**1. Introduction**

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In developed countries, the elderly population increases at an accelerated rate due to a decrease in the birth rate and the prolongation of life through medical development. Moreover, increases in the elderly population allow the prediction of an increase in hyperprolactinemia caused by aging. It is well known that hyperprolactinemia decreases libido and causes oligozoospermia [1]. On the other hand, hyperprolactinemia is caused by or associated with, a variety of pathogenic stages: pituitary adenoma, hypothalamic disorders, hypogonadism and hypothyroidism, and is detected in patients with infertility [2, 3], impotence and hypogonadism [4]. PRL is a polypeptide hormone that is synthesized and secreted from mammotropes in the anterior lobe of the pituitary gland [5]. Many studies have documented a critical role of PRL in the maintenance of lactation in women and female animals [6, 7] as well as in immunregulation in both, males and females [8], however, its role in sexual behavior is not entirely clear [9-16].

It has been also shown that DAerg agonists facilitate several aspects of copulatory behavior and *ex copula* genital responses [10]. DAerg neurons, locating in the medial preoptic area (MPOA), and the zona incerta (incertohypothalamic DAerg system) are the key centers in the stimulatory control of sexual functions [17-18]. (R)-salsolinol (SAL), a DA related and derived tetrahydroisoquinoline, has been recently identified as a strong candidate for being the endogenous PRL releasing factor (PRF) synthesized in both the hypothalamus and the neurointermediate lobe (NIL) of the pituitary gland. Analysis of SAL concentrations revealed parallel increase and decrease with the elevation and reduction of plasma PRL, respectively. SAL is sufficiently potent and selective *in vivo* to account for the massive discharge of PRL that occurs after physiological changes. At the same time, parallel with its DA depleting effect in sympathetically innervated peripheral organs, SAL can reduce testosterone secretion both *in vivo* and *in vitro* from Leydig cells [19-20]. Based upon all of

© 2013 Nagy et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

these data, the aim of our present studies was to confirm the suppressive effects of hyperprolactemia induced by grafting pituitary glands under the kidney capsule and to investigate the effect of a single injection of SAL on the elevation of plasma PRL and on sexual behavior in male rats.

The Role of Prolactin in the Regulation of Male Copulatory Behavior 141

Prolactin (ng/ml)

Testosterone (ng/ml)

All data are presented as mean ± SEs. The results from the copulatory behavior testing were analyzed using Fisher's exact probability test and the Mann-Whitney U test and the data for

The results of serum hormone levels in two pituitaries grafted and sham animals are shown in Table 1. Prolactin (PRL) concentration in rats having two pituitary grafts was significantly higher than sham operated animals (p < 0.05). There were no significant differences in the serum levels of luteinizing hormone (LH), folliculostimulating hormone (FSH) and

> FSH (μg/ml)

Graft 1.3 ± 0.16 65.3 ± 13.60 31.1 ± 3.40a 2.1 ± 0.29

Sham 1.3 ±0.20 97.4 ± 9.84 7.2 ± 1.51b 2.6 ± 0.24

As it is shown on Fig. 1, the mean number of mount in rats having one-, two grafted pituitaries and non was 76.5 ± 5.35, 66.8 ± 6.37 and 40.2 ± 3.43, respectively. The mount frequency (MF) showed a tendency to be higher in grafted, compared with sham-operated animals. The mean frequency of intromission (IF) was lower for one (18.0 ± 1.27) and two pituitaries grafted males (10.0 ± 2.00), compared to sham-operated controls (29.0 ± 2.00). There were significant differences in IF between two pituitaries grafted and sham-operated males (p < 0.05). Ejaculation could be detected in all one-pituitary grafted and shamoperated males, while did not in 4 out of 6 two-pituitaries grafted males. The mean ejaculation frequency (EF) of one, two pituitaries grafted and sham males was 3.5 ± 0.33, 0.8 ± 0.20 and 5.2 ± 0.20, respectively. The EF of two pituitaries grafted males is significantly

As shown on Fig. 2, having two pituitary grafts resulted in a significant prolongation in the mean latency of intromission, compared with sham-operated animals (1,014.4 ± 206.12 versus 67.8 ± 7.72 sec, p < 0.05). The mean latency to the first ejaculation showed a tendency to extend in animals having one (652.1 ± 56.06 sec), two (1,367.5 sec) pituitary grafts, compared with sham-operated males (369.7 ± 25.28 sec). The post-ejaculatory interval (PEI) tended to extend in animals having one (425.7 ± 27.40 sec) or two (487.5 sec) pituitary grafts,

hormone levels and organs weights were analyzed by Duncan's multiple t-test.

