In vitro membrane desialylation in porcine granulosa cells unmasks functional receptors for follicle-stimulating hormone.
To evaluate the possible existence of masked gonadotropin-binding sites and the functional role of the binding sites in porcine granulosa cells (GCs), we characterized the effects of neuraminidase (NA) pretreatment on [125I]human LH and [125I]human FSH binding to porcine GCs and the functionality of the unmasked receptors exposed by NA pretreatment. NA pretreatment at 37 C for 30 min increased specific FSH binding (P less than 0.01) and had no effect on LH binding to the GCs harvested from follicles of different sizes. Analysis of equilibrium binding experiments revealed that NA increased the number of FSH-binding sites without altering the affinity. In addition, pretreatment with NA resulted in progressive loss of cell membrane-bound sialic acid (P less than 0.01). To evaluate whether the unmasked FSH receptors constitute functional gonadotropin-binding sites, we measured the amounts of cAMP and 17 beta-estradiol produced by cultured GCs pretreated with NA. cAMP formation by GCs pretreated with NA was significantly (P less than 0.02) greater than that by GCs pretreated with medium alone in the presence of ovine FSH. Moreover, 17 beta-estradiol formation significantly increased (P less than 0.05) in GCs pretreated with NA compared with that in GCs pretreated with medium alone. However, there was no difference between medium-treated and NA-treated GCs in cAMP formation stimulated by forskolin. We suggest that porcine GCs contain a population of masked FSH-binding sites exposed by in vitro pretreatment of NA, and the binding sites constitute functional receptors capable of increasing cAMP and 17 beta-estradiol formation with FSH stimulation. Moreover, a possible involvement of plasma membrane-associated sialic acid in the masking/unmasking mechanism of FSH-binding sites was suggested.
Nishimori
K
K
Department of Obstetrics and Gynecology, Wakayama Medical College, Japan.
Yamoto
M
M
Nakano
R
R
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006107:Granulosa Cells / Q000378:metabolism*; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology*; D011962:Receptors, FSH / Q000378:metabolism*; D011974:Receptors, LH / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism; D013552:Swine
D011962:Receptors, FSH; D011974:Receptors, LH; D012794:Sialic Acids; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D009439:Neuraminidase