Metabolic and antioxidant status during transition is associated with changes in the granulosa cell transcriptome in the preovulatory follicle in high-producing dairy cows at the time of breeding

Abstract

In this study, we hypothesized that early postpartum (pp) metabolic and oxidative stress conditions in dairy cows (particularly those with severe negative energy balance, NEB) are associated with long-term changes in granulosa cell (GC) functions in the preovulatory follicle at the time of breeding. Blood samples were collected at wk 2 and wk 8 pp from 47 healthy multiparous cows. Follicular fluid (FF) and GC were collected from the preovulatory follicle after estrous synchronization at wk 8. Several metabolic and antioxidant parameters were measured in blood and FF, and their correlations were studied. Subsequently, 27 representative GC samples were selected for RNA sequencing analysis. The GC gene expression data of LH-responsive genes and the estradiol: progesterone ratio in FF were used to identify pre-and post-LH surge cohorts. We compared the transcriptomic profile of subgroups of cows within the highest and lowest quartiles (Q4 vs. Q1) of each parameter, focusing on the pre-LH surge cohort (n = 16, at least 3 in each subgroup). Differentially expressed genes (DEG: adjusted P-value < 0.05, 5% false discovery rate) were determined using DESeq2 analysis and were functionally annotated. Blood and FF β-carotene and vitamin E concentrations at wk 2, but not at wk 8, were associated with the most pronounced transcriptomic differences in the GC, with up to 341 DEG indicative for lower catabolism, increased oxidoreductase activity and signaling cascades that are known to enhance oocyte developmental competence, increased responsiveness to LH, and a higher steroidogenic activity. In contrast, elevated blood NEFA concentrations at wk 2 (and not at wk 8) were associated with a long-term carryover effect detectable in the GC transcriptome at wk 8 (64 DEG). These genes are related to response to lipids and ketones, oxidative stress, and immune responses, which suggests persistent cellular stress and oxidative damage. This effect was more pronounced in cows with antioxidant deficiencies at wk 8 (up to 148 DEG), with more genes involved in oxidative stress-dependent responses, apoptosis, autophagy and catabolic processes, and mitochondrial damage. Interestingly, within the severe NEB cows (high blood NEFA at wk 2), blood antioxidant concentrations (high vs. low) at wk 8 were associated with up to 194 DEG involved in activation of meiosis and other signaling pathways, indicating a better oocyte supportive capacity. This suggests that the cow antioxidant profile at the time of breeding might alleviate, at least in part, the effect of NEB on GC functions. In conclusion, these results provide further evidence that the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on GC functions in preovulatory follicles at the time of breeding. The interplay between the effects of antioxidants and NEFA illustrated here might be useful to develop intervention strategies to minimize the effect of severe NEB on fertility.

The high fertility cycle

“…In addition, NEFA concentrations remained elevated 2-fold at 75 to 81 DIM in cows that lost versus cows that maintained or gained BCS during early lactation. Another recent study ( Marei et al, 2022 ) reported differences in granulosa cell gene expression for cows that lose weight and experience severe negative energy balance postpartum. In agreement with the Britt hypothesis, the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on granulosa cell function in preovulatory follicles at the time of insemination.…”

The impact of offspring and maternal obesogenic diets on adult offspring oocyte mitochondrial morphology in primordial and preantral follicles

“…According to the Britt’s hypothesis [ 33 ], this suggests that early preantral stages of follicular development are also affected. Recent data could provide strong evidence that is in line with this notion [ 34 ]. Similarly, studies from our laboratory recently indicated that an increased rate of mitochondrial ultrastructural abnormalities is still detectable in diet-induced obese mice after 4–6 weeks of diet normalization or caloric restriction, implying that the dormant follicle pool may indeed be affected [ 35 ].…”

Maternal contributions to pregnancy success: from gamete quality to uterine environment

“…Association studies in cattle confirm that severe negative energy balance during the transition period affects fertility; a recent study demonstrated several differences in the transcriptomic profile of pre-ovulatory follicle granulosa cells at eight weeks post-partum when they originated from cows with high or low concentration of non-esterified fatty acid in blood at two weeks post-partum (Marei et al, 2022). Although a reliable in vitro system that allows the growth of bovine preantral follicles to the pre-ovulatory stage and beyond is not yet available, short-term follicle or ovarian cortex culture have unraveled inflammatory processes, oxidative stress and apoptosis among the factors potentially involved in impairment of oocyte viability (Leroy et al, 2005;Marei et al, 2019;Pedroza et al, 2022).…”

“…However, studies examining the long-term effects of altered physiological states on oocyte quality and fertility demonstrate that the importance of a healthy environment for oocyte growth extends much beyond the antral follicle wave. It has been demonstrated in cattle that the occurrence of severe negative energy balance or disease (reproductive or otherwise) during the early postpartum period affects the quality of the ovulatory oocyte up to 60 days later (Garverick et al, 2013;Ribeiro et al, 2016;Marei et al, 2022). Culture of murine preantral follicles at the early secondary stage (starting follicle diameter of 100-130 µm) in the presence of non-esterified fatty acids altered follicular development as evidenced by lower rate of antrum formation, changes in gene expression and steroidogenic profile.…”