Reproduction

Mann, G. E.; Lamming, G. E.

doi: 10.1530/jrf.0.1040001pmid: 7636790

Mated cows that subsequently undergo luteolysis have significantly lower plasma concentrations of progesterone than do cows in which pregnancy is maintained. Two groups of ovariectomized cows were treated with progesterone and oestradiol to recreate luteal phases exhibiting the extremes of plasma concentrations of progesterone (6.0 ± 0.4 and 12.4 ± 0.8 ng ml−1) normally seen in intact cows. The effect of this difference in progesterone on the development of the luteolytic signal was determined by monitoring the basal and oxytocin-stimulated plasma concentrations of the principal metabolite of prostaglandin F2α, PGFM on days 12–16 of the simulated luteal phases. Basal PGFM concentrations were similar in the low and high progesterone groups on all days. The mean plasma concentration of PGFM showed a larger increase following oxytocin in the low progesterone group on days 13 and 14 (P < 0.05) and on days 15 and 16 (P < 0.1). These results demonstrate that a low plasma concentration of progesterone results in the development of a stronger luteolytic signal, and provide an explanation for the fact that cows with lower plasma concentrations of progesterone are more prone to embryo loss.

Auclair, D.; Sowerbutts, S. F.; Setchell, B. P.

doi: 10.1530/jrf.0.1040007pmid: 7636808

Merino ram lambs were actively immunized against oestradiol-6 (o-carboxy methyl) oxime–BSA conjugate at 14 weeks of age and received a booster injection 4 weeks later. This treatment led to an increase in plasma concentrations of gonadotrophin and tended to enhance the increase in testicular volume until 26 weeks of age; however, testis size and mass at time of castration (30 weeks of age) were similar to values in BSA-immunized lambs. Detrimental effects were observed in some oestradiol-immunized ram lambs, for example a steep decline in testicular volume towards the end of the experiment, the presence of large vacuoles within the seminiferous epithelium and, in one lamb, few germ cells at 30 weeks of age. Testicular blood plasma flow was significantly reduced in oestradiol-immunized lambs (P < 0.01). The steroidogenic function of the testis was markedly enhanced in oestradiol-immunized lambs as reflected by high plasma concentrations of testosterone measured at 22, 26 and 30 weeks of age and by high testosterone production calculated from blood flow and venous–arterial differences at 30 weeks of age. Nevertheless, total live mass gain over the 16 week study was not increased in oestradiol-immunized lambs. Testicular biopsies were taken at 22 and 26 weeks of age in half of the lambs in each treatment group. Testicular volume measured at castration was decreased in control lambs in which biopsies were taken (P < 0.05), and plasma concentrations of testosterone measured at 30 weeks of age were significantly lower in oestradiol-immunized lambs in which biopsies were taken (P < 0.02) compared with lambs in which no biopsy had been taken. It is concluded that active immunization against oestradiol in ram lambs does not advance the time of onset of puberty and does not confer any reproductive or maturational advantages.

Auclair, D.; Sowerbutts, S. F.; Setchell, B. P.

doi: 10.1530/jrf.0.1040017pmid: 7636799

The long-term effects of active immunization against testosterone were studied in rams, with particular reference to blood concentrations of gonadotrophin and testosterone, spermatogenesis, testis blood flow and mating behaviour. Ten 18-month-old Merino rams, kept on pasture, were studied for 1 year. Every 2 months, five rams received injections of BSA in Freund's adjuvant and five other rams were treated with testosterone-3(o-carboxymethyl)oxime–BSA as immunogen. Anti-testosterone antibodies (mean titre: 1:4484 ± 582, after boosters) were maintained in the circulation, with the help of regular booster injections. In time, immunization reduced live mass in testosterone-immunized rams; however, there was no effect on testicular volume throughout the whole study. In testosterone-immunized rams, significantly higher concentrations of gonadotrophins were found in jugular venous plasma, as well as increased concentrations of total plasma testosterone. LH pulse frequency, amplitude and nadir were increased significantly in testosterone-immunized rams. After 12 months of immunization, no differences were found in the number of spermatozoa per ejaculate, in daily sperm production or in testis mass between the two groups of rams; however, testicular blood flow (per testis) and epididymis mass were significantly reduced in testosterone-immunized rams. Testosterone immunoneutralization also resulted in a significant reduction in the number of mounts culminating in ejaculation performed during a 10 min trial carried out on a number of occasions during the experiment. Additional information on these rams was collected 3 months after castration. However, there were no significant differences in mean plasma LH and FSH concentrations, either before, or after, a single GnRH injection between the two groups of rams at this time.

