Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

Abstract

BackgroundSince noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance.MethodsPrimary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs.ResultsGCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8) M), a non-selective beta-adrenergic antagonist.ConclusionsThe present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

An immortalized steroidogenic goat granulosa cell line as a model system to study the effect of the endoplasmic reticulum (ER)-stress response on steroidogenesis

“…For example, the culture conditions for granulosa cells supplemented with serum can induce granulosa cell differentiation. The serum itself already contains unknown ingredients including a variety of hormones, nutrients, and undefined growth factors that mainly cause granulosa cells to proliferate and luteinize [ 53 ]. Therefore, GGC culture conditions without serum need to be formally explored in future studies.…”

Long-term α1B-adrenergic receptor activation shortens lifespan, while α1A-adrenergic receptor stimulation prolongs lifespan in association with decreased cancer incidence

“…In the current study, shortened lifespan in the female CAM-α 1B AR animals may have involved hormonal changes mediated by the α 1B AR. There are noradrenergic innervations to the breast and ovarian follicles, where norepinephrine release can increase the levels of estradiol and progesterone and a decrease of sympathetic input can slow tumor growth (Romeo et al 1991;Piccinato et al 2012). Furthermore, the norepinephrine reuptake inhibitor desipramine promotes breast cancer progression with α 2 AR activation; however, phenylephrine, a nonselective α 1 AR agonist, did not have an effect (Szpunar et al 2013).…”

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

“…A serum-free condition in vitro is crucial for understanding and control of the identity of all the constituents of serum that affect cultured cells ( 8 , 22 ). Serum-free medium can be achieved by replacing serum with PVA, a synthetic polymer that does not have any negative effects on cells in cultures ( 23 ).…”

“…The media used for FWS culture were: 1) NDM: TCM-199 with Earl's salts (Invitrogen-Gibco/BRL, USA), supplemented with 10% fetal calf serum, 11 mg/mL pyruvic acid, 200 ng/mL FSH (Sigma-Aldrich, USA), 5 IU/mL penicillin and streptomycin as described by Channing ( 5 ); or 2) serum-free DM: minimum essential medium alpha (Invitrogen-Gibco/BRL), supplemented with polyvinyl alcohol (PVA; Sigma-Aldrich), bovine insulin (Sigma-Aldrich), human recombinant IGF-1 (Invitrogen Life Technologies, USA), androstenedione (Sigma-Aldrich), nonessential amino acids (Invitrogen-Gibco/BRL), human transferrin (Invitrogen-Gibco), sodium selenium (AcrosOrganics, USA), 10 mM sodium bicarbonate (Invitrogen-Gibco/BRL), 0.02 M HEPES, and 10,000 IU penicillin and streptomycin (Sigma-Aldrich), as described by Gutierrez et al ( 6 ) and modified as described previously ( 13 , 14 ) (Patent PI No. 0803140-1, deposit date December 5, 2008, applicant: Fundação Universidade de Brasília).…”

“…Nondefined medium (NDM) is a commercial, luteinizing medium that includes serum ( 4 , 5 ), and DM contains growth factors and antioxidants that positively affect cultured cells ( 6 ). Other DMs, as developed by Gutierrez et al ( 6 ), allow bovine GC to respond, even in absence of FSH, and produce high levels of steroids ( 8 ), characterizing an ideal model for the study of factors intervening in steroidogenesis, markers of follicle fate; and, consequently, the dominance, atresia, or luteinization processes in vitro . For these reasons, we studied the effect of a DM previously known to induce estrogen production in short-term culture of bovine follicle walls.…”