Attainment of puberty, cycle lengths, ovulation rate and endocrinology during the periovulatory period were studied in Meishan (MS) and European Large-White hybrid (LW) gilts. The mean age at onset of puberty of 115 days in MS (n = 20) gilts was younger (P < 0.001) than the 235 days in LW (n = 23). In the MS population studied, ovulation rate was not different (P > 0.1) during the third and fourth oestrous cycles, nor were there differences (P > 0.1) in the mean cycle length over the first three cycles. Overall changes in plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestradiol did not differ significantly (P > 0.1) between the breeds (MS, n = 6; LW, n = 5) during the periovulatory period, but plasma inhibin concentrations were significantly (P < 0.05) higher in the MS. The time intervals from the oestradiol peak concentration and the onset of the LH surge until the onset of behavioural oestrus were significantly different (P < 0.005) between the breeds, with oestrus occurring earlier in the MS. However, no difference (P > 0.1) was found between the groups when the intervals from the peak oestradiol concentration to the onset of the LH surge were compared. These results indicate differences between the breeds, particularly in terms of the age of attainment of puberty and the timing of the onset of behavioural oestrus relative to the oestradiol and LH surges.
M. G. Hunter, C. Biggs, G. R. Foxcroft, A. S. McNeilly and J. E. Tilton
B. K. Campbell, H. Engelhardt, A. S. McNeilly, L. M. Harkness, M. Fukuoka and D. T. Baird
The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin α and βA′ βB subunits, P450 aromatase and P450 17α-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 μg cloprostenol on days 10–12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n=7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin–activin βB subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin–activin α and βA′ subunit or P450 17α-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin βB and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.
D. P. Gilmore, H. G. Dobbie, A. S. McNeilly and C. H. Mortimer
Summary. Immunoreactive LH-RH was present in all the hypothalamic and cortical extracts of mid-term human fetuses studied and in the cortical tissue removed from the two youngest fetuses. Gonadotrophin-releasing activity of hypothalamic and cortical extracts was demonstrated by the significant rises of circulating LH after infusion into oestrogen and progesterone-primed ovariectomized rats.
R. B. Land, W. R. Carr, A. S. McNeilly and R. D. Preece
Summary. The effects of pharmacological reduction of the high plasma prolactin concentration typical of seasonal anoestrus in sheep were assessed with respect to positive feedback of oestrogen on LH release, ovulation, and progesterone secretion. Treatment of 16 Scottish Blackface ewes with 1 mg bromocriptine, i.m. twice daily for 12 days, reduced prolactin concentrations in peripheral plasma from 64 ± 10 ng/ml before treatment to <4 ng/ml. This treatment had no effect on the proportion of ewes discharging LH and FSH in response to 12·5 μg oestradiol benzoate (3/8 before compared with 5/16 during treatment) or the proportion of ewes ovulating in response to oestrogen treatment. Plasma progesterone concentrations remained low even in ovulating ewes.
It is concluded that treatment with bromocriptine alone is unlikely to restore oestrous cycles to ewes in seasonal anoestrus.
P. M. Collins, W. P. Collins, A. S. McNeilly and W. N. Tsang
Summary. Rats were treated by exposure of the scrotum to a temperature of 43°C for 30 min or bilateral ligation of the vasa efferentia and bled at 0, 3, 7, 14 and 21 days after treatment. In heat-treated rats FSH levels rose linearly from pretreatment levels while those in efferentiectomized animals remained unchanged for 3 days before increasing. In both groups FSH concentrations reached similar maximum values after 7 days and were significantly higher than those of intact controls at 7, 14 and 21 days. LH levels, although not generally different from those in the controls, rose from pretreatment levels in parallel with FSH. No differences were found in testosterone concentrations in any of the groups. Histological examination at 3 weeks after treatment confirmed that the germinal epithelium consisted mainly of spermatogonia and Sertoli cells. The cytological appearance and lipid content of the Leydig cells of the aspermatogenic testes were indistinguishable from those of the controls and the weight and histological appearance of the accessory sex organs and the fructose content of the coagulating glands were also normal. It is concluded that the sterilizing effects of heat treatment and efferentiectomy are independent of changes in Leydig cell function and that the increase in gonadotrophin levels is related to the germ cell degeneration.
B. K. Campbell, H. M. Picton, G. E. Mann, A. S. McNeilly and D. T. Baird
Summary. Treatment of ewes with steroid-free ovine follicular fluid (oFF) during the follicular phase of the oestrous cycle results in the immediate inhibition of the ovarian secretion of oestradiol, inhibin and androgens. An experiment was conducted to determine whether this effect of oFF was due to inhibin, or to direct inhibition of ovarian function by other factors in oFF.
Eight ewes in which the left ovary and vascular pedicle had been autotransplanted to a site in the neck were studied during the breeding season. Luteal regression was induced in all animals by injection of cloprostenol (100 μg i.m.; PG) on Day 10 of the luteal phase. The animals were divided into two groups (n = 4) and treated with either steroid-free oFF (oFF; 3 ml s.c.; 3·2 μg p1-26α inhibin/ml) or steroid-free oFF in which the inhibin content had been reduced by >90% (IFoFF; 3 ml s.c.; 0·3 μg p1-26α inhibin/ml) by affinity chromatography, 24 and 36 h after PG. Samples of ovarian and jugular venous blood were collected at (i) intervals of 4 h from 16 h before until 120 h after PG and (ii) intervals of 10 min from 48 to 52 h after injection of PG to investigate the pattern of pulsatile secretion of ovarian hormones. All ewes had previously been monitored during a normal PG-induced follicular phase.