Phillips, D. J.; Moore, L. G.; Hudson, N. L.; Lun, S.; McNatty, K. P.

doi: 10.1530/jrf.0.1040027pmid: 7636801

This study investigated the effects of short-term (20 days) ovariectomy, the effects of FSH assay (radioimmunoassay, receptor assay or in vitro bioassay) and of FecBB genotype on the characteristics of pituitary FSH from Booroola ewes. Pituitary extracts were obtained from ovariectomized homozygous carriers (BB) and non-carriers (++, n = 8 per genotype) and ovary-intact controls (n = 4 per genotype). The extracts (n = 4 per genotype per treatment) were subjected to agarose suspension electrophoresis and the eluates were assayed by the three FSH methods. There were significant effects of ovariectomy (P < 0.01) and assay system (P < 0.05) but not of genotype on the median charge of FSH isoforms. The mean ± sem migration rates for FSH in intact and ovariectomized ewes were 0.469 ± 0.006 and 0.439 ± 0.006 albumin mobility units, respectively (P < 0.01), indicating a shift to more basic isoforms after short-term ovariectomy. When the pituitary extracts were subjected to anion-exchange HPLC, there was a significant (P < 0.01) shift to more basic isoforms in the ovariectomized ewes as shown using agarose electrophoresis, and no gene effects were noted. When the pituitary extracts (n = 4–8 per group) were injected into mature female mice, there were no significant effects of ovariectomy or genotype on the circulating half-lifes of the pituitary FSH isoforms. These results indicate that after short-term ovariectomy, the increase in plasma FSH concentrations is accompanied by a shift in the median charge of pituitary FSH isoforms without any observable change in their metabolic clearance rates. Moreover, the FecBB gene has little effect on the median charge or half-life of pituitary FSH.

Rhodes, F. M.; Fitzpatrick, L. A.; Entwistle, K. W.; Kinder, J. E.

doi: 10.1530/jrf.0.1040033pmid: 7636802

Changes in pulsatile secretion of LH, FSH, oestradiol and progesterone were related to the growth phase, early plateau phase and regression phase of the first ovarian dominant follicle of the oestrous cycle in Bos indicus heifers. Relationships between these hormones during the three phases were also investigated. Accurate measurements of episodic ovarian steroid secretion were obtained by catheterizing the caudal vena cava via the lateral saphenous vein; the tip of the catheter was positioned just cranial to the ovarian vein using transrectal ultrasonography. Pulsatile secretion of oestradiol was increased only during the growth phase of the dominant follicle and was associated with high frequency release of LH pulses. However, mean concentrations of oestradiol were reduced when the dominant follicle attained its maximum diameter. Between the growth and plateau phases, as the amount of progesterone released increased and oestradiol released decreased, LH pulse frequency and mean concentration of LH decreased. Pulses of LH released were followed within 15 min by increases in mean concentrations of oestradiol (P < 0.001); however, there was no apparent relationship between LH and progesterone release (P = 0.19). Although there was little evidence of pulsatile release of FSH, mean concentrations of FSH were increased by 0.2 ng ml−1 (P = 0.04) during the plateau phase, which was on average 2.1 days before the day of emergence of the second dominant follicle of the oestrous cycle. This increase in FSH, in conjunction with the decrease in secretion of oestradiol, may be an indication of the loss of functional dominance by the first dominant follicle of the oestrous cycle.