**2. Effect of pituitary transplants induced hyperprolactinaemia on** 

**copulatory behavior** 

Group LH

All data represent mean ± S.E.

lower than sham males (P < 0.01).

compared with sham-operated animals (341.7 ± 3.27 sec).

\*p<0.05 a vs. b

testosterone between the same groups of animals.

(ng/ml)

**Table 1.** Serum hormone levels in grafted male rats

Sexually experienced male rats of the Wistar-Imamichi strain (Imamichi Institute for Animal Reproduction, Tsuchiura, Japan), approximately 10 weeks old at the start of the experiments, were used. The animals were kept in a room with a temperature of 22-26 Celsius and subjected to a light-schedule of 14 hrs light and 10 hrs darkness (lights off at 19:00). They were provided with pellet diet CRF-1 (Charles River Laboratories Japan, Atsugi, Japan) and water ad libitum. Stimulus females of the same strain were rendered sexually receptive by treatment with estradiol benzoate (10μg/0.1 ml sesame oil, Sigma Chemical Co. Ltd., St. Louis, USA) 48 hrs prior to, and progesterone (500μg/0.1 ml sesame oil, Sigma Chemical Co. Ltd., St. Louis, USA) 4 hrs prior to exposure to males.

One group of experimental animals were anesthetized with Nembutal (40 mg/kg, i.p.) and implanted one and two whole pituitaries from male donors with the same age in the same strain under the left kidney capsule [21]. Rats having 1 or 2 pituitary grafts were sacrificed by decapitation between 19:30 and 20:30 in a week after the test. It was carried out in 30 seconds after taking animals out from their cages [22-23].

In a separate group of animals, intravenous (i.v.) cannula have been inserted into the jugular vein of male Sprague-Dawley rats for injection of SAL, and being able to take blood samples. Saline or SAL (4 mg/kg body weight i.v.) have been injected to the animals prior to expose them to females being in estrus.

Copulatory behavior test have been conducted four weeks after the surgery. After a male rat was placed in the semi-circular observation cage (radius 40, height 50 cm) faced with Plexiglass under low-level red-light illumination for a few minutes, a sexually receptive female was introduced to its cage. Tests lasted 60 min from the introduction of the female. Behavioral testing was conducted between 19:30 and 20:30. The behavior categories scored included the following [24]. *Mounting frequency* (MF): number of mounts without intromission preceding ejaculation. *Intromission frequency* (IF): number of mounts with intromission preceding ejaculation. *Ejaculation frequency* (EF): number of ejaculations during 60 min. *Mount latency* (ML): time from the presentation of the female to the male's first mount. *Intromission latency* (IL): time from the presentation of the male's first intromission. *Ejaculation latency* (EL): latency from the first intromission until ejaculation. *Post-ejaculatory interval* (PEI): latency from ejaculation to the next intromission.

Blood collected and centrifuged at 3,000 g for 15 minutes for the analysis of serum hormone. The serum was stored at -80 Celsius until analyzed by RIA for determination of serum PRL, LH and FSH. Serum concentrations of PRL, LH and FSH were measured by RIA using the method of Furudate et al. [25] with reagents provided from NIADDK. The standard references used were, rPRL-RP-3 for PRL, rLH-RP-2 for LH and rFSH-RP-2 for FSH. The intra- and inter-assay coefficients of variation were 9.6 and 15.9 for PRL, 3.5 and 5.3 for LH, and 5.3 and 9.8 for FSH, respectively. Testosterone levels were also measured by direct RIA.

All data are presented as mean ± SEs. The results from the copulatory behavior testing were analyzed using Fisher's exact probability test and the Mann-Whitney U test and the data for hormone levels and organs weights were analyzed by Duncan's multiple t-test.
