TY - JOUR
T1 - An optimized model for hCG stimulation of human mural granulosa cell culture
AU - Ophir, Libby
AU - Yung, Yuval
AU - Yerushalmi, Gil Mordechai
AU - Baum, Micha
AU - Machtinger, Ronit
AU - Maman, Ettie
AU - Hourvitz, Ariel
N1 - Publisher Copyright:
© 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn
PY - 2019/3
Y1 - 2019/3
N2 - Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion. Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6–8 h after hCG, while 24 h of hCG stimulation was needed for late induced genes. Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later.
AB - Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion. Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6–8 h after hCG, while 24 h of hCG stimulation was needed for late induced genes. Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later.
KW - Follicle
KW - Granulosa cells
KW - Ovary
KW - Primary cell culture
KW - hCG
UR - http://www.scopus.com/inward/record.url?scp=85060083589&partnerID=8YFLogxK
U2 - 10.1016/j.repbio.2019.01.002
DO - 10.1016/j.repbio.2019.01.002
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C2 - 30661769
AN - SCOPUS:85060083589
SN - 1642-431X
VL - 19
SP - 67
EP - 74
JO - Reproductive Biology
JF - Reproductive Biology
IS - 1
ER -