Establishment of steroidogenic granulosa cell lines expressing follicle stimulating hormone receptors

Keren-Tal, Iris; Dantes, Ada; Sprengel, Rolf; Amsterdam, Abraham

doi:10.1016/0303-7207(93)90042-i

Cited by 67 publications

(25 citation statements)

References 43 publications

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“…The addition of the appropriate gonadotropins to these cells has previously been shown to stimulate cAMP production, activation of PKA, and induction of steroidogenesis (Ref. 18 and data not shown). Since the ERK cascade was implicated in the signaling of G protein-coupled receptors (23), we first examined whether the ERK cascade is also activated in the rLHR-4 and rFSHR-17 cell lines.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

Seger¹,

Hanoch²,

Rosenberg

et al. 2001

Journal of Biological Chemistry

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212

110

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The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH-and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.Gonadotropic hormones, follicle-stimulating hormone (FSH) 1 and luteinizing hormone (LH), which are released from the pituitary, play a crucial role in controlling reproductive function in males and females. The pleotropic effects of gonadotropins are manifested in various cells of the reproductive system including LH and FSH in ovarian granulosa cells, LH in theca interna cells, FSH in testicular Sertoli cells, and LH in Leydig cells (1-3). One of the main effects of both LH and FSH on the ovary is the stimulation of the production of estradiol and progesterone, which play important roles in ovarian function and control of the reproductive cycle (reviewed in Ref. 4). The mechanisms involved in the regulation of progesterone production by ovarian granulosa cells have been characterized in detail. Gonadotropins exert their stimulatory activity via interaction with specific seven-transmembrane receptors, the LH receptor and FSH receptor. Upon binding of the gonadotropins, both receptors stimulate the G s protein, which activates the membrane-associated adenylyl cyclase, causing an elevation of intracellular cAMP (5). This cyclic nucleotide serves as a second messenger for the up-regulation of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 (P450scc) enzyme system (reviewed in Refs. 6 and 7).Activation of alternative signaling pathways by the gonadotropin receptors was described in the last decade, including calcium ion mobilization, activation of the phosphoinositol pathway, and stimulation of chloride ion influx (reviewed in Ref. 8). However, these gonadotropin-induced signaling processes were not previously implicated in the modulation of s...

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Section: Resultsmentioning

confidence: 99%

“…The rFSHR-17 cell line was established by immortalization of preovulatory rat granulosa cells via cotransfection of primary cells with SV40 DNA and an HA-ras gene. Cells were transfected with plasmid expressing the rat FSH receptor (18). The cells were maintained in F-12/DMEM medium (1:1) containing 5% fetal calf serum.…”

Section: Methodsmentioning

confidence: 99%

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

Seger¹,

Hanoch²,

Rosenberg

et al. 2001

Journal of Biological Chemistry

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212

110

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“…For the bioassays, we used rat immortalized granulosa cells expressing the rat receptor of FSH or LH as previously described (denoted as FSHr and LHr cells) (Keren-Tal et al, 1993;Suh et al, 1992). Following stimulation with gonadotropins from various species including the bovine, adenylyl cyclase is activated and the cells produce and secrete progesterone (Keren-Tal et al, 1993;Suh et al, 1992).…”

Section: Immortalized Rat Granulosa Bioassaymentioning

confidence: 99%

“…Following stimulation with gonadotropins from various species including the bovine, adenylyl cyclase is activated and the cells produce and secrete progesterone (Keren-Tal et al, 1993;Suh et al, 1992). The cells were grown on 24-well culture dishes (40,000 cells/well) in DMEM/F12 medium (1:1) and supplemented with 5% FCS and antibiotics.…”

Section: Immortalized Rat Granulosa Bioassaymentioning

confidence: 99%

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

Asraf

Amsterdam

Ben-Menahem

2015

General and Comparative Endocrinology

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“…The levels of rhFSH and rbFSH produced were, 32 IU/ml and 5 lg/ml, respectively, as measured by radioimmunoassay (RIA), according to Dobson [31]. The biological activity of these recombinant FSH products has been assessed in vitro, using the rat pre-ovulatory follicle cell line (GFSHR-17), which over-expresses the rat FSH receptor and responds to FSH stimulus by progesterone production [32]. The potency of the recombinant FSH preparations was assessed in vivo by assaying the follicle hypertrophy activity of the recombinant hormone when injected into pre-pubertal female rats [33].…”

Section: Recombinant Follicle-stimulating Hormone For Human Fertilitymentioning

confidence: 99%

NUCEL (Cell and Molecular Therapy Center): A Multidisciplinary Center for Translational Research in Brazil

et al. 2008

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Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil's scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the São Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.

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Establishment of steroidogenic granulosa cell lines expressing follicle stimulating hormone receptors

Cited by 67 publications

References 43 publications

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

NUCEL (Cell and Molecular Therapy Center): A Multidisciplinary Center for Translational Research in Brazil

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