Retinol administration to superovulated ewes improves in vitro embryonic viability.

Retinol and its metabolites, all-trans retinoic acid and 9-cis retinoid acid, are regulators of cellular growth, differentiation, and development and have been implicated in reproductive processes including folliculogenesis and embryonic survival. Three experiments were conducted to identify effects of retinoid treatment of superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS), or vehicle (Control) on the first and last day of FSH treatment. Embryos were recovered at the morula stage, cultured in vitro for 96 h, and observed for blastocyst formation. Embryos from ROH-treated animals had a higher (p < 0.01) incidence of blastocyst formation than RA-, CIS-, or vehicle-treated animals (72% vs. 27%, 33% and 32%, respectively). In experiment 2, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs. 22%, p < 0.05). In experiment 3, ewes were treated with ROH or vehicle, and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs. 5%, p < 0.05). The majority of embryos (60% vs. 6%; p < 0.01)) from vehicle-treated animals failed to develop beyond the 8-cell stage in comparison with those from ROH animals. ROH treatment of superovulated ewes increased embryonic viability and positively impacted embryonic development.

Steroid regulation of retinol-binding protein in the ovine oviduct.

Two studies were conducted to identify retinol-binding protein (RBP) expression in the ovine oviduct and to determine the role of ovarian steroids in its regulation. Ewes were salpingectomized on Days 1, 5, or 10 of their respective estrous cycles, and oviducts were homogenized for RNA analysis, fixed for immunocytochemistry (ICC), or cultured for 24 h for protein analysis. ICC localized RBP to the epithelium of all oviducts. RBP synthesis was demonstrated by immunoprecipitation of radiolabeled RBP from the medium of oviductal explant cultures. Explant culture medium from oviducts harvested on Day 1 contained significantly more RBP than medium from oviducts collected on Days 5 or 10. Slot-blot analysis demonstrated that steady-state RBP mRNA levels were significantly higher on Day 1 than Day 5 or 10. In the second experiment, ovariectomized ewes were treated with estradiol-17beta (E2), progesterone (P4), E2+P4 (E2+P4), or vehicle control, and oviducts were analyzed as above. P4 alone or in combination with E2 significantly reduced steady-state RBP mRNA levels compared to those in E2-treated animals. Oviductal explants from E2- and E2+P4-treated animals released 3- to 5-fold more RBP into the medium than control and P4 treatments as determined by ELISA. RBP synthesis of metabolically labeled RBP was increased by E2 and E2+P4 treatments. This study demonstrates that P4 applied on an estradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.

Immunolocalization of retinol-binding protein and profiles of its mRNA as related to testicular development in the beef bull.

A study was conducted to identify the cell types that express retinol-binding protein (RBP) in the bovine testis and to compare relative steady-state levels of RBP mRNA expression at different times of testicular development. At the ages of 10 (n = 3), 20 (n = 8), and 34 (n = 7) wk, Angus bulls were bled three times at 1.5-hr intervals, then surgically castrated. Blood samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T), by radioimmunoassay and the degree of seminiferous tubule development was evaluated histologically in sections of fixed tissue samples stained with hematoxylin and eosin. Immunolocalization of RBP was based on the biotin-strepavidin-horseradish peroxidase method. Testis weight and concentrations of LH and T increased with age (P < 0.05), but those of FSH did not change (P > 0.05) between 10 and 34 wk. Seminiferous tubules at 10 wk contained immature Sertoli cells and gonocytes whereas, at 20 wk, spermatogonia and few spermatocytes were detected. At 34 wk, Sertoli cells appeared differentiated and spermatids were observed. RBP was immunolocalized in Sertoli, Leydig, and peritubular cells at the ages of 10, 20, and 34 wk. Furthermore, no differences in staining between Sertoli cells from tubules with or without germ cells were detected. Northern hybridization of testicular RNA with an RBP cDNA probe revealed the presence of a 1.4-Kb mRNA, which was similar to previous RBP transcripts found in other bovine tissues. Quantitative slot blot analysis revealed that steady-state RBP mRNA levels were 50% higher at 10 wk (P < 0.05) than at 20 and 34 wk of age.

Oviductal fluid and growth factors failed to enhance development of porcine embryos.

Porcine embryos (1-, 2- and 4-cell) were cultured in a basal medium consisting of Krebs-Ringer bicarbonate buffer supplemented with oviductal fluid and several growth factors and observed for further development. Oviducts were flushed at either 48 h (Experiment 1) or 96 h (Experiment 2) after the onset of estrus. Observations were made every 48 h (Experiment 1) or 12 h (Experiment 2) until failure of the embryos to develop for 2 consecutive observations. Embryos were scored 0 = no development, 1 = cleavage, 2 = morula, 3 = blastocyst, or 4 = hatched blastocyst. In the first experiment, development of 1-, 2- and 4-cell embryos (n=282) in the basal medium supplemented with oviductal fluid (4:1) or 3 sets of growth factors, was less or equal to one cleavage stage. Those embryos cultured in the basal medium supplemented with bovine serum albumin (fatty acid free) (BSA) advanced to the blastocyst stage. In the second experiment, 96 h aged embryos (n=142) were cultured in the basal medium supplemented with IGF-1 and - 2 and EGF, or with BSA alone or with BSA and the three growth factors. In the treatments without BSA, the embryonic development was less than one cleavage, whereas in those treatments with BSA, embryos advanced beyond hatching and began to expand. We conclude that for culture of porcine embryos, supplementation with several growth factors or with oviductal fluid, in the concentration used in this study, was of little benefit at this stage of development. However, the type of BSA significantly affected development. More than 90% of the embryos reached the morula and blastocyst stages in medium than included BSA (fatty acid free).

Identification and content of insulin-like growth factors in porcine oviductal fluid.

Oviductal fluid (OvF) was collected from gilts by indwelling catheters during the estrous cycle and analyzed for content of insulin-like growth factors (IGF-I, IGF-II). Group 1, composed of 9 gilts in a summer environment near a boar, yielded mean daily fluid volumes of 1.18 +/- 0.16 ml during estrus and 0.69 +/- 0.03 ml post-estrus. Group 2, composed of 7 gilts in a moderate-temperature, light-regulated room, yielded 1.20 +/- 0.18 ml during estrus and then OvF flow essentially stopped. Serum samples were also collected 2 times daily during the cycle and analyzed along with the OvF for IGF-I and IGF-II. Serum was also analyzed for estradiol-17 beta (E2). For group 1, OvF content (concentration x fluid volume) of IGF-I and IGF-II was greater (p less than 0.05) at estrus than pre- and post-estrus. Daily mean content values (ng/day) for IGF-I and IGF-II during peri-estrus were 30.9 +/- 6.3 and 62.2 +/- 12.3, respectively. During nonestrus, values were 6.8 +/- 6.3 and 11.7 +/- 12.3, respectively. For group 2, OvF content of IGF-I and IGF-II during estrus was similar to that of group 1. Whereas IGF content differed between estrus and nonestrus periods, IGF concentrations were similar (p greater than 0.05), a finding that results from the difference in OvF produced. Compared with OvF concentrations of IGF for a given pig, blood plasma concentrations of IGF-I and IGF-II were 2- to 5-fold higher in the plasma sample collected the same day.(ABSTRACT TRUNCATED AT 250 WORDS)