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M. WEBB

Summary.

In virgin female rats, subcutaneously injected Cd2+ accumulates to high levels and is retained in the livers and kidneys. When such animals become pregnant, this stored Cd2+ is not mobilized and, thus, in contrast to `free' Cd2+, does not induce toxaemia or excessive fetal malformations.

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M. WEBB

Summary.

Biochemical changes that occur in the rat or mouse testis after a single subcutaneous injection of a sub-toxic dose of CdCl2 (2·2 μmol/100 g body wt) include decreases in the contents of DNA, RNA and in the activities of certain Zn2+-containing enzymes. The protein content is increased, although the rate of protein synthesis is reduced. Uptake of Cd2+ by the testis is small and does not cause the displacement of Zn2 +. In both species, the concentration of Zn2 + remains constant in the testis for a short time after the parenteral administration of Cd2 +, and then increases progressively. Initially, this increase appears to be due to the decrease in weight of the damaged testis, which is not accompanied by the loss of Zn2+. Later, when the testicular weight becomes constant, Zn2+ continues to accumulate to a level nine to ten times greater than in the normal testis.

In the male rat, subcutaneously injected Cd2+ accumulates to high levels and is retained in the liver and kidneys. In both of these organs also, the Zn2+ content increases with the uptake of Cd2+. Bound Cd2+ is present in the soluble components (cell sap) of these tissues as a single fraction, probably metallothionein. This Cd2+-binding protein also accumulates Zn2+ and is responsible for its increased uptake.

Competitive antagonism between the toxic Cd2+ cation and the essential Zn2 + ion may occur in the epididymis since, in this organ, the content of the latter cation decreases with the uptake of the former.

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M. A. Driancourt, I. K. Gauld, M. Terqui and R. Webb

Summary. Prolific breeds of sheep (Romanov, Finn and Booroola Romanov crosses heterozygous for the Booroola gene (F +) were compared with breeds of lower prolificacy (Ile-de-France, Finn × Scottish Blackface, Merino × Blackface and Booroola × Romanov not carrying a copy of Booroola gene (++)) by in-vivo monitoring of follicular kinetics by ink labelling during the late luteal phase and follicular phase of the oestrous cycle followed by histological examination of the ovaries or follicle dissection. At each of 3 successive laparotomies, the 3 largest follicles of each ovary were measured and ink labelled. At the final laparotomy, around the beginning of oestrus, all ewes were ovariectomized.

High ovulation rate was not associated with the total number of antral follicles in any of the breeds. However, there were more follicles > 2 mm in diameter in Romanov and Booroola × Romanov crosses (F +) compared to their respective controls. Such a feature was not observed in Finnish Landrace compared to Finn × Blackface and Merino × Blackface ewes. A more numerous population of recruitable follicles, together with a similar incidence of selection through atresia, were the features associated with the high ovulation rate of Romanov compared to Ile-de-France ewes. The high ovulatory potential of the Finn ewes resulted from a markedly reduced incidence of selection through atresia. Booroola × Romanov ewes carrying a copy of the Booroola gene (F +) appeared to possess features of both parental breeds, including high numbers of recruitable follicles, smaller follicular size when recruitment occurs and an extended time for recruitment. Booroola × Romanov (++) ewes, not carrying the gene, appeared to have lost part of the 'Romanov characteristics' of a more numerous population of recruitable follicles. The variability in the kinetics of preovulatory enlargement, seen in these breeds of sheep, demonstrates that there are a number of pathways through which high ovulation rate can be achieved and hence through which ovulation rate might be manipulated.

