Feed restriction affects follicular development by regulating cell cycle progression in porcine mural granulosa cells

Mammalian reproductive outcomes are influenced by whole-body energy status, especially in high-prolific sows. During lactation, high energy demands often lead to a severe negative energy balance (NEB), which compromises subsequent reproductive performance. The molecular processes underlying the sustained impact of lactational energy insufficiency on post-weaning follicular development are not well understood. We hypothesize that this energy shortage disrupts the development of mural granulosa cells (GCs), ultimately compromising oocyte developmental competence. We aimed to delineate the molecular processes by which energy status regulates mural GC development employing a nutritional intervention.

Over the 14 days of feed restriction, sows receiving a restricted diet (RES; 3.25 kg/d) lost double the amount of body weight and loin muscle depth compared to full-fed sows (FF; diet 6.5 kg/d). Both ovarian weight and average size of the largest healthy follicle were significantly reduced. RNA-seq analysis identified 2,282 differentially expressed genes in RES GCs, of which 1,531 were upregulated and 751 were downregulated. The most enriched pathway was the cell cycle, corroborated by CDK1 immunostaining. RES GCs showed upregulation of key positive cell cycle regulators (e.g., CCNA1/2, CCNB1-3, CCNE2), while cell cycle inhibitor CDKN1A (P21) was downregulated. In contrast to FF GCs, in RES GCs several members of the IGFBP family, especially IGFBP7, strongly correlated with most cell cycle-related genes including FOXO1, CDKN1A, and follicle size exclusively in RES GCs, but not in FF sows. Proliferation markers (PCNA and MKI67) were also upregulated, while differentiation marker CYP19A1 was downregulated.

Our data suggest that a severe lactational NEB has a lasting impact on mural GCs after weaning, delaying their transition from proliferation to the terminal differentiation. We propose a potential regulatory axis in which IGFBPs, especially IGFBP7, may link energy metabolism and follicular development through the AKT-FOXO1-CDKN1A-cell cycle regulatory axis. These molecular alterations likely contribute to the compromised oocyte competence observed following energy restriction. Furthermore, our findings identified IGFBP7 as a candidate biomarker linking metabolic status to follicular development.