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Identification of luteinizing hormone receptor binding inhibitor in bovine corpora lutea
P Rathnam1, S Q Lin, B B Saxena
1Department of Obstetrics and Gynecology, Cornell University Medical College, New York 10021.
Insights
Researchers isolated luteinizing hormone (LH) receptor binding inhibitors (LH-RBI) from bovine corpora lutea. These inhibitors, in both high and low molecular weight forms, effectively blocked LH receptor binding and testosterone production.
Area of Science:
- Reproductive biology
- Endocrinology
- Biochemistry
Background:
- Luteinizing hormone (LH) receptor plays a crucial role in reproductive functions.
- Bovine corpora lutea are a rich source of reproductive hormones and related factors.
- Understanding LH receptor regulation is key to reproductive health research.
Purpose of the Study:
- To isolate and characterize luteinizing hormone (LH) receptor binding inhibitors (LH-RBI) from bovine corpora lutea.
- To determine the molecular weight and stability of LH-RBI.
- To investigate the inhibitory effects of LH-RBI on hormone binding and steroidogenesis.
Main Methods:
- Ultrafiltration of bovine corpora lutea homogenate to concentrate LH-RBI activity.
- Sequential filtration using Amicon PM-10, PM-30, and UM-2 filters to fractionate LH-RBI by molecular weight.
- Purification of high- and low-molecular-weight LH-RBI fractions using Sephadex G-25 and G-50 chromatography, respectively.
- Assay of LH-RBI activity by measuring inhibition of 125I-labeled human chorionic gonadotropin (hCG) binding to receptors and testosterone production in rat Leydig cells.
Main Results:
- LH-RBI activity was identified in fractions with molecular weights between 10,000-30,000 and 1,000-10,000.
- Both high- and low-molecular-weight LH-RBI fractions inhibited hCG binding to bovine and rat receptors.
- Both fractions dose-dependently inhibited testosterone production in hCG-stimulated rat Leydig cells.
- Low-molecular-weight LH-RBI showed greater thermal stability than the high-molecular-weight form.
- The 7000 g supernatant provided a substantial source for LH-RBI isolation.
Conclusions:
- LH-RBI exists in multiple molecular weight forms with significant biological activity.
- These LH-RBI fractions represent a valuable resource for further structural and functional studies of LH receptor regulation.
- The characterized LH-RBI could have implications for understanding and potentially modulating reproductive processes.
Abstract:
A 7000 g supernatant, obtained during the purification of luteinizing hormone (LH) receptor from bovine corpora lutea homogenate, was concentrated by ultrafiltration. The filtrate, containing < 50,000 molecular weight material, exhibited LH receptor binding inhibitor (LH-RBI) activity. The filtrate was ultrafiltered sequentially through Amicon PM-10, PM-30 and UM-2 filters to yield a LH-RBI-containing fraction in the higher molecular weight range of 30,000-10,000 and a LH-RBI-containing fraction in the lower molecular weight range of 10,000-1000. The higher molecular weight LH-RBI fraction was purified on Sephadex G-25 and the lower molecular weight LH-RBI fraction was purified on Sephadex G-50. Both the high- and the low-molecular-weight LH-RBI species inhibited the binding of 125I-labeled human chorionic gonadotropin (hCG) to bovine corpora lutea and to rat Leydig cell membrane receptors. Similarly, the production of testosterone by hCG-stimulated rat Leydig cells was inhibited in a dose-response manner by both the high- and the low-molecular-weight LH-RBI species. The LH-RBI activity in the low-molecular-weight species was stable at 4 degrees C for up to 6 months and at temperatures up to 90 degrees C for 15 mins, whereas the LH-RBI activity of the high-molecular-weight species was stable at 4 degrees C for 15 months and unstable at 60 degrees C after 15 min. The 7000 g supernatant provided a much-needed source to obtain larger than previously reported quantities of LH-RBI for isolation as well as for structure and function studies.
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