Rhodes, F. M.; Fitzpatrick, L. A.; Entwistle, K. W.; De'ath, G.

doi: 10.1530/jrf.0.1040041pmid: 7636803

This study examined the effects of live mass on ovarian follicular dynamics in heifers before and after the onset of nutritional anoestrus. The effects of decreased live mass on secretion of progesterone, LH and FSH were also investigated. Ovarian characteristics were monitored in 16 postpubertal Brahman heifers using daily transrectal ultrasonography. After two normal oestrous cycles, 11 heifers (treatment group) were allocated to a restricted diet, until the cessation of their oestrous cycles, that resulted in a decrease in live mass of 0.8 kg day−1. Heifers were then fed an ad libitum diet, resulting in increasing live mass until resumption of oestrous cycles. Five heifers (control group) were fed an unrestricted diet throughout the experimental period. The concentrations of progesterone in plasma were monitored twice a week and the concentrations of LH and FSH were determined on day 6 after ovulation, before initiation of dietary restriction, after a 17% decrease in live mass and after the onset of anoestrus in the treatment group. Onset of anoestrus was preceded by linear decreases in the size of ovarian follicles and corpora lutea, and in the persistence of the first dominant follicles of oestrous cycles. These changes were proportional to the decrease in live mass (P < 0.001). The frequency of pulses of LH or mean concentration of FSH on day 6 of the oestrous cycle were not changed after a 17% decrease in live mass, and did not increase following the onset of anoestrus, although concentrations of progesterone were < 0.2 ng ml−1, suggesting that release of LH was suppressed at this time. Increasing live mass following anoestrus resulted in a linear increase in dominant follicle size and persistence, which were correlated with changing mass (P < 0.001); ovulation resumed when dominant follicles were 105 ± 4% of the size of initial ovulatory follicles.

Parkinson, T. J.; Douthwaite, J. A.; Follett, B. K.

doi: 10.1530/jrf.0.1040051pmid: 7636804

Thyroidectomy of seasonally breeding birds and mammals prevents the return to a state of sexual quiescence at the end of the breeding season. In starlings, thyroidectomy also causes premature sexual maturity. In this study, the effect of thyroidectomy upon the time of sexual maturity of prepubertal (8 week-old) ram lambs was examined. Thyroidectomy of four prepubertal and six mature rams was performed early in the spring. These and sham-operated controls were maintained in ambient photoperiods (south-west England). Scrotal circumference and serum LH, FSH, prolactin and thyroxine were measured every 2 weeks. In both the prepubertal lambs and the mature rams, scrotal circumference increased significantly within 5 weeks of thyroidectomy. FSH concentrations increased significantly in the mature rams after thyroidectomy. The relatively high FSH concentrations of thyroidectomized animals at the start of the experiment were maintained, but the FSH concentrations of intact lambs decreased during the late spring. These results provide the first indication that the timing of puberty in seasonally breeding mammals is a thyroid-dependent phenomenon.

Bany, B. M.; Kennedy, T. G.

doi: 10.1530/jrf.0.1040057pmid: 7636805

The effect of epidermal growth factor on prostaglandin production and cyclooxygenase activity in endometrial stromal cells isolated from the uteri of ovariectomized rats sensitized for the decidual cell reaction was examined. Treatment with epidermal growth factor (40 ng ml−1) for 24 h caused approximately 6.9-, 3.4-, and 4.1-fold increases in prostaglandin E2, prostaglandin F2α and 13,14-dihydro-15-keto-prostaglandin F2α accumulation in the media of cultured cells. The increase in prostaglandin E2 accumulation induced by epidermal growth factor was inhibited by α-amanitin (2 μg ml−1), cycloheximide (0.5 μg ml−1) and dexamethasone (5 μmol l−1). Epidermal growth factor increased cyclooxygenase activity in the stromal cells in a time-dependent fashion and this increase in activity was also inhibited by α-amanitin, cycloheximide and dexamethasone. These results provide evidence that epidermal growth factor stimulates prostaglandin production in rat endometrial stromal cells from uteri sensitized for the decidual cell reaction through a mechanism that involves an increase in cyclooxygenase activity.

