Annual-Report-2019

PLEOTROPISM OF GONADOTROPIN ACTION
Prof. Manuela Simoni
The goal is to investigate how the two human gonadotropins influence
each other in granulosa cells expressing both receptors, or by co-culturing
cells expressing either the LHCGR or the FSHR (as a model granulosa/
theca interaction). To reach this goal the production of several cell lines is
necessary. These cell lines will then be used to perform in vitro experiments
with LH, FSH, hCG and combinations thereof and the data used for in
silico modeling to gain insight onto the dynamics of the intertwined cell
response to FSH and LH. Hypotheses about the underlying mechanisms
will be proposed and tested through a systems biology approach combining
computational modelling and experimental measurements.
LE STUDIUM / Marie Skłodowska-Curie
Research Fellow
Smart Loire Valley General Programme
From: University of Modena and Reggio
Emilia - IT
In residence at: Physiology of Reproduction
and Behaviour (PRC)​- Tours
Nationality: Italian
Dates: April 2019 to April 2020
Manuela Simoni, MD, PhD, trained as clinical
endocrinologist at the Unit of Endocrinology of the
University of Modena, Italy between 1982 and 1990
and, thereafter, as molecular endocrinologist
at the Institute of Reproductive Medicine of the
University of Münster, Germany, where she was
Professor for Endocrinology and Molecular
Biology of Reproduction from 1998 to 2008. Since
2008 she is full professor of Endocrinology at the
University of Modena & Reggio Emilia, Italy.
Her research interests are gonadotropin and
androgen action, testicular function, male
infertility, endocrinology and pathophysiology of
reproduction.
She is Editor-in-Chief of ANDROLOGY, the official
journal of the European Academy of Andrology
and the American Society of Andrology.
Bibliometry (Scopus): 239 articles, 11519 citations,
55 H-index.
Dr Pascale Crépieux
Host scientist
Pascale Crepieux is a CNRS Research Director at
the Laboratory of Physiology of Reproduction and
Behaviors in Nouzilly. Her main research area is
on the mechanisms whereby extracellular signals
are integrated into an adapted cell response, from
gonadotropin receptors membrane receptors
to gene expression. She acquired a strong
background in molecular biology during her PhD at
the Pasteur Institute in Lille, then in cell signaling
during a 3-year post-doctoral training at McGill
University, Montreal. Since 2012, she has been coheading
the Biology and Bioinformatics of Signaling
Systems (BIOS) group, a pluridisciplinary group
that gathers 8 research scientists. Among other
management activities, she has been participating
to the Specialized Scientific Commission of Animal
Physiology at INRA since 2011, and has been
recently reviewer for the Biomedecine Agency, for
the « SYSBios» of the Cancer ITMO, for the MRC
and for the ESF. She also teaches in Masters of «
Biology of Reproduction », « Biology, Health », «
Antibodies », at the University of Tours.
The achievements to date are the construction of plasmids (one still
ongoing) and the production of three cell lines. The last plasmid and
two more cell lines are expected to be completed before the end of the
stay. Further experiments will be continued both at the host site and
the University of Modena at the end of the fellowship, though continued,
intensive collaboration and exchanges.
To control both temporally and quantitatively the expression of the FSHR
and the LHCGR in the KGN human granulosa cell line, cell lines stably
expressing both receptors are needed. Three plasmids expressing FSHR,
LHCGR, Luciferase (as positive control) and an empty plasmid (as negative
control) with the TetOn system (induction of expression of the inserted gene
in the presence of Tetracycline), were constructed.
The basic structure consists of Sleeping Beauty recombinase recognition
site (SB) - response element activated by the Tet-sensitive transactivator
- minimal promoter - gene of interest (RFSH/RLHCG/Luciferase) - GFP
- tetracycline-sensitive transactivator - resistance to Puromycin - SB
recognition site - Ampicillin resistance. Both receptors were tagged in the
extracellular portion to be easily identified, c-myc-tagged-LHCGR and the
FLAG-tagged FSHR. Transfection conditions and conditions of tetracyclin
induction were optimised and several clones of KGN cells were obtained,
which have stably integrated the plasmid both FSHR and LHCGR. We are
currently proceeding with the characterisation of the clones and production
of the cell lines still outstanding. The cell line stably expressing LHCGR
is responsive in terms of cAMP production after stimulation by hCG.
Some intriguing results have been obtained with the FSHR-KGN cell line,
containing but apparently not expressing the FSHR, and this is currently
under further investigation.
Finally, further experiments with the new induction system based on
Coumarin are ongoing. The production of the double inducible system
(coumarin, tetracyclin) will complete the cell lines expressing both FSHR
and LHCGR.
Life & Health Sciences 2019
43