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A. J. Ziecik, K. L. Esbenshade and J. H. Britt

Summary. Male (N = 8) and female (N = 8) pigs were assigned to receive saline or a potent GnRH antagonist ([Ac-d 2Nal1,d 4-Cl-Phe2,d-Trp3,d-Arg6, d-Ala10]GnRH*HOAc; 1 mg/kg body weight) at 14 days of age. The GnRH antagonist caused LH to decline (P < 0·01) from 1·7 ng/ml at 0 h to <0·5 ng/ml during 4–32 h in males and females. Concentrations of FSH in gilts declined slowly from 75 ± 8 to 56 ± 5 ng/ml (P < 0·05) at 32 h. In males FSH was low (5·7 ± 0·5 ng/ml) at 0 h and did not change significantly.

To observe the effect of long-term treatment with GnRH antagonist, 10 male and 10 female pigs, 3 days of age, were treated with saline or 1 mg GnRH antagonist per kg body weight every 36 h for 21 days. Concentrations of LH were reduced (P < 0·01) to 0·2–0·4 ng/ml throughout the experimental period in male and female piglets treated with GnRH antagonist. Plasma FSH increased in control females, but remained suppressed (P < 0·001) in females treated with GnRH antagonist. Treatment with the GnRH antagonist suppressed FSH levels in males on Days 8 and 16 (P < 0·05), not on Day 24. Treatment of females with the GnRH antagonist did not influence (P > 0·10) oestradiol-17β concentrations. Administration of GnRH antagonist to males suppressed testosterone and oestradiol-17β values (P < 0·01) and reduced testicular weight (P < 0·01). Concentration of LH/hCG receptors in testes of boars treated with GnRH antagonist was lower (P < 0·10) than in controls, but concentration of FSH receptors was not affected. Basal and potassium-stimulated release of GnRH from the stalk median eminence and medial basal hypothalamus in vitro did not differ between treatment groups. The amount of residual GnRH in hypothalamic tissue was not different in control gilts and in gilts receiving the GnRH antagonist, but it was lower (P < 0·05) in boars treated with GnRH antagonist than in control boars.

Keywords: pig; GnRH antagonist; LH; FSH

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J B Kerr, R Duckett, M Myers, K L Britt, T Mladenovska and J K Findlay

Proliferation and partial meiotic maturation of germ cells in fetal ovaries is believed to establish a finite, non-renewable pool of primordial follicles at birth. The supply of primordial follicles in postnatal life should be depleted during folliculogenesis, either undergoing atresia or surviving to ovulation. Recent studies of mouse ovaries propose that intra- and extraovarian germline stem cells replenish oocytes and form new primordial follicles. We quantified all healthy follicles in C57BL/6 mouse ovaries from day 1 to 200 using unbiased stereological methods, immunolabelling of oocyte meiosis (germ cell nuclear antigen (GCNA)) and ovarian cell proliferation (proliferating cell nuclear antigen (PCNA)) and electronmicroscopy. Day 1 ovaries contained 7924±1564 (s.e.m.) oocytes or primordial follicles, declining on day 7 to 1987±203, with 200–800 oocytes ejected from individual ovaries on that day and day 12. Discarded oocytes and those subjacent to the surface epithelium were GCNA-positive indicating their incomplete meiotic maturation. From day 7 to 100 mean numbers of primordial follicles per ovary were not significantly depleted but declined at 200 days to 254±71. Mean numbers of all healthy follicles per ovary were not significantly different from day 7 to 100 (range 2332±349–3007±322). Primordial follicle oocytes were PCNA-negative. Occasional unidentified cells were PCNA-positive with mitotic figures observed in the cortex of day 1 and 12 ovaries. Although we found no evidence for ovarian germline stem cells, our data support the hypothesis of postnatal follicle renewal in postnatal and adult ovaries of C57BL/6 mice.

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B. F. King, J. H. Britt, K. L. Esbenshade, W. L. Flowers and J. J. Ireland

The aim of this study was to determine whether immunoneutralization of inhibin altered compensatory ovarian hypertrophy. Crossbred postpubertal gilts actively immunized with a synthetic bovine inhibin peptide fragment (bINH) conjugated to human alpha globulins (HAG, n = 4 gilts) or HAG alone (control; n = 5) were unilaterally ovariectomized at mid-cycle. After unilateral ovariectomy, the remaining ovary was removed between day 8 and day 12 of the subsequent oestrous cycle. The number of corpora lutea per ovary was determined at each ovariectomy. Blood samples were collected at frequent intervals beginning 1 h before and continuing until the first oestrus after unilateral ovariectomy, and serum concentrations of FSH, LH, progesterone and oestradiol were determined. Inhibin antibody titres were estimated from the percentage of125I-labelled bINH bound to serum diluted 1:4000. At unilateral ovariectomy, the number of corpora lutea per ovary was similar for bINH:HAG-immunized and control gilts (8.6 ±0.7 versus 7.6 ± 0.6). During the next oestrous cycle after unilateral ovariectomy, the number of corpora lutea on each remaining ovary had doubled (P < 0.05) in controls compared with the number of corpora lutea per ovary in the previous cycle. In contrast, the number of corpora lutea remained unchanged in bINH:HAG-immunized gilts. Titre of anti-inhibin antibodies in bINH:HAG-immunized gilts was 9 ± 1% at unilateral ovariectomy compared with 0% for controls. Alterations in serum concentrations of hormones after unilateral ovariectomy did not differ between treatment groups. Compensatory ovarian hypertrophy was blocked after unilateral ovariectomy in immunized gilts independent of alterations in serum hormones, duration of oestrous cycle, or normal ovulation rate per ovary. Thus, it is concluded that inhibin or inhibin α subunits are positive local stimulators of compensatory ovarian hypertrophy in postpubertal gilts.

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M Myers, K L Britt, N G M Wreford, F J P Ebling and J B Kerr

Accurate estimation of the number of ovarian follicles at various stages of development is an important indicator of the process of folliculogenesis in relation to the endocrine signals and paracrine/autocrine mechanisms that control the growth and maturation of the oocytes and their supporting follicular cells. There are 10-fold or greater differences in follicular numbers per ovary at similar ages and/or strains reported in earlier studies using various methods, leading to difficulties with interpretation of ovarian function in control vs experimental conditions. This study describes unbiased, assumption-free stereological methods for quantification of early and growing follicular numbers in the mouse ovary. A fractionator approach was used to sample a defined fraction of histological sections of adult wild-type ovaries. Primordial and primary follicles were counted independently with the optical and physical disector methods. The fractionator/disector methods, which are independent of follicular size or shape, gave estimations of 1930 ± 286 (S.E.M.) and 2227 ± 101 primordial follicles, and 137 ± 25 and 265 ± 32 primary follicles per ovary at 70 and 100 days of age respectively. From exact counts on serial sections, secondary and later follicular numbers at 100 days of age were estimated at 135 per ovary. Remnants of zona pellucidae (a marker of previous follicular atresia) were estimated using a fractionator/physical disector approach and were approximately 500 per ovary. The application of the quantitative methods described will facilitate an improved understanding of follicular dynamics and the factors that mediate their growth and maturation and allow for a better comparison between different studies.