**8. Methodologies used to analyze the participation of neural signals**

The participation of the peripheral nervous system in the regulation of adrenal, ovarian and testicular functions is studied using two main experimental approaches: *in vitro* and *in vivo* methods. Studies *in vitro* allow for the understanding of the isolated participation of one, two or even three neurotransmitters in the regulation of hormones secretion by one type of cell, or even an organ tissue. Studies *in vitro* have certain advantages, such as the possibility of analyzing the cellular mechanisms regulated by neurotransmitters, identifying the receptors participating in the regulation, and the molecular changes that occur. *In vitro* methods also have disadvantages, since in many studies the amount of neurotransmitters added to the culture medium is much higher than the normal concentration measured in the organ. Another problem of *in vitro* studies is the loss of the interplay occurring between different kinds of cells.

*In vivo* methods include the analysis of nerve stimulation and/or sectioning, the extirpation of one or both organs, the denervation of the *in situ* organ, as well as the local or systemic injection of neurotransmitters or blocking agents. The information obtained from *in vivo*  studies gives an idea about the animal's response to such manipulations (changes in hormone levels; metabolic modifications, etc.). In general, the cellular mechanisms participating in the modifications resulting from such manipulations are not clearly evident.

### **8.1** *In vitro* **methods**

Taken together, the results of *in vitro* and *in vivo* studies give an idea about the participation of neurotransmitters in regulating steroid hormone secretion. Studies on the participation of different systems regulating the secretion of steroid hormones analyze the effects of directly injecting neurotransmitters or substances known to block its receptors. Incubating steroidhormones producing cells, with or without specific neurotransmitters, or in neurotransmitters "cocktails", is the main methodology used for studying the participation of neural signals regulating the secretion of steroid hormones.

Serotonin inhibits testosterone, dihydrotestosterone, and androstane-3alpha, 17beta-diol production from testicles of peripubertal and adult hamsters maintained in long or short photoperiods. Serotonin also inhibits the stimulation induced by hCG, cAMP and testosterone production, by its union to 5-HT1A and 5-HT2A receptors subtypes. The testicular activity of the serotoninergic system is mediated by the corticotrophin releasing

The ovaries receive motor innervations from the sympathetic and the parasympathetic system via the vagus nerve, and possess afferent fibers travelling sympathetic and vagal routes (Burden et al 1983; Klein and Burden, 1988, Gerendai et al., 2000, 2009). The vagus nerve connects the ovaries with the area postrema, the nucleus of the solitary tract, the dorsal vagal complex, the parapyramidal nucleus, A1, A5, and A7 -cell groups, the caudal raphe nuclei, the hypothalamic paraventricular nucleus, the lateral hypothalamus, the Barrington's nucleus, the locus coeruleus, the periaqueductal gray, and the dorsal hypothalamus. All of these areas form a neural circuit that directly participates in the neural communication between the CNS and the ovaries (Gerendai et al., 2000; Tóth et al., 2007). As in the adrenals, the ovaries have micro-ganglia with tyrosine hidroxilase positive neurons (D'Albora & Barcia, 1996; Dees et al., 1995; D'Albora et al., 2002), and along some

capillaries there are neurons resting on the basal (D'Albora & Barcia, 1996).

different kinds of cells.

**8.1** *In vitro* **methods** 

regulating the secretion of steroid hormones.

**8. Methodologies used to analyze the participation of neural signals** 

The participation of the peripheral nervous system in the regulation of adrenal, ovarian and testicular functions is studied using two main experimental approaches: *in vitro* and *in vivo* methods. Studies *in vitro* allow for the understanding of the isolated participation of one, two or even three neurotransmitters in the regulation of hormones secretion by one type of cell, or even an organ tissue. Studies *in vitro* have certain advantages, such as the possibility of analyzing the cellular mechanisms regulated by neurotransmitters, identifying the receptors participating in the regulation, and the molecular changes that occur. *In vitro* methods also have disadvantages, since in many studies the amount of neurotransmitters added to the culture medium is much higher than the normal concentration measured in the organ. Another problem of *in vitro* studies is the loss of the interplay occurring between

*In vivo* methods include the analysis of nerve stimulation and/or sectioning, the extirpation of one or both organs, the denervation of the *in situ* organ, as well as the local or systemic injection of neurotransmitters or blocking agents. The information obtained from *in vivo*  studies gives an idea about the animal's response to such manipulations (changes in hormone levels; metabolic modifications, etc.). In general, the cellular mechanisms participating in the modifications resulting from such manipulations are not clearly evident.

