The actions of the intraovarian catecholaminergic system have been the focus of a number of studies since the discovery of noradrenergic innervation in the ovarian follicle. The present investigation describes the effects of NE in the steroid hormone profile of a serum-free GC culture system in the context of follicular development and dominance. Our initial aims were to evaluate how effectively the system maintains GC features and follicular steroidogenesis and also, to identify the beta-adrenergic modulation of hormone production. The high E2-producing culture described here represents the ovarian follicle that is growing towards dominance, given that such culture system had greater production of P3, high positive correlation between pro-dominance hormones E2 and P3, and tended to have high E2:P4 ratio. In this physiologically relevant system, it was possible to observe NE stimulation on P4 production, which suggests a pro-luteinization function of NE in the growing follicle.
To our knowledge, the present study is the first to examine the effects of NE on gonadotropin-independent steroidogenesis by primary serum-free GC cultures with high E2 production. The finding that NE is a modulator of follicular steroidogenesis is relevant for a better understanding of local intrafollicular regulatory mechanisms because NE is released by terminal innervations in the outer theca cells and there is evidence of de novo synthesis of NE by oocytes  and interstitial neuron-like cells . Furthermore, NE binds to adrenergic receptors which are mostly concentrated in other compartments, such as at interstitial and theca cells [23–25]. Thus, NE has the potential to function as an intrafollicular paracrine regulator of GCs. Our finding that NE increased P4 production by bovine GCs in a dose-dependent manner agrees with previous reports that exogenous delivering of NE to cows during the luteal phase  and in vitro to granulosa-luteal cells  enhanced P4 production. However, in both in vivo and in vitro studies mentioned above, GCs were evaluated during the luteal phase. In cows, during this phase, circulating levels of P4 are already high and follicular E2 production is low. Moreover, these models also presented interfering factors (gonadotropins and serum contaminants, respectively) that prevent a proper evaluation of gonadotropin-independent hormone production. These limitations highlighted the need for a proper and controlled in vitro model that allows the investigation of the effects of NE and other putative intraovarian factors in the follicles in a more physiologically relevant state in regards to the modulation of follicular development and dominance.
Steroid hormone production by GCs may vary under different culture conditions. Most serum-based GC culture systems are not adequate for studying the actions of intrafollicular factors because the serum itself already contains an undefined combination of hormones, nutrients, and both growth and attachment factors that frequently leads to GC proliferation  and/or luteinization . In fact, E2 production, a pro-dominance marker of GC, declines during culture in serum-coated dishes [18, 21]. In the serum-free culture system described here, GCs cultivated under chemically defined medium supplemented with PVA continued to secrete E2 throughout the 144h of culture under gonadotropin-independent conditions. High E2 production by GCs cultured in the presence of bovine serum albumin or other serum replacers has also been described in earlier studies [16, 28]. Although attempts to propagate bovine GCs using serum replacers have succeeded in some cases [16, 28, 29], some of those serum replacers were contaminated with steroids and other interfering factors . Previous experiments from our group suggest that replacement with PVA (and additional defined supplements) allows for greater and earlier E2 production by GCs when compared with bovine serum albumin, a widely used serum replacer .
Pregnenolone is an important intermediary metabolite for E2 production and is synthesized at both follicular compartments: granulosa and theca cell . However, only theca cells express the enzymes that convert P3 into androgens, which, in turn, are the direct precursors of E2. Also, the concomitant expression of aromatase (the main E2 synthesizing enzyme) and of cytochrome P450 11A1 (enzyme that converts cholesterol in P3) have been associated to healthy growing follicles greater than 4mm in diameter . The present investigation did not use the expression of steroidogenic enzymes as markers of healthy growing follicles, however a parallel evaluation is proposed here based on the production of E2 and P3, which are the resulting hormones synthesized by these key steroidogenic enzymes associated to growing follicles. In fact, P3 production is a useful marker of active follicular steroidogenesis , and an important characteristic of the serum-free culture model described here is the constant increase in P3 and a high significant correlation between E2 and P3 production.
In contrast to findings reported from other bovine GC primary cultures [28, 29, 32] and even buffalo GC serum-free culture , P4 was low during all time points evaluated in our cell cultures and a significant increase was observed only after 96h. A low although increasing P4 production is an important feature of the ideal GC culture system that represents physiological events of an E2 active follicle that is growing towards dominance. Also, the lack of positive correlation between E2 and P4 production reinforces the adequacy of the present culture system.
Our ultrastructural findings show that GCs cultured under the present conditions preserve morphological characteristics similar to those of in vivo and in vitro E2-producing GCs. Because cellular architecture directly reflects cellular function, culture systems that preserve normal GC morphology are more physiologically relevant . For instance, the maintenance of epithelial characteristics is only possible due to the presence of gap and tight intercellular junctional complexes responsible for intercellular communication . Tight junctions were commonly observed in the GCs in our culture system. Furthermore, elongated mitochondria with tubulovesicular cristae, cytoplasmic lipid droplets, and abundant rough/smooth endoplasmic reticulum were also detected in the GCs and are consistent with features of cells involved in cholesterol and steroid metabolism . Taken together, the steroidogenic profile and the ultrastructural findings of the GC reveal the functional status of the present culture conditions, suggesting direct similarities to the follicle in vivo that is growing towards dominance.
Norepinephrine has frequently been studied as an intraovarian modulator of gonadotropins [36, 37], prostaglandin , and oxytocin . The purpose of the present study, however, was to examine the direct and gonadotropin-independent actions of NE. Our results show a dose-dependent effect of NE in P4 production of high E2-producing bovine GCs. In agreement with this finding, dose-dependent increments in P4 and cAMP were observed in a rat GC model in response to the non-selective beta-adrenergic agonist isoproterenol . Similarly, catecholamines promoted P4 production in in vitro porcine GCs . In addition, isoproterenol induced expression of the P4 synthesizing enzyme, 3-beta-hydroxysteroid dehydrogenase, in primary cultures of porcine GC . However, both rat and porcine in vitro systems used serum supplementation and consequently, GCs tended to luteinize spontaneously. Thus, those systems present limitations on evaluating the adrenergic effects in the health growing follicle. In contrast, beta-adrenergic agonists minimally stimulated P4 production in hypophysectomized rats , perhaps because of in vivo interfering factors.
To further substantiate our findings, we evaluated the ability of the non-selective beta-adrenergic antagonist, propranolol, to reverse NE-induced P4 production. It is well established that propranolol acts through beta-1 and beta-2 adrenergic receptors to block catecholaminergic actions. Unexpectedly, propranolol was not able to block P4 production in the lower molar concentrations despite being added at 10-fold molar excess in relation to NE. Although it is possible that propranolol was not added in sufficiently high concentration, the lack of effect seems surprising considering that propranolol reverses isoproterenol-induced increments in P4 production in porcine GCs , as well as adrenaline (epinephrine)-dependent P4 stimulation in human GCs . Although an extended dose–response experiment still need to be performed in order to optimize propranolol blockage in the present conditions, a possible explanation to the lack of propranolol effect could be the involvement of alpha-adrenergic receptors mediating NE effects in GCs. Most studies about the effects of intraovarian NE in the presence of non-selective adrenergic agonists and antagonists have suggested that beta-2 adrenergic receptors mediate the effects of NE on P4 production by GCs and luteal cells [9, 36, 37]. However, NE may also modulate steroidogenesis via alpha-adrenergic receptors , or interactions between alpha- and beta-adrenergic receptors. Since most NE-mediated P4 responses have been described in luteal cells or during the luteal phase, we speculate that GCs from developing growing follicles (and not from luteal or luteinized tissues) may have a greater response to NE via alpha-adrenergic receptors. This might be a distinct mechanism from the one previously described in luteal cells. However, this hypothesis still needs to be formally tested in future investigations.