Injection of oFF resulted in an increase (P < 0·05) in the concentration of inhibin in jugular venous plasma and a profound (P < 0·001) and prolonged decrease in the peripheral concentration of follicle-stimulating hormone (FSH). Injection of IFoFF had no significant effect on peripheral concentrations of inhibin or FSH in the first 24 h after treatment; thereafter inhibin concentrations fell (P < 0·01) progressively until 40 h and then increased (P < 0·01) until 72 h after treatment. In both treatment groups, however, within 24–36 h of treatment the concentration of FSH increased 5–10-fold (P < 0·001) to a peak that occurred within 48–60 h and then declined to basal concentrations within 72–84 h of treatment. The concentration of luteinizing hormone (LH) in jugular venous plasma increased in both groups after treatment (P < 0·01), although the rise after injection of oFF only started after 24 h. Thereafter, there was a progressive increase in the concentration of LH, peaks occurring 48–60 h after treatment. The preovulatory LH surge was delayed by ∼48 h in both groups of ewes, being detected at 114 ± 5 h and 110 ± 4 h after PG in ewes treated with oFF and IFoFF, respectively (P < 0·001). The effects of oFF and IFoFF on ovarian hormone secretion were similar, the secretion rate of oestradiol, androstenedione and inhibin beginning to decline within 8 h of the first injection (P < 0·001). The rate of decrease was more marked for the steroids, a mean nadir of < 1 ng/min being reached within 12–24 h of treatment whereas inhibin secretion did not reach a nadir until 36–44 h after treatment.
Androstenedione secretion began to increase (P < 0·05) within 24 h of treatment whereas inhibin and oestradiol secretion did not begin to increase significantly (P < 0·05) until 48–60 h after treatment. Generally, ovarian hormone secretion tended to increase until 72 h after treatment, after which mean secretion rates reached a plateau. In ewes treated with oFF or IFoFF the episodic secretion of oestradiol and inhibin was markedly suppressed and the amplitude of androstenedione pulses reduced, although the amplitude of LH pulses was greater than in normal untreated ewes. Injection of oFF or IFoFF caused a marked decline (P < 0·01) in the number of large follicles within 48 h of treatment and, concomitant with the rebound release of FSH, an increase (P < 0·01) in the number of small follicles/ovary. By 96 h after treatment, the number of large follicles/ewe had increased (P < 0·05) to pretreatment levels, concomitant with a decline (P < 0·05) in the number of small follicles. In individual animals, changes in gonadotrophin concentrations, ovarian hormone secretion rates and ovarian follicle populations were temporally related.
We conclude that oFF contains a factor that acts directly on the ovary to induce atresia of large preovulatory follicles and results in immediate suppression of ovarian inhibin, oestradiol and androstenedione secretion. The lack of feedback by ovarian hormones results in a rebound release of FSH, which stimulates the growth of ovarian follicles, and the re-establishment of ovarian hormone secretion and normal cyclicity.
Keywords: follicle-stimulating hormone; inhibin; oestradiol; androgen; follicular fluid; sheep
A. S. McNeilly, R. D. Martin, J. K. Hodges and G. L. Smuts
Summary. No seasonal variation in any of the hormones measured was apparent in males or females. Testosterone levels in males increased around puberty (10–11 years) and remained significantly higher in adult than prepubertal males. This was not accompanied by any significant change in levels of LH, FSH or prolactin.
In non-pregnant females there was no apparent difference in levels of LH, FSH or prolactin with age. There was a significant increase in progesterone around puberty (12 years) but there was considerable overlap in values between prepubertal and adult females.
During pregnancy, progesterone levels were significantly higher than in non-pregnant females with maximum levels occurring at mid-pregnancy (9–12 months). However, there was considerable overlap in values between non-pregnancy and pregnancy. Concentrations of LH and FSH decreased significantly during mid-pregnancy while prolactin levels increased dramatically during pregnancy; after 7 months of gestation until term levels were always at least 8 ng/ml greater than in any non-pregnant female. It is suggested that this consistent increase in plasma/serum levels of prolactin can be used to diagnose pregnancy in the elephant.
Rachel E Dickinson, Lynn Hryhorskyj, Hannah Tremewan, Kirsten Hogg, Axel A Thomson, Alan S McNeilly and W Colin Duncan
In humans and domestic mammals, pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life; however, they remain poorly understood at the mechanistic level. In mammals, the SLITs (SLIT1, SLIT2 and SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1 and ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition, the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identified SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied that ROBO2 expression and ROBO4 expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (P<0.05). Immunohistochemistry examination demonstrated that ROBO1 was localised to the pre-granulosa cells, while ROBO2, ROBO4 and SLIT2 were expressed in the oocytes of the developing primordial follicle. This indicates that in the fetal ovary, SLIT–ROBO signalling may require an autocrine and paracrine interaction. Furthermore, at the time of increased SLIT–ROBO expression, there was a significant reduction in the number of proliferating oocytes in the developing ovary (P<0.0001). Overall, these results suggest, for the first time, that the SLIT–ROBO pathway is expressed at the time of follicle formation during fetal ovary development.