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M. A. Driancourt, R. Webb and R. C. Fry

Summary. The process by which a single follicle is selected to ovulate while others regress is unknown in ewes. If the dominant follicle secretes substances that directly inhibit the growth of other follicles, the superovulatory response to the administration of exogenous gonadotrophins may be blunted. Administration of 1250 iu pregnant mares' serum gonadotrophin (PMSG) before or after the emergence of the dominant follicle in the follicular phase, or 1000 iu PMSG in the presence or absence of a large healthy or atretic follicle during the luteal phase did not affect the induced ovulatory response. Comparisons between the ovary with or without the dominant follicle did not reveal any differences in ovulatory response to PMSG. The in-vitro features (i.e. mitotic index, oestradiol and testosterone production) of follicles ipsilateral or contralateral to the dominant follicle during the early and late follicular phases were also similar.

If the dominant follicle secretes substances detrimental to the other follicles, this could be mimicked in vitro. Co-culture of small follicles with the largest follicles in a closed system did not reduce their incorporation of 3H thymidine in granulosa cells, compared with small follicles cultured alone.

These data suggest that dominance is probably not operative in sheep. The administration of 500 iu of PMSG during the midfollicular phase increased ovulation rate in Merino ewes, indicating that dominance is essentially passive in ewes and can easily be overcome by raising gonadotrophin concentration.

Keywords: follicle; ovulation; gonadotrophin; paracrine regulation; sheep

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R. Webb, I. K. Gauld and M. A. Driancourt

Summary. Morphological and functional features of large ovarian follicles from three breeds of sheep, with different ovulation rates (Finnish Landrace N = 12, Finnish Landrace × Scottish Blackface N = 16, Merino × Scottish Blackface N = 16) were compared by integrating three techniques; ink labelling, in-vitro oestradiol production and morphological classification. The follicles were removed at two stages of the follicular phase, 1 (PG + 1) or 2 (PG +2) days after PGF-2α treatment and compared after monitoring their rates of growth with the use of ink labelling. After ovariectomy all follicles ≥ 1 mm in diameter were dissected, and the 8 largest were incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. After incubation the follicles were processed for histological examination and checked for atresia. An analysis of the follicle population was based on in-vitro oestradiol secretion rates in all three breeds; an oestrogen-active population producing 500–8100 pg oestradiol/ml/h and an oestrogen-inactive population producing 0–499 pg oestradiol/ml/h. A comparison of the 3 approaches demonstrated agreement on 94·3 ± 1 ·2% of occasions. Ink-labelling demonstrated that all follicles identified as oestrogen-active were increasing in size. Within oestrogen-active follicles significant correlations were detected between oestradiol production and testosterone production (r = 0·42), oestradiol production and granulosa cell number (r = 0·45) and between oestradiol production and mitotic index (r = −0·38). A regression model fitting breed, stage of atresia, granulosa cell number, in-vitro testosterone production and mitotic index demonstrated that granulosa cell number is a characteristic which contributes significantly to the variation of in-vitro oestradiol production in oestrogen-active and oestrogen-inactive follicles. There was no significant difference between breeds in the mean number of ink-labelled follicles growing from Day PG − 1 to Day PG +1. There was a significant difference between the breeds in the number of ink-labelled follicles growing between Days PG + 1 and PG +2 (Days 1 and 2 of the follicular phase), the number being similar to the ovulation rate for the breed. The majority of the oestrogen-active follicles had been recruited by Day PG − 1, although in the Finnish Landrace genotypes more than 30% were recruited on or after Day PG + 1 compared to less than 10% in Merino × Scottish Blackface ewes. Although mean oestradiol production by oestrogen-active follicles was similar in all three breeds, oestrogen-active follicles in Finnish Landrace ewes had significantly reduced granulosa cell number and mitotic index compared to the other breeds.

