GnRH Yields Membrane Particles Containing Annexin A5 (ANXA5) from Gonadotrophs and Their Enhancement of LH Release

Presentation Number: SUN 492
Date of Presentation: April 2nd, 2017

Numfa Fungbun*, Makoto Sugiyama, Ryota Terashima, Shiro Kurusu and Mitsumori Kawaminami
Kitasato University, Aomori, Japan

Abstract

Annexin A5 (ANXA5) is a member of annexin family protein that is characterized by calcium-dependent binding to phospholipid membranes. The expression of ANXA5 is stimulated by GnRH in gonadotropes and ANXA5 augments gonadotropin secretion. While abundant ANXA5 exists within gonadotropes, extracellular ANXA5 was also demonstrated even though it does not contain a signal sequence in the transcript. Hence, it is not known so far how and where ANXA5 exerts its stimulatory effect on LH release at gonadotrophs. In the present study, we first confirmed the stimulatory effect of ANXA5 on LH release and then examined our hypothesis that GnRH would affect ANXA5 localization in the gonadotrope. Recombinant rat ANXA5 augmented LH release in LβT2 gonadotrope cells in a dose dependent manner. GnRH agonist stimulation of LH release was also augmented by adding human or rat recombinant ANXA5 to the primary culture of pituitary cells. Immunocytochemistry of LβT2 cells showed that GnRH agonist induced membrane buddings containing ANXA5 within ten minutes. When primary pituitary cells were double stained with anti-ANXA5 and LHβ, ANXA5 containing membrane buddings were also observed on gonadotropes. Organ culture of hemi-pituitary gland with GnRH agonist was performed for 30 minutes and tissues were subjected to electron microscope observation. It was detected that the border of gonadotrope became obscure with bubble like structure. Particulate fractions of conditioned medium after incubation of LβT2 cells with GnRH agonist were obtained by sequential centrifugation. LβT2 cells were incubated with GnRH agonist for 10, 30 or 180 minutes. Conditioned medium was sequentially centrifuged at 2,000, 20,000 and 100,000 xg. Each precipitates were suspended with SDS-PAGE sample buffer and subjected to SDS-PAGE and Western-blotting with anti-ANXA5. ANXA5 was detected profoundly in the 20,000 xg pellets of GnRH agonist stimulated culture. Finally, 20,000 xg pellet fraction was shown to stimulate LH release of LβT2 cells. These results reveal that GnRH yields extracellular vesicles, probably ectosomes, containing ANXA5 and the vesicle is beneficial for LH release. It is suggested that this occurs at the gonadotrope under the control by GnRH.

 

Nothing to Disclose: NF, MS, RT, SK, MK