory progesterone indicative of the onset of functional luteolysis was not affected by systemic administration of alpha-tocopherol; however, corpora lutea consequently (beyond 24 h) rebounded from the steroidogenic insult. Luteal tissues obtained at 24 h after PGF2 alpha revealed that internucleosomal DNA fragmentation and cellular collapse were inhibited by alpha-tocopherol. These observations indicate that regressive corpora lutea can be spared from terminal involution by diminishing the apoptotic influence of luteolytic hormone with an antioxidant.

Oestradiol inhibits spontaneous and cisplatin-induced apoptosis in epithelial ovarian cancer cells: relationship to DNA repair capacity.

A prospective role of sex steroid hormones in the pathogenesis of common epithelial ovarian cancer remains equivocal. We hypothesized that oestradiol can protect ovarian cells from apoptosis by augmenting their DNA repair capacity. Two established oestrogen receptor-positive human cancer cell lines of ovarian surface epithelial origin (OVCAR-3, SKOV-3) were studied during short-term (24 h) subculture in the absence or presence of oestradiol-17β and/or the DNA-damaging chemotherapeutic agent cisplatin. Apoptosis was monitored among individual cells by in situ DNA fragmentation analysis. Basal rates of apoptosis were diminished by exposure to oestradiol (progesterone or testosterone were without effect). Oestradiol also suppressed apoptosis induced by cisplatin and enhanced the repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene transfected into ovarian cells. The ability of oestrogen-responsive ovarian cancer cells to efficiently repair DNA and thereby avoid apoptosis may be related to propensity for clonal expansion and drug resistance.

Toward regulation of gonadal function by a synthetic hybrid molecule composed of gonadotropin and Fc fragment of immunoglobulin G.

ABSTRACT: A conjugate of human chorionic gonadotropin (HCG) and Fc fragment of immunoglobulin G was prepared by covalent cross‐linking using the heterobifunctional reagent, N‐succini‐midyl 3‐(2‐pyridyldithio) propionate. Mouse Leydig tumor cells expressing receptors for luteinizing hormone were specifically lysed in vitro as a consequence of complement fixation via the Fc component of the hybrid molecule. Furthermore, administration of HCG‐Fc to rams caused an acute depression in circulatory testosterone. This novel concept of targeted inhibition of gonadal function could prove to have future applications in control of reproductive processes.

Effects of feeding high-linoleate safflower seeds on postpartum reproduction in beef cows.

Two experiments were conducted to evaluate reproductive responses to supplemental high-linoleate safflower seeds in postpartum beef cows. In Exp. 1, 18 primiparous, crossbred beef cows (411 +/- 24.3 kg of BW) were fed Foxtail millet hay starting 1 d postpartum at 1.68% of BW (DM basis) and a low-fat control (control: 63.7% cracked corn, 33.4% safflower seed meal, and 2.9% liquid molasses; DM basis) at 0.35% of BW (n = 9) or a supplement (linoleate) containing 95.3% cracked high-linoleate (79% 18:2n-6) safflower seeds and 4.7% liquid molasses (DM basis) at 0.23% of BW (n = 9). Beginning 1 d postpartum, blood was collected every 3 d for sera. Cows were slaughtered at 37 +/- 3 d postpartum for collection of hypothalami, anterior pituitary glands, liver, ovarian follicles, and uterine tissue. By 37 +/- 3 d postpartum, dietary treatment did not influence ovarian follicular development (P >or= 0.17), hypophyseal concentrations of LH (P = 0.14), or concentrations of IGF-I in liver (P = 0.15). In contrast, anterior pituitary glands from linoleate cows contained more FSH (P = 0.02) than control cows and linoleate cows had less IGF-I in the medial basal hypothalamus (P = 0.05), preoptic area (P = 0.06), and in follicular fluid (P <or= 0.03) from follicles less than 15 mm in diameter. In Exp. 2, twenty-four 3-yr-old multiparous beef cows (initial BW 473.9 +/- 9.2 kg) were fed chopped bromegrass hay at 2.1% of initial BW starting 1 d postpartum and a low-fat supplement (control) fed at 0.6% of initial BW or a high-linoleate supplement (linoleate) fed at 0.4% of initial BW until 80 d postpartum. Cows were observed for estrus twice daily from d 30 to 80 postpartum and treated with GnRH between 40 and 45 d postpartum. Seven days after GnRH administration cows were given PGF(2alpha) and were checked for estrus and artificially inseminated until 80 d postpartum. The magnitude of GnRH-induced release of LH (P = 0.82) or FSH (P = 0.86) did not differ between treatments. However, peak serum concentrations of estradiol during proestrus after treatment with PGF(2alpha) were less (P = 0.04) in linoleate than control cows. In conclusion, fat supplementation with high-linoleate safflower seeds did not improve the development of ovarian follicles and detrimentally affected early postpartum fertility possibly because of a reduction in IGF-I concentrations in tissues essential to reproduction.