These results demonstrate: (1) a good agreement between the three criteria used to characterize follicles, (2) that the mechanisms controlling follicular recruitment into the population of gonadotrophin-dependent follicles are different from the mechanisms controlling the selection of the follicles destined to ovulate and (3) that despite no

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R. G. Gosden, M. I. Boulton, K. Grant and R. Webb

Cortical slices of either cat or sheep ovaries were grafted under the renal capsules of ovariectomized SCID mice. The grafts became vascularized and were still surviving with large follicles present at autopsy up to nine months later. As developing follicles undergo atresia during the period of ischaemia after ovarian grafting, those found in long-term grafts at autopsy had presumably started to grow from the primordial stage after transplantation. Some follicles had reached a diameter of 3 mm with a normal antrum and appeared to be cytologically normal, but the latent period for the emergence of antral follicles was shorter in cat compared with sheep grafts. Oestradiol production from grafts, as indicated by vaginal cornification and plasma measurements collected at autopsy, was not constant and circulating concentrations varied among animals, and were sometimes far in excess of the normal physiological range of the host. The vaginal smears never presented cytological patterns like those of the normal mouse oestrous cycle, and ovulation had not occurred in any of the grafts. These results demonstrate that ovarian xenografts in SCID mice can serve as experimental models for investigating follicle development in species in which follicle growth in vitro and studies of the parent animal are impracticable.

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M. Puig-Domingo, J. M. Guerrero, R. J. Reiter, M. A. Peinado, A. Menendez-Pelaez, C. Santana and S. M. Webb

Summary. Harderian glands of Syrian hamsters contained measurable levels of immunoreactive somatostatin. After an extraction procedure, serial dilutions of tissue were assayed and showed parallelism in the displacement curve with dilutions of purified somatostatin standard in the radioimmunoassay. Somatostatin concentrations were higher in female hamsters (10·0 ± 2·1 ng/mg protein) than in males (2·6 ± 0·4 ng/mg protein). Castrated males had somatostatin values in the range of females (12·4 ± 2·3 ng/mg protein) at 1 month after gonadectomy. Testosterone implants prevented the rise of Harderian gland somatostatin in castrated males. Gonadectomized males had lower somatostatin content in the gland than did control males (1·0 ± 0·2 ng/mg protein) at 2 months after castration. Somatostatin values in females were unaffected by gonadectomy, but there were variations during the oestrous cycle, with a nadir dectected at dioestrus-1 and maximal values coincident with the day of the ovulation.

Keywords: somatostatin; Harderian gland; androgens; Syrian hamster

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J. R. McNeilly, M. Fordyce, R. B. Land, G. B. Martin, A. J. Springbett and R. Webb

Summary. The dynamics of FSH and LH secretion were studied in sheep genetically selected for High (H) and Low (L) rates of testis growth. Gonadotrophin secretion had previously been shown to be affected in the ram lamb with H-line lambs more sensitive to steroid feedback than L. While there were significant differences in mean LH concentrations during the luteal and follicular phases of the oestrous cycle, mean LH values were essentially similar in the two lines in response to ovariectomy, the effect of oestradiol implants on the response to ovariectomy and the response to LHRH. However, the frequency of LH pulses in the H line was similar during both phases of the oestrous cycle, showing a surprising insensitivity to steroid feedback. By contrast, LH pulse frequency was markedly lower in the L-line ewes in the luteal than the follicular phase (0·6 vs 1·1 pulses/h) as expected from the literature. Mean FSH concentrations were significantly higher in the L-line ewes during the follicular phase of the oestrous cycle and after ovariectomy but no significant differences were detected at the other sampling periods. There were no differences in ovulation rate between the lines. It was concluded that selection for testis size had affected the feedback control of gonadotrophin release in the ewe, as in the ram, and hence the expression of the genes controlling this is not sex limited.

Keywords: ewes; gonadotrophins; selection; testis diameter

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M. I. Boulton, C. S. Haley, A. J. Springbett and R. Webb

