Expression of atresia biomarkers in granulosa cells after ovarian stimulation in heifers

in Reproduction
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The use of younger gamete donors in dairy cattle genetic selection programs significantly accelerates genetic gains by decreasing the interval between generations. Ovarian stimulation (OS) and the practice of follicle-stimulating hormone (FSH) withdrawal, also known as coasting, are intensively used in pre-pubertal heifers without detrimental effects on subsequent reproductive performance but generally with lower embryo yields. However, recent data from embryo transfer programs showed similar embryo yields in younger and sexually mature animals but with a significant difference in the coasting period. The aim of the present study was to identify a set of granulosa cell biomarkers capable of distinguishing optimal follicle differentiation from late differentiation and atresia in order to assess the differences in coasting dynamics between pre- and post-pubertal donors. We integrated transcriptomic data sets from a public depository and used vote counting meta-analysis in order to elucidate the molecular changes occurring in granulosa cells during late follicle differentiation and atresia. The meta-analysis revealed the gene expression associated with follicle demise, and most importantly, identified potential biomarkers of that status in bovine granulosa cells. The comparison of the expression of six biomarkers between pre- and post-pubertal donors revealed that younger donors had more signs of atresia after the same period of coasting. We found different follicular dynamics following coasting in younger donors. It is possible that younger donors are less capable to sustain follicular survival most likely due to insufficient luteinizing hormone signaling. In summary, the pre-pubertal status influences follicular dynamics and reduces the oocyte developmental competence curve following OS and FSH withdrawal in heifers.

Downloadable materials

  • Supplemental Table 1: Primers used in real-time PCR experiments
  • Supplemental Table 2: Dataset of the 193 differentially expressed genes (DEGs) (Gene symbol, average P-value, and average fold change) from the vote counting meta-analysis in granulosa cells associated with late differentiated and atretic follicles.


    Society for Reproduction and Fertility

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    Relationship between oocyte donor age and coasting period length during ovarian stimulation regimens for (A) 170 pre-pubertal heifers and (B) 161 post-pubertal cows. For the same animals, (C) mean embryo percentages 7 days following in vitro fertilization of all oocytes matured.

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    The five most significant biological functions from the meta-analysis datasets of the differentially expressed genes (DEGs) in granulosa cells associated with follicle late differentiation and atresia. The threshold line represents the significant P value.

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    Gene expression levels of six selected biomarkers were used to assess the atresia level at 30 h and 43 h of coasting in pre- and post-pubertal bovine granulosa cells. RT-qPCR values are mean relative expression with standard error of the mean compared to the reference groups (black bar). Significant differences were calculated using unpaired T test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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    Schematization of follicle dynamics following coasting in pre- and post-pubertal donors. The oocyte developmental competence is represented by the black curve across the coasting period. Each specific phase of follicle dynamics is described inside the curve as follows: early, optimal and late windows of oocyte developmental competence. The arrows represent the expression of the selected biomarkers, a plus or negative (+/−) sign represents an increase or decrease in gene expression compared to the other group for the same period of coasting.

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