**2. Effect of the reproductive system on the immune system: Roles of GnRH and sex steroids**

Distinctive features of GnRH-producing neurons include their small population size (800– 2000 cells), diffuse distribution in the septo-preoptic hypothalamic region, and extracerebral origin during ontogeny. Most these neurons have axons terminating in the median eminence, where GnRH is released in the portal circulation and regulates the synthesis and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by gonadotrope cells. GnRH isolated from the mammalian brain was initially regarded only as the hormone controlling the reproductive function, but subsequent studies have demonstrated that GnRH occurs in a variety of functionally different forms. To date, 23 such forms have been identified in vertebrates. Three of them—GnRH1, GnRH2, and GnRH3 are the most frequent, being expressed in groups of neurons differing in origin, location, and functional role. In adult animals, GnRH1 is expressed mainly in the hypothalamus; GnRH2, in the midbrain and tectum; and GnRH3, in the rostral regions of the forebrain. The population of hypothalamic neurons expressing GnRH1 is usually referred to as the GnRH1 system, and its basic function is to regulate the release of gonadotropins. GnRH2 and GnRH3 supposedly function as neuromodulators, the former being involved in the regulation of sexual behavior and the latter, in the integration of olfactory signals and other processes related to reproduction. Extracerebral synthesis is characteristic mainly of GnRH2, which has been revealed in the thymus, spleen, ovaries, testes, prostate, mammary gland, and placenta (Jacobson et al., 2000; Zakharova et al., 2005). Along with hypothalamic GnRH, extracerebral GnRH2 plays a role in the development and functioning of the immune system at different stages of ontogