Antral follicle size might be a valuable additive predictive marker for IVF outcome. However, while some studies show positive relations between follicle size and reproductive outcome, others have not been successful in establishing this relation. To better understand consequences of antral follicle size for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates, needed for follicular growth and oocyte development. Using pigs as a model, we compared gene and protein expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool at the start of the follicular phase of the estrous cycle. In sows, the early antral follicle pool is very heterogeneous when e.g. size and steroid content of the follicular fluid are considered. To which extent this variety contributes to the developmental competence of the follicles is not clear. Therefore, sows with high variation in antral follicle size (HighVAR) as well as sows with low variation in antral follicle size (LowVAR) were used. Granulosa cells of smaller antral follicles in the healthy antral follicle pool show increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of AR and EGFR which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool were more proliferative, indicative of higher follicular growth, granulosa cells of larger follicles in the pool showed less proliferation and were more differentiated, as they showed a higher expression of follicular maturation marker IGF1 and ANGPT1. Our results imply that the inclusion of strict criteria of antral follicle size in IVF protocols might improve reproductive outcome. In addition, we have granulosa cell gene expression of healthy follicles to unravel underlying mechanisms of differences in COC morphology. We compared gene expression in sows with low vs. high-COC-health and found a decreased expression of genes involved in ovarian steroidogenesis (e.g. CYP19A1, ADM, SPP1) and higher expression of genes involved in follicular atresia (e.g. GADD45A, INHBB) in sows with low-COC-health. Thereby we have identified several genes which may serve as markers for follicle developmental competence.
Overall design: A total of 29 multiparous Dutch Landrace sows (parity 3 to 5; Topigs Norsvin, Vught, the Netherlands) were used. The sows were fed a standard lactation diet (ca. 12.5MJ NE/kg, 154 g/kg CP, 9.3 g/kg lysine; Lacto Excellent, Agrifirm, Apeldoorn, The Netherlands). Within 24 hours after parturition, piglets were cross-fostered to ensure 13 suckling piglets per sow. The sows had a lactation period of 26.1±0.2 (25 to 27) days and weaned 13.0±0.9 piglets. The sows were slaughtered at the slaughterhouse by stunning and exsanguination within 2 hours after weaning. Follicle size, follicle health status and cumulus-oocyte complex (COC) morphology were determined. For each sow, the variation in follicle size of the 10 largest healthy follicles for each right ovary was determined, as well as the percentage healthy COCs. The variation in follicle size was used to select a total of eight sows for further analysis; four sows with a large variation (SD) in follicle size of the 10 largest healthy follicles (HighVAR; 1.56±0.18 mm) and four sows with a small variation in follicle size (LowVAR; 0.54±0.16 mm). For each sow, granulosa cells of the 3 largest (large follicles 1-3) and 3 smallest (small follicles 4-6) of the 10 largest healthy follicles were isolated and indiviudally hybridized on microarrays. Differences in gene expression between large and small follicles were determined, for both HighVAR and LowVAR sows. As an alternative approach, which will be described elsewhere, the percentage healthy COCs was used to group 4 of the previously mentioned sows as sows with Low-COChealth (% healthy COCs < 70%) and 4 sows with High-COChealth (% healthy COCs > 70%). Granulosa cell gene expression profiles of sows with Low_COChealthy and high_COChealth were compared, by analysing 6 follicles for each sow (3 large follicles and 3 small follicles).
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