Taken together, the results of *in vitro* and *in vivo* studies give an idea about the participation of neurotransmitters in regulating steroid hormone secretion. Studies on the participation of different systems regulating the secretion of steroid hormones analyze the effects of directly injecting neurotransmitters or substances known to block its receptors. Incubating steroidhormones producing cells, with or without specific neurotransmitters, or in neurotransmitters "cocktails", is the main methodology used for studying the participation of neural signals

Serotonin inhibits testosterone, dihydrotestosterone, and androstane-3alpha, 17beta-diol production from testicles of peripubertal and adult hamsters maintained in long or short photoperiods. Serotonin also inhibits the stimulation induced by hCG, cAMP and testosterone production, by its union to 5-HT1A and 5-HT2A receptors subtypes. The testicular activity of the serotoninergic system is mediated by the corticotrophin releasing hormone (CRH) and by the noradrenergic system. CRH has an inhibitory modulation of testosterone, dihydro-testosterone, and androstane-3alpha, 17beta-diol secretion, while epinephrine and norepinephrine have a stimulatory effect through alpha1/beta1-adrenergic receptors (Frungeri et al., 2002).

Stress induced by sleep deprivation results in lower testosterone levels in serum and lower testicular StAR protein expression, while serotonin and corticosterone serum levels are elevated (Wu et al., 2011). These results suggest that serotonin regulation of steroid hormones release depends on the cells where such sterols originate.

Acting through -1 and -2 receptors, NA stimulates progesterone secretion from luteal slices of heifers, and increases cytochrome P-450scc and 3 beta-HSD activity (Miszkiel & Kotwica, 2001). Nitric oxide (NO) inhibits the activity of cytochrome P450 aromatase and the secretion of estradiol by granulosa cells in culture (Ishimaru et al., 2001). *In vitro* studies show that in the rat, the participation of neurotransmitters regulating the secretion of ovarian progesterone varies throughout the day of the estrous cycle. In diestrus-1, NPY, NA, and VIP inhibit progesterone secretion by the ovaries, while on diestrus-2 these neurotransmitters stimulate progesterone secretion. In diestrus 1 and 2, NA+VIP or NA+NPY had a synergic effect on progesterone secretion, since measured concentrations were higher than VIP or NPY treatment alone (Aguado, 2002). In the rat, ovarian denervation reduces the synthesis and secretion of progesterone by inhibiting 3-betaHSD activity (Burden & Lawrence, 1977). Sectioning the plexus nerve and the SON of pigs led to lower LH, progesterone, androstenedione (A4), testosterone, estrone and estradiol-17beta plasma levels. In addition, a significant increase in the immune-expression of cholesterol side-chain cleavage cytochrome P450 occurs in follicles, as well as a decrease in 3-betaHSD activity, and in LH, progesterone, androstenedione (A4), testosterone, estrone and estrogen plasma levels (Jana et al., 2007).

Using an *ex vivo* celiac ganglion (CG)-SON-ovary (CG-SON-O) system, Aguado's research group has contributed to the understanding of the participation of the SON, the plexus ovarian nerve and the vagus nerve in regulating the secretion of ovarian hormones. In *in vitro* studies, the release of ovarian hormones is modulated by the stimulation/inhibition of neurons present in the CG.

According to Morán et al., (2005), the CG form a bilateral structure with the superior mesenteric ganglia in the rat, receiving the name of celiac-superior mesenteric ganglion (CSMG) which is composed of noradrenergic neurons called principal neurons, small intensely fluorescent cells, and peptidergic interneurons.

In *in vitro* studies, adding NPY, VIP or substance P (SP) to the ovaries obtained from rats in diestrus 1 resulted in lower release of progesterone, while the same treatment to ovaries obtained from rats in diestrus 2 increased it. Adding these three neuropeptides to the CG from rats in diestrus 2 resulted in higher progesterone secretion (Garraza et al., 2004). These results suggest that the way neural signals participate in the regulation of steroid secretions depends on the day of the estrous cycle and the type of cells receiving the signal.

Adding NA to the CG obtained from rats on diestrus 1 resulted in ovarian dopaminergic and noradrenergic activity increases, while adding NA to the CG system from rats on diestrus 2 only increased noradrenergic activity. Such changes in dopaminergic and noradrenergic ovarian activities resulted in lower release of androstenedione in systems obtained from rats on diestrus 1, and higher release of androstenedione in systems obtained from rats on diestrus 2 (Bronzi et al., 2011).

