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R. B. L. Gwatkin, G. H. Rasmusson and D. T. Williams

Summary.

Fusion of capacitated spermatozoa with the vitelline membrane, but not actual penetration, appears to initiate the cortical reaction in hamster eggs. The reaction can be artificially induced by the application of positively charged particles to the vitelline surface, a situation which may normally be prevented by the zona pellucida. Exposure of hamster eggs to neuraminidase, to lectins (concanavalin A and phytohaemagglutinin-P), to a monovalent ionophore (boromycin) and to 1,3-bis(4chlorocinnamylideneamino)guanidine elicits a cortical granule discharge resulting in a block to fertilization. These agents all appear to act by inducing depolarization of the vitelline membrane.

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H. O. Hoppen, D. M. Williams and J. K. Findlay

Summary.

Eight ewes each with an autotransplanted ovary received infusions of tritium-labelled pregnenolone (41 μCi/hr) for 8 hr into the artery supplying the ovary, together with prostaglandin (PG) F-2α (30 μg/hr) for 3 hr beginning 2 hr after the start of the pregnenolone infusion. All animals exhibited oestrus 2-3 days after the start of the experiment. During the PGF-2α infusion blood flow through the ovaries was increased by 13%, but subsequently returned to pre-infusion levels. Secretion rates of endogenous progesterone and 20α-hydroxypregn-4-en-3-one dropped rapidly 5 hr after the PGF-2α infusion had started from values of 250 μg/hr and 25 μg/hr to values below 60 μg/hr and 8 μg/hr, respectively. At this time the conversion of radioactive pregnenolone to progesterone was reduced by 50% of its initial value, but the secretion of endogenous pregnenolone and the formation of radioactive metabolites other than progesterone were not diminished. In 4 control animals, which received pregnenolone only, no changes in ovarian blood flow, steroid secretion rates, or in the conversion of labelled pregnenolone were observed. These results suggest a possible involvement of PGF-2α in the regulation of progesterone biosynthesis by an action on the 3β-hydroxysteroid oxidoreductase-Δ5−4 isomerase enzyme system.

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H O Goyal, T D Braden, C S Williams and J W Williams

In this review, we report permanent dysmorphogenesis of the penis and loss of fertility in adult rats treated neonatally with estrogen. Specifically, we report replacement of smooth muscle cells and cavernous spaces by fat cells in the corpus cavernosum penis, but not in the adjoining corpus spongiosum. Induction of these novel, region-specific phenotypes is dose-dependent, requires a critical window of exposure and associated with decreased testosterone and up-regulation of estrogen receptor α (ERα). The resistance of ERα knockout mice to develop these abnormalities implies an unequivocal role for ERα in mediating maldevelopment of the penis. Additionally, the prevention of estrogen-inducible penile abnormalities by ER antagonist ICI 182 780 implies that a functional ER-mediated pathway is essential for inducing penile abnormalities. Likewise, the ability of testosterone or dihydrotestosterone to negate these abnormalities suggests a role for an androgen receptor (AR)-mediated pathway. Taken together, these observations led us to hypothesize that neonatal estrogen exposure, via an ER-mediated pathway (direct action) or an AR-mediated pathway (indirect action through decreased testosterone) or both pathways, up-regulates ERα expression in stromal cells of the penis, which are then reprogrammed such that their differentiation into smooth muscle cells is inhibited and their differentiation into adipocytes is stimulated.

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D. Goulding, D. H. Williams, J. F. Roche and M. P. Boland

Superovulation in cattle normally involves the administration of gonadotrophins at specific times of the oestrous cycle, followed by the induction of luteolysis and insemination with high quality semen. The first aim of this experiment was to examine the effect of supplementary progesterone when used in conjunction with porcine FSH (pFSH) to induce superovulation in heifers. The methods compared were PGF given at mid-cycle or a progesterone-releasing intravaginal device (PRID) inserted at different phases of the cycle. The second aim was to determine whether site of insemination or use of fresh or frozen semen affected embryo production. A factorial design was used involving 185 beef heifers. The main factors were (i) synchronization methods PGF or PRID); (ii) semen type (fresh or frozen); (iii) insemination regimens (involving two inseminations and variations in the sites) and number of straws used (one or two) at the second insemination. Eight injections of pFSH were given twice a day for 4 days starting either on days 9, 10 or 11 of the oestrous cycle or on the fourth day after insertion of a PRID. Heifers were checked for oestrus, inseminated twice and embryos were recovered on day 7 of the superovulated cycle. There was no difference between heifers given either PRID or PGF in the oestrous response (93% versus 96%), number of ovulations (15.9 ± 1.11 versus 13.4 ± 1.06), large follicles (2.5 ± 0.24 versus 2.3 ± 0.23) or embryos recovered (9.1 ± 0.77 versus 9.1 ± 0.74). The number of embryos that could be frozen was lower (P = 0.05) in heifers given PRID. The stage of the cycle at which the PRID was inserted affected the number of ovulations, large follicles and embryos recovered (P < 0.04). The use of fresh or frozen semen had no effect on the number of embryos recovered, but the use of frozen semen resulted in fewer grade 1 and 2 embryos and more grade 4 and 5 embryos in PRID-treated heifers. The number of straws did not affect the number or quality of embryos recovered. In conclusion, the use of a PRID, inserted at different stages of the cycle, in conjunction with PGF and pFSH resulted in fewer freezable embryos recovered compared with the use of PGF and pFSH given at mid-cycle. The use of frozen semen did not affect the number or quality of embryos recovered following the use of PGF and pFSH at mid-cycle, but it did decrease the number of grade 1 and 2 embryos recovered following the use of PRID and pFSH; the number (two versus three) of straws used did not affect the yield or quality of embryos recovered.

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H O Goyal, T D Braden, P S Cooke, M A Szewczykowski, C S Williams, P Dalvi and J W Williams

Previously, we reported an association between estrogen receptor-α (ERα) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERα knockout (ERαKO) mouse model to test the hypothesis that ERα mediates DES effects in the developing penis. ERαKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 μg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80–240 days of age and tissues were examined at 96–255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERαKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERαKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERαKO mice when compared with controls, although testosterone concentration was much higher in the ERαKO mice. Hence, the resistance of ERαKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERα in mediating the harmful effects of neonatal DES exposure in the developing penis.

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F. Ellendorff, M. Forsling, N. Parvizi, H. Williams, M. Taverne and D. Smidt

Summary. A single i.m. injection of 5 mg PGF-2α evoked a significant elevation of plasma oxytocin values in sows 6 days post partum and during dioestrus. Plasma vasopressin levels in dioestrous sows were not significantly affected by PGF-2α. It is concluded that circulating steroid levels do not interfere with the response of oxytocin levels to PGF-2α.

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A. R. Scanlon, S. J. Sunderland, T. L. Martin, D. Goulding, D. O'Callaghan, D. H. Williams, D. R. Headon, M. P. Boland, J. J. Ireland and J. F. Roche

Two experiments were conducted in cyclic beef heifers to determine whether active immunization against bovine inhibin α 1–26 Gly-Tyr (bINH) affected follicular dynamics, hormone concentration or ovulation rate. In Expt 1, heifers (n = 9) were actively immunized against bINH conjugated to human α globulins (HAG) using bisdiazotized benzidine in non-ulcerative Freund's adjuvant (NUFA; primary on day 0; booster injections on days 53, 84 and 116 using conjugated bINH and on days 176 and 366 using unconjugated bINH; ten heifers were used as controls). Ovaries were examined daily using ultrasound scanning (days 70–155 and 384–391) and corresponding blood samples were collected for bINH antibody titre, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestradiol determinations. Four treated and four control heifers were injected with 10 μg gonadotrophin-releasing hormone (GnRH) on day 386 (day 2 of the oestrous cycle). Although bINH-immunized heifers had variable antibody titres ranging from 4 to 50% I125-labelled bINH bound to serum diluted 1:2000, ovulation rate was unaffected. In oestrous cycles with three dominant follicles, the ovulatory follicles grew faster (2.5 ± 0.2 versus 1.6 ± 0.3 mm day−1; mean ± sem), had shorter durations of growth (5.7 ± 0.8 versus 9.6 ± 1.6 days) and duration of detection (7.5 ± 0.8 versus 12.0 ± 2.4 days) in immunized heifers. Mean concentrations of FSH, LH and oestradiol were unaltered in most cases during oestrous cycles in bINH-immunized compared with control heifers. There was no significant difference in the percentage increase in FSH or LH, after GnRH injection, between control and immunized heifers. As ovulation rate was unaltered in the first experiment, a second similar study was designed using a different immunization protocol. In Expt 2, heifers were immunized with bINH conjugated to human serum albumin using glutaraldehyde with the following doses: 0.0 (control; n = 7), 0.33 (n = 7), 1.0 (n = 8) and 3.0 (n = 7) mg. Three booster immunizations were given 33, 66 and 209 days after primary immunization. Immunization increased the number of oestrous cycles with multiple ovulations (42 of 132 (32%) oestrous cycles examined) compared with controls (1 of 30 (3.3%) oestrous cycles examined). Neither titre nor ovulation rate was affected by dose of bINH used. In summary, following bINH immunization, ovulation rate was not increased despite changes in follicular dynamics in Expt 1, but was increased in 32% of oestrous cycles in Expt 2. We conclude that immunization protocols can affect responsiveness of heifers to bINH immunization, and that immunization against inhibin can increase ovulation rate.

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M Amstalden, D A Zieba, M R Garcia, R L Stanko, T H Welsh Jr, W H Hansel and G L Williams

Experiments were performed to test the hypothesis that lamprey GnRH-III (lGnRH-III) selectively releases FSH. Primary cultures of bovine adenohypophyseal cells were treated with mammalian GnRH (mGnRH) and lGnRH-III (10−9, 10−8, 10−7 and 10−6 M) or control media in Experiment 1. All doses of mGnRH and the two highest doses of lGnRH-III stimulated (P < 0.001) a non-selective release of LH and FSH. In Experiments 2–4, Latin Square designs were utilized in vivo to examine whether physiological and hormonal milieu regulate putative selective effects of lGnRH-III. In Experiments 2 and 3, ovariectomized cows with basal levels of estradiol only (Experiment 2) or in combination with luteal phase levels of progester-one (Experiment 3) were injected with mGnRH and lGnRH-III (0.055, 0.11, 0.165 and 1.1 μg/kg body weight (BW) and saline. All doses of mGnRH released (P < 0.001) LH and FSH, but only the highest dose of lGnRH-III stimulated (P < 0.001) a non-selective release of both LH and FSH (Experiment 3). For Experiments 4A and 4B, intact, mid-luteal phase cows were injected with mGnRH and lGnRH-III (1.1 μg/kg BW; Experiment 4A), lGnRH-III (1.1 and 4.4 μg/kg BW; Experiment 4B) and saline. As before, mGnRH released (P < 0.001) both LH and FSH at all doses. In contrast, lGnRH-III at the highest dose released (P < 0.001) LH but not FSH. These findings suggest that lGnRH-III may act as a weak competitor for the mGnRH receptor and do not support the hypothesis that it selectively releases FSH in cattle.