Effects of porcine relaxin on induced parturition in beef heifers

Two-year-old crossbred beef heifers were used to test the effects of porcine relaxin (pRelaxin) alone, or in combination with dexamethasone, on the induction of parturition, the incidence of dystocia, and retained placentas. Effects of treatment on pelvic area, postpartum interval, milk production, colostrum quality, calf birth weight, calf vigor, and calf performance were also evaluated. On Day 275 of gestation, heifers from two fetal-sire groups were randomly assigned to one of four groups in a 2 x 2 factorial design and received; no treatment (controls, n = 19), 20 mg of dexamethasone intramuscularly (im) (n = 22), 5 mg of pRelaxin (3,000 U/mg) im (n = 19), or 20 mg of dexamethasone plus 5 mg of pRelaxin (n = 17). Length of gestation (in days) was less (P < 0.05) in heifers treated with dexamethasone (279.8 +/- 1.0) than in controls (286.6 +/- 0.9), but was not influenced (P > 0.05) by treatment with pRelaxin. The incidence of retained placentas in heifers treated only with dexamethasone (27.3%) was not reduced by concomitant treatment with pRelaxin (35.3%). Retained placentas were not observed in any control heifers and in only one heifer (5.2%) treated solely with pRelaxin. Ease of calving (1 = unassisted, 5 = abnormal presentation) was not influenced by treatment (P > 0.05), even though birth weights (in kilograms) of calves from heifers treated with dexamethasone (36.4 +/- 0.8) were less (P < .01) than those of calves from nondexa-methasone-treated heifers (39.2 +/- 0.8). Dexamethasone tended to reduce (P < 0.07) calf vigor (1 = healthy and strong, 5 = dead on arrival; 1.48 +/- 0.11 vs. 1.18 +/- 0.11), but was not (P > 0.05) influenced by pRelaxin. The duration of the postpartum anestrous interval (73.1 +/- 1.8 d across groups) and pelvic areas following treatment and parturition were not influenced (P > 0.05) by dexamethasone or pRelaxin. Although determinants of colostrum quality (P < 0.01) and quantity (P < 0.08) of milk produced were influenced by dexamethasone, adjusted 205-d weights of calves did not differ (P > 0.05) among groups. In conclusion, treatment with pRelaxin alone failed to induce parturition or, when combined with dexamethasone, to reduce the incidence of retained placentas.

Effects of season and estradiol on concentrations of histamine within discrete brain regions of ovariectomized ewes.

Histamine has been implicated as a neuromodulator of secretion of gonadotropins in several species. Concentrations of histamine were analyzed within discrete brain regions and endocrine tissues to help determine whether this amine has the potential to exert a similar function in ewes expected to have dramatically different serum concentrations of LH. Following collection of blood samples at 12-min intervals for 4-hr, ovariectomized (OVX) and OVX-estradiol treated (OVX-E) ewes were slaughtered during the breeding and anestrous seasons (five animals/group). Concentrations of LH were depressed by treatment with estradiol (E; P < .01), but to a greater extent (P < .05) during the anestrous season compared to the breeding season. Concentrations of histamine in tissues (ng/mg) differed (P < .01) between the breeding and anestrous seasons, in the medial thalamus (39.2 +/- 14.1 vs 109.9 +/- 13.0), posterior pituitary gland (247.6 +/- 50.7 vs 23.0 +/- 9.1) and midbrain tegmentum (10.4 +/- 5.6 vs 50.7 +/- 3.9). Estradiol containing implants decreased (P < .05) concentrations of histamine in the midbrain tegmentum (20.3 +/- 7.1 vs 37.7 +/- 7.8) and posterior pituitary gland (87.3 +/- 24.0 vs 258.2 +2- 67.5) compared to non-estradiol treated controls. Histamine concentrations in the pineal and anterior pituitary glands and brain regions; stalk-median eminence, medial basal hypothalamus, preoptic area, cerebellum, parietal neocortex, were not (P > .05) affected solely by either season or E. An interaction between effects of season and estradiol on concentrations of histamine occurred (P < .05) in the posterior pituitary gland and the preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)