Hormonal and Neural Mechanisms Regulating Hormone Steroids Secretion 17

vagus centers seem to be the place where information from other regions of the CNS converges. Both centers send and receive neural information that modulates the reactivity of the endocrine cells to hormonal signals from the ovaries and adrenals. The existence of neural communication between the right and left CMSG implies the existence of neural communication between the ovaries. The modulation exerted by the neural signals over the ovaries and the adrenals varies during the estrous cycle (ovaries) and along the day (adrenals and ovaries). Such modulation is asymmetric, and the asymmetry varies during

Extirpating one ovary or one adrenal result in acute neural stimulation of the *in situ* ovary and/or adrenal, that modifies the response of endocrine cells to the hormonal signals. Sectioning one nerve also results in an acute neural stimulation of the denervated organ**,**  though such stimulation is more restricted. For the ovaries and adrenals, the partially denervated organ still has neural pathways regulating its functions and, in theory, the

To study the role played by the ovarian innervations in regulating progesterone, testosterone and estradiol levels in serum we have used five experimental models. The experimental models were performed on cyclic rats and our studies analyzed the influence

The unilateral perforation of the dorsal or ventral abdominal wall results in different changes in progesterone, testosterone and estradiol serum levels. Irrespective of the day of the estrous cycle surgery was performed, rats with a ventral sectioning of the abdominal wall showed higher progesterone and testosterone levels in serum than control rats and rats with dorsal sectioning of the abdominal wall (Figure 2). The increase in hormone release could be explained by an increase in StAR protein phosphorylation and/or synthesis stimulated by the neurotransmitters released by the neural terminals arriving to the ovaries and adrenals. Changes in ACTH, LH and FSH serum levels induced by sectioning the abdominal wall cannot be ruled out. Since estradiol levels were not modified, we presume that P-450 aromatase activity is not influenced by the neural information arising from the

Uchida et al. (2005) showed the existence of asymmetry in the neural reflexes arising from the abdominal skin that arrive to the ovaries and affect the ovarian blood flow and the activity of the SON. Stimulating the left abdomen produced a much stronger effect on the activity of the left ovarian sympathetic nerve than stimulating the right abdomen. The magnitude of the changes in hormone levels induced by ventral or dorsal surgery depend on both, the dorsal or ventral side (left or right) of the surgery and the day of the estrous cycle when surgery is performed. These results suggest the existence of a multisynaptic neural pathway between the abdominal wall, the adrenals and the ovaries, a pathway that is mediated through the innervations of the adrenals and ovaries (Flores et

1. The effects of dorsal and ventral surgery to reach the ovaries and their innervation.

the estrous cycle and the hour of the day.

innervated organ received only one different neural signal.

2. The effects of bilateral ovariectomy or adrenalectomy

4. The effects of unilateral or bilateral section of the SON

**9.1 The effects of dorsal and ventral surgical approaches** 

of the day of the estrous cycle on treatment results.

3. The effects of unilateral ovariectomy

abdominal wall.

al., 2008).

5. The effects of unilateral adrenalectomy.

The results presented above suggest that the adrenergic activation of the CG plays a role in regulating ovarian androgen secretion, and that this role varies along the estrous cycle. Therefore, steroidogenesis appears to be controlled by a balance between the stimulatory effects of hormones secreted by the pituitary, the inhibitory effects of other hormones, and the modulating participation of the ovarian innervations.

Pituitary hormones and innervations, including sympathetic and sensory nerves, also regulate the adrenal cortex secretion of hormones. The nerves innervating the adrenal cortex include heterogeneous populations containing various different neuropeptides (Kondo, 1985). The sympathetic innervation is composed of cholinergic preganglionic fibers and catecholaminergic postganglionic fibers that are positive for tyrosine hydroxylase (TH) and NPY (Kondo, 1985; Holgert et al., 1998). Sensory innervations consist of primary afferent fibers that are positive for calcitonin gene–related peptide (CGRP) and SP (Kuramoto et al. 1987). Intrinsic innervations on the adrenal cortex arise from two types of medullar ganglion cells: Type I cells are noradrenergic and NPY-positive, whereas Type II cells produce neuronal nitric oxide synthase and VIP (Holgert et al., 1998). Preganglionic sympathetic and primary afferent fibers are carried in the thoracic splanchnic nerve (Ulrich-Lai & Engeland 2000).