The aim of this study was to investigate the role of FSH in the control of ovulation rate by the Booroola gene. Three Booroola genotypes (FecBFecB, FecBFec+ and Fec+Fec+) of the F2 population, from a cross between Booroola Merino and Scottish Blackface, and two Booroola genotypes (FecBFec+ and Fec+Fec+; 25% Booroola Merino and 75% Scottish Blackface), from the backcross of FecBFec+ sires to Scottish Blackface ewes, were compared. During seasonal anoestrus significant differences (P < 0.05) in hCG-stimulated ovulation rates were obtained between FecBFecB and Fec+Fec+ ewes from the F2 population, and FecBFec+ ewes were intermediate. No significant difference in hCG-stimulated ovulation rate was observed in the backcross population between FecBFec+ ewes and Fec+Fec+ ewes. There were no significant differences between genotypes in mean serum FSH concentrations during seasonal anoestrus in either backcross or F2 populations. During the breeding season, two separate experiments confirmed the expected ovulation rate differences between genotypes (FecBFecB > FecBFec+ > Fec+Fec+). In both experiments, mean peripheral FSH concentrations in the F2 population were similar in FecBFec+ and Fec+Fec+ ewes, but were significantly higher (P < 0.05) in FecBFecB ewes. In the backcross population, mean peripheral FSH concentrations during the oestrous cycle were not significantly different between FecBFec+ and Fec+Fec+ ewes, despite significant differences in ovulation rate. Ovariectomy during the breeding season resulted in significantly higher (P < 0.001) mean peripheral FSH concentrations in all three genotypes. After ovariectomy, mean FSH concentrations between FecBFec+ and Fec+Fec+ ewes, from both the backcross and F2 populations, were not significantly different. However, mean FSH concentrations in the F2 population were significantly higher in FecBFecB ewes than in the other two genotypes. The pattern of differences between genotype in peripheral FSH concentrations and ovulation rates suggest that FSH is not wholly responsible for differences in ovulation rate between genotypes. The results support the hypothesis that the FecB gene is operating both within the ovary and at the level of the hypothalamus and pituitary gland.

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R. Webb, G. Baxter, D. McBride, M. Ritchie and A. J. Springbett

Summary. Three experiments were carried out during seasonal anoestrus in Finnish Landrace and Scottish Blackface ewes, to establish whether the differences between the breeds in ovulation rate are functional during the non-breeding season and are therefore independent of the mechanism controlling ovulation.

In Expt 1, follicles ⩾2 mm in diameter were dissected from the ovaries of both breeds and incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. In both breeds, follicles producing ⩾500 pg oestrogen/ml/h (oestrogen-active) were readily identifiable from a population producing less (oestrogen-inactive). The number of oestrogen-active follicles in each breed was similar to the number of ovulations near the end of the breeding season. Oestrogen-active follicles also had more luteinizing hormone (LH) receptors and larger diameters than oestrogen-inactive follicles. There were, however, no significant differences between the two follicle types in follicular fluid or in-vitro testosterone concentrations.

In Expt 2, seasonally anoestrous Scottish Blackface ewes were unilaterally ovariectomized; the second ovary was removed 7 days later. Follicles from both ovaries were processed as described for Expt 1; oestrogen-active follicles were categorized according to their ability to produce >500 pg/ml/h. There were twice as many oestrogen-active follicles in the second ovary as in the first ovary; the number of oestrogen-active follicles in the second ovary was also similar to the total number of oestrogen-active follicles in both ovaries of the Scottish Blackface ewes in Expt 1. There were no significant differences between the first and second ovaries for any of the other parameters measured in oestrogen-active follicles. There were no significant changes in peripheral gonadotrophin concentrations measured 24 h after removal of the first ovary.

In Expt 3, seasonally anoestrous ewes of both breeds were challenged with an ovulatory dose of human chorionic gonadotrophin (hCG) (750 iu). There was a significant difference in the mean number of ovulations between the breeds and it was representative for the breed (Finnish Landrace 2·6 ± 0·2; Scottish Blackface 1·6 ± 0·2 mean ovulations per ewe). None of the saline-treated controls ovulated.

The results demonstrated that the mechanism controlling the number of mature, oestrogen-active follicles, and hence ovulation rate, is functional during seasonal anoestrus. This conclusion was confirmed by the observation that compensatory ovarian hypertrophy also occurs during seasonal anoestrus.

Keywords: sheep; follicle; ovulation rate; steroidogenesis; seasonal anoestrus