Morphological and functional characterization of large antral follicles in three breeds of sheep with different ovulation rates

Abstract

Morphological and functional features of large ovarian follicles from three breeds of sheep, with different ovulation rates (Finnish Landrace N = 12, Finnish Landrace x Scottish Blackface N = 16, Merino x 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 and 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 x 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 between-breed differences for in-vitro oestradiol production by oestrogen-active follicles, the follicles of Finnish Landrace ewes had significantly lower numbers of granulosa cells and mitotic index, indicating alterations in morphological and functional differentiation associated with prolificacy.