Wakayama, T.; Suto, J.; Matubara, Y.; Imamura, K.; Fukuta, K.; Toyoda, Y.; Kurohmaru, M.; Hayashi, Y.

doi: 10.1530/jrf.0.1040063pmid: 7636806

Optimal conditions for in vitro fertilization of Japanese field voles (Microtus montebelli) were analysed. The medium used was a modified Krebs–Ringer bicarbonate devised for in vitro fertilization in rats. Ovulated eggs and epididymal spermatozoa were co-incubated in vitro at 37°C under 5% CO2 in air for 6 h, and the eggs were fixed with 2.5% (w/v) glutaraldehyde, stained with 0.25% (v/v) acetolacmoid and examined for evidence of fertilization at the pronuclear stage. Although the fertilization rate with spermatozoa preincubated at 1–2 × 108 cells ml−1 for 2 h was very low (1–13%), it was significantly increased (43–51%, P < 0.05) when spermatozoa were preincubated at a lower concentration (1–2 × 107 cells ml−1). Furthermore, the fertilization rate was significantly higher with 1 mmol hypotaurine l−1 (74.0%) than without hypotaurine (44.4%, P < 0.05). Fertilization rates of spermatozoa preincubated at 1–2 × 107 cells ml−1 for 0.5 or 2 h were similar (69.0% and 73.6%), but a longer preincubation (10 h) resulted in a significantly lower fertilization rate (56.8%, P < 0.01). Vole spermatozoa preincubated for 2 h penetrated the zona pellucida 2 h after insemination, and the sperm heads became decondensed 3 h after insemination. At 6 h after insemination, male and female pronuclei were found in most penetrated eggs. When the eggs were left in the fertilization medium without washing and cultured for 96 h after insemination, they developed to two-cell (82.6%), four-cell (60.9%), eight-cell (23.2%) and morula/blastocyst (8.7%) stages in modified Krebs–Ringer bicarbonate supplemented with 1 mmol hypotaurine l−1.

Manes, C.; Lai, N. C.

doi: 10.1530/jrf.0.1040069pmid: 7636807

A minimum value for nonmitochondrial oxygen utilization in rabbit blastocysts at day 6 post coitum was determined by measuring oxygen consumption in the presence of cyanide. A microcathode oxygen electrode was used to monitor oxygen concentration continuously during blastocyst incubation in a newly devised culture medium, and the uninhibited blastocyst was found to consume 2.79 ± 0.09 μl O2 h−1 cm−2. This rate was reduced by 51% in the presence of 1 mmol KCN l−1. The addition of nitroblue tetrazolium to the cyanide-containing medium reduced net oxygen consumption by an additional 23% as the nitroblue tetrazolium was reduced to formazan. The ability of rabbit blastocysts to reduce nitroblue tetrazolium in the presence of cyanide was investigated using a spectrophotometric assay. Fractionation of blastocyst cells revealed that the enzymatic activity chiefly responsible for formazan production partitioned with the membrane/particulate fraction and could be solubilized by the detergent NP40. The enzyme was NAD(P)H-dependent, did not require divalent cations for activity, and appeared to contain no haeme moiety. The rate of formazan production in the spectrophotometric assay was markedly reduced by the presence of superoxide dismutase. The oxygen electrode and spectrophotometer data indicate that there is a superoxide-generating NAD(P)H oxidase on the blastocyst surface. Calculations based on the average surface area of rabbit blastocysts at day 6 show that these embryos can produce at least 8 nmoles of superoxide per embryo h−1. Potential deciduogenic effects of blastocyst-derived superoxide and its dismutated product, hydrogen peroxide, are discussed.