Gonadotrophin-binding components in porcine follicular fluid

Abstract

The follicular fluid is an important milieu for the growing and maturing oocyte and granulosa cells. In this study we investigated: (1) the properties of gonadotrophin-binding sites in the supernatant fraction of porcine follicular fluid (pFF) and compared them with those of membrane-bound receptors, and (2) the relative changes that occur in pFF and granulosa cell receptor-binding activity following hormone priming of gilts. 125I-Labelled human chorionic gonadotrophin (hCG) and 125I-labelled ovine FSH (oFSH) binding to particulate and supernatant fractions of pFF were hormone-specific and saturable. The concentration of 125I-labelled hCG-binding sites was roughly 50-fold higher in particulate than in supernatant fractions of pFF. However, 30-40% of the total 125I-labelled hCG-binding activity in pFF was present in the supernatant fraction of commercial batches of pFF. 125I-Labelled oFSH binding to pFF membranes was markedly higher than to supernatant fractions. Binding of 125I-labelled hCG and 125I-labelled oFSH to granulosa cells and supernatants of pFF showed a time-dependent variation in response to hormone priming. The results suggest that gonadotrophin-binding sites in the supernatant fraction of pFF have properties similar to those of their membrane-bound counterparts. 125I-Labelled hCG-binding activity in the supernatant fraction of pFF was shown to be more stable than detergent-solubilized LH/hCG receptors, even in glycerol-preserved preparations. Based on a number of criteria, we have speculated that pFF may have components which may be similar in structure to the extracellular domain of the LH/hCG receptor.

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

“…Cross-Linking of ,2il-oFSH to Membrane Receptor. As specific oFSH binding was detected in every batch of the particulate fraction (Sebok et al, 1987;Yarney et al, 1988) studied thus far, it was of interest to ascertain the approximate size(s) of the molecular species involved in this interaction.…”

“…As specific oFSH binding was detected in every batch of the particulate fraction (Sebok et al, 1987;Yarney et al, 1988) studied thus far, it was of interest to ascertain the approximate size(s) of the molecular species involved in this interaction. This was examined by employing the same cross-linking methods as utilized for IZI-hCG in Figure I, with the exception that 1 mM DSS was used.…”

“…There are no reports in the literature on attempts to purify the LH/hCG receptor from granulosa cells which contain FSH receptors as well (Channing & Ledwitz-Rigby, 1975 0006-2960/90/0429-375 1 $02.50/0 or loosely attached in the mature graafian follicle, it occurred to us that they may be an appropriately clean source of starting material from which to purify LH/hCG and/or FSH receptor(s), which would allow more reliable comparisons to be made between structures of these receptors. In a preliminary study, we have recently reported on the relative abundance of specific LH/hCG binding sites (Yarney et al, 1988) as well as FSH binding sites (Sebok et al, 1987;Yarney et al, 1988) in particulate fractions of pig follicular fluid collected from commercial suppliers. In the present report, we describe the purification and characterization of a single LH/hCG receptor protein from this source and compare it to the FSH receptor found in the same source.…”

Purification of high-molecular-weight follicle-stimulating hormone binding inhibitor in porcine follicular fluids

“…the fraction not retained on Blue Sepharose CL6B (A), the fraction eluted from Mono Q by 0.9-1.3 mol/I NaCl ( ) and the fraction obtained by gel filtration on Sephacryl S300HR (O) were assayed at multiple doses in the FSH radioligand-receptor assay described in the text. Yarney et al (27) have reported that the supernatant fraction of porcine follicular fluids might contain FSH-binding components. Sephacryl S300HR a BI50: 50% inhibition of -labelled human follicle-stimulating hormone binding to porcine granulosa cells.…”

Differences in properties of the sheep testicular LH and FSH receptors

“…Aliquots of mem¬ brane pellets were stored at -70°C until assayed. Granulosa cell membranes were prepared from particulate fractions of porcine follicular fluid as described by Yarney, Sairam, Bhargavi et al (1990a). Membranes intended for cross-linking studies were prepared with buffers containing 100 pg bacitracin/ml and 100 pM PMSF.…”