**Development of Male Sexual Function After Prenatal Modulation of Cholinergic System**

Alekber Bairamov1, Alina Babenko1, Galina Yukina2, Elena Grineva1, Boris Komikov2, Petr Shabanov3 and Nikolay Sapronov3 *1Almazov Federal Heart, Blood and Endocrinology Centre, St. Petersburg, 2Saint Petersburg State Medical Academy named after I. I. Mechnikov, 3Institute Experimental Medicine, Nord-West Division of the Russian Academy of Medical Science, St. Petersburg, Russia* 

### **1. Introduction**

92 Sexual Dysfunctions – Special Issue

Wallen, K.1990Desire and ability: hormones and the regulation of female sexual

Wilson, L. A., Wayman, C. P., and Jackson, V. M.2009Neuropeptide modulation of a lumbar spinal reflex: potential implications for female sexual functionJ.Sex Med.64947957

behaviorNeurosci.Biobehav.Rev.1422332410149-7634

Embryonal period of ontogenesis plays an important role in the brain development which is defined, first of all, by genetical factors. Normal flow of the process can be disturbed also under the influence of many environmental factors which affect, both a differentiation of neurones, and on a neurotransmitter choice in them used for communications with the proximate cells (Le Douarin, 1981; Pendleton, 1998). The majority of the factors attacking developing brain during this period, break a normal ontogenesis of neurotransmitter systems: NA, 5-HT, DA and ACh that shows high sensitivity of a brain in critical periods of the development (Williams, 1992; Oliff, 1999; Qiao, 2004).

A variety of neurochemical changes in the embryonic brain, induced by exposure to neurotropic compounds during the prenatal period, result in the development of functional impairments and behavioral disorders in the adult offspring. The mechanisms of action of many chemical factors on the developing fetal brain during early ontogenesis are in most cases mediated by alterations in the formation and functioning of brain neurotransmitter systems, including the cholinergic system, whose CNS function is associated with memory, learning, and behavioral processes (Yamada et al., 1986; Buzsaki, 1989; Everitt & Robbins, 1998; Levin & Slotkin, 1988; Zoli et al., 1999). During the period of neuron development, actions on cholinergic mechanisms lead to delays in cell differentiation which correlate with cognitive and behavioral deficits in fertile offspring (Yamada et al., 1986; Levin & Simon, 1998; Beer et al., 2005).

Prenatal exposure to neurotoxins (nicotine, organochlorine compounds, barbiturates), which have cholinotropic properties, produces long-lasting changes in neurotransmitter functions in early ontogenesis with the subsequent development of neurobehavioral anomalies and affective disorders in pubescent individuals (Seidler et al., 1992; Barinaga, 1996; Slotkin, 2004). Thus, embryonic exposure to nicotine leads to alterations to cell proliferation and differentiation, resulting in long-term changes to synaptic function (Peters, 1986; Lichtensteiger, 1988). Binding to N-cholinergic receptors in catecholamine-containing neurons in the fetal brain, nicotine disrupts the expression of these transmitters

Development of Male Sexual Function After Prenatal Modulation of Cholinergic System 95

performed. Female rats with a known date of pregnancy were obtained by mating females in proestrus-estrus with males. The day on which sperm were seen in vaginal smears was taken as the first day of pregnancy. Pregnant females, at different stages of gestation (9–11, 12–14, and 17–19 days of pregnancy), were given three i.m. injections (once daily) of the Ncholinoblocker ganglerone (10 mg/kg), while other groups received injections of the Mcholinoblocker methylbenactyzine (2 mg/kg) at the same time points. Doses were determined on the basis of the selectivities of cholinolytic actions and the absence of nonspecific actions. Control groups of females received injections of physiological saline. Experiments offspring groups (12–14 individuals per group) were formed in accord with the timing of prenatal administration of ganglerone (groups G10, G13, and G18, respectively) and methylbenactyzine (groups M10, M13, and M18). The offspring of intact rats served as

