Production and validation of a polyclonal serum against bovine FSH receptor.
MARELLI, B.; REY F.; RAMIREZ, C.; FAILLA, J.; FLORES BRUN, R.B.; MATILLER, V.; LEIVA, C; AMWEG, A.; ORTEGA, H.H.
INST ANIMAL REPRODUCTION FOOD RESEARCH
In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiationevents required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through theFSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications ofthis receptor in reproductive disorders have been extensively studied. However, the limited availability ofspecific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developedan anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserumobtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primarycultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretionand cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein,consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, twoimmunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would berelated to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHRin the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial tolarge preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity andspecificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies ofthe biological function of FSHR in physiological conditions as well as of the molecular mechanism and functionalinvolvement of FSHR in reproductive disorders.