Abstract

The beta-nerve growth factor (β-NGF), a seminal plasma protein with established luteotrophic effects in camelids and cattle, has been proposed as a modulator of periovulatory follicular function. The optimization of the steroidogenic environment of the preovulatory follicle is critical for oocyte maturation, corpus luteum formation, and embryo quality. However, the molecular actions of β-NGF on granulosa cells (GCs) during the periovulatory period in dairy heifers remain poorly defined. This study evaluated the effects of systemic administration of heterologous β-NGF (purified from llama seminal plasma) on the expression of steroidogenic enzymes in GCs from preovulatory follicles in estrus-synchronized Holstein heifers. Animals received 1 mg of β-NGF intramuscularly on Day 9 (estradiol benzoate administration) or Day 10 (fixed-time artificial insemination) of a progesterone (P4)/estradiol (E2)-based synchronization protocol. The follicular fluid and GCs were collected via transvaginal ultrasound-guided aspiration for hormone quantification and gene expression analysis. β-NGF treatment on Day 9 significantly upregulated Lutenizing hormone/choriogonadotropin receptor (LHCGR) and key steroidogenic enzymes, including 3β-hydroxysteroid dehydrogenase (HSD3B), Cytochrome P450 family 11 subfamily A member 1 (CYP11A1), 17β-hydroxysteroid dehydrogenase (HSD17B), and Cytochrome P450 family 19 subfamily A member 1 (CYP19A1), whereas no transcriptional effects were observed when NGF was administered on Day 10, indicating a timing-dependent regulation of GCs differentiation. In contrast, Growth hormone receptor GHR and Steroidogenic acute regulatory protein expression levels remained unaffected, supporting the selectivity of NGF-induced transcriptional modulation. The follicular fluid P4/E2 ratios were not significantly altered by NGF treatment. These findings indicate that β-NGF increased steroidogenic and LHCGR gene abundance of GCs in a timing-dependent manner, suggesting that NGF may represent a potential modulator of the preovulatory follicle function in dairy cattle.