Investigating the Physiological Role of Relaxin-3 in Poultry Reproduction

Abstract

Layers (L), given unrestricted access to feed, regulate their feed intake and maintain organized ovaries, while broiler breeders (BB), prone to overeating, experience disorganized ovaries, erratic ovulations, and oviposition. The relationship between excess nutrition and ovarian dysregulation is known, but the physiological mechanism remains unclear. Recently, relaxin-3 (RLN3), an orexigenic neuropeptide hormone, was discovered in granulosa cells (GC) of ovarian follicles and the pituitary gland, while its receptors RXFP1 and RXFP3 were expressed in theca tissue (TH) and the ovarian cortex, respectively. The objective of the present study was to determine if there are differences between layers and broiler breeders in the gene and protein expression of RLN3, RXFP1, and RXFP3 in the cellular compartments of ovarian follicles, the pituitary gland, and the hypothalamus. Pre-recruitment follicles (3-8 mm diameter) recently recruited follicles (9-12 mm), ovarian cortex, pituitary gland, and hypothalamus were collected from age-matched (36 to 40-week-old) layers (L) and broiler breeders (BB; n=4/group) into 10% neutral buffered formalin for protein localization or into liquid nitrogen for gene expression. Immunofluorescence staining for RLN3, RXFP1, and RXFP3 revealed that RLN3 was predominantly localized in the GC of all follicles but was most prominent in 6-8 mm follicles. RLN3 was also localized in the TH interna and externa of all follicles but not in cortical follicles. Surprisingly, RLN3 was localized in small vesicles in the ooplasm of 3-5mm and 6-8 mm but not 9-12 mm follicles. The RXFP1 receptor was localized in the GC of 3-5mm, 6-8mm, and 9-12 mm follicles, and the TH interna and externa of 3-5mm and 6-8mm follicles, while RXFP1 was not localized in the cortex. RXFP3 was localized in the GC of all follicle sizes but most prominently in the TH interna and externa of 3-5 mm and 6-8 mm follicles. RXFP3 was also localized in the stromal cells of the cortex but not in cortical follicles. Surprisingly, RXFP3 was localized in the nucleus. Gene expression analysis revealed RLN3 was highest in the GC of 9-12 mm follicles L and BB compared to other follicles, but BB had the highest expression (p=0.0031). In the pituitary, RLN3 was higher in L compared to BB (p=0.0051) and very minimally expressed in the TH of follicles, cortex, and hypothalamus. RXFP1 was expressed in the TH of all follicles, very minimally in the GC, and hypothalamus, but not in the cortex. RXFP3 was expressed in the GC of follicles with the highest expression in the 6-8mm follicles. Within these follicles L had a higher expression compared to BB (p= 0.0036). TH expressed very low levels of RXFP3 whereas the stromal cells of the cortex had the highest expression. The hypothalamus had the highest expression of RXFP3 across all tissues. We conclude that RLN3 may have a paracrine effect during follicular development in the ovary and may help regulate the hypothalamic-pituitary-ovarian. Further, RLN3 may play a role in excess nutrition and ovarian dysregulation. Future studies will focus on further elucidating these roles.