**Behavioral studies** were performed on rat offspring aged 3.5–4 months. Sexual experience was acquired in four sequential tests with receptive females. Sexual activity parameters were assessed using a standard sexual behavior test (3). Adult rats were kept in individual cages with food and water available *ad libitum*, in a room with controlled temperature and under an inverted 12 × 12 h light cycle (light off at 09:00 h). Tests for sexual behavior were done during the dark phase of the cycle and under dim red light illumination The test male was placed in the study chamber, of size 40 x 40 x 30 cm, for 5 min prior to presentation of a sexually susceptible female. Experiments were performed in dim red illumination. Receptivity in sterilized females was induced by sequential administration of estradiol dipropionate (25 mg, 48 h before the experiment) and progesterone (500 g, 4 h before the experiment). Components of sexual activity were recorded visually for 15 min in tests 1 and 4. The numbers of components of sexual behavior (mountings, intromissions, and ejaculations) and their latent periods were registered. During each behavioral test, the behavioral components recorded were mount latency (time from the introduction of a receptive female to the first mount), intromission latency (time from the introduction of a receptive female to the first intromission), ejaculation latency (interval between the first intromission and ejaculation), and postejaculatory interval (interval between the first

**Neurochemical studies** were performed using brains from 20-day embryos and brain structures (hypothalamus, hippocampus) from rat offspring two month age. The concentrations of the neurotransmitters dopamine (DA), noradrenaline (NA), and serotonin (5-HT) in brain tissues were measured by high-performance liquid chromatography using a Beckman System Gold with an LC-4C electrochemical detector. Brain structures were extracted on a cryostat at –20°C and were stored in liquid nitrogen until chromatographic analysis. Peaks were separated on a SphereClone 5 μ ODS 2 chromatography column (250 × 4.60 mm) with a Phenomenex precolumn. The mobile phase consisted of citratephosphate buffer pH 3.5, acetonitrile (88 ml/liter), and octanesulfonic acid (43 mg/liter). Chromatographic peaks were identified and assessed quantitatively in relation to peaks

Serum hormone levels were assayed by immunoenzyme analysis using standard biochemical kits (Chema, Access) on a Uniplan immunoenzyme analyzer. Statistical analysis. Results were compared with control data and analyzed statistically by analysis of

the control group.

ejaculation and the next intromission).

obtained from internal standards.

variance (ANOVA) on Origin 7.0.

(Lichtensteiger et al., 1988; Dani & Heinemann, 1996). Prenatal exposure to nicotine predominantly damages cholinergic, noradrennergic, and dopaminergic projections in the brain during postnatal life, with later cognitive and behavioral dysfunction in adult offspring (Naeye & Peters, 1984; Milberger et al., 1996; Fergusson et al., 1998; Orlebeke et al., 1997; Weissman et al., 1999; Slotkin et al., 2001).

The neurobehavioral teratogenic actions of barbiturates are also mediated by disrupted functioning of septohippocampal cholinergic conducting pathways, which is accompanied by a deficiency of synaptic transmission and accompanying hippocampus-related behavioral deficit (Wallace, 1984; Smith et al, 1986; Yanai, 1984, 1996; Steingart et al,, 2000; Azmitia, 2001; Beer et al. 2005; Beer et al., 2005). Phenobarbital, previously used for the prophylaxis of neonatal hyperbilirubinemia and bleeding in neonatal children, is a teratogenic factor in relation to behavior in both humans and animals (Yanai, 1984; Wallace, 1984; Smith et al, 1986). Experimental exposure to organochlorine compounds, like other neurotoxins with cholinotropic properties, damages cholinergic conducting pathways and leads to long-term alterations to the cholinergic system (Lauder, 1985; Dreyfus, 1998; Qiao et al., 2002; Slotkin et al., 2002; Slotkin, 2004). Dysfunction of cholinergic neurons plays a significant role in behavioral disorders seen in adult rats given organochlorine compounds during the prenatal period (Sherman et al., 1981).

Behavioral abnormalities as long-term consequences of prenatal exposure to various factors are generally difficult to observe because of the large phenotypic variability of the developing organism (Nicholls, 2000). There is great value in studying sexual behavior in these situations, as sexual functions are the most sensitive and susceptible aspects of reproduction in males and, as has been demonstrated, are regulated by the activities of several neurotransmitter systems, including the cholinergic (Bitran & Hull, 1987; Mas et al., 1987; Hull et al., 1988; Retana et al., 1993; Gladkova, 2000).

In addition, despite many studies of substances with cholinotropic properties and adverse influences on the developing brain during the prenatal period, the literature lacks reports of the behavioral effects of prenatally administered selective cholinolytics.

Taking into consideration all these statements, the purpose of the present research is study of known selective blockers of M- and N-cholinergic systems, prescribed in various terms of a prenatal period, on development dynamics of neurotransmitter systems of the rats embryos brain, and track their dynamics in an adolescent period and in adulthood in comparison to the behavioural sexual status in the paste for a sexual behavior at rats males. Tasks of the present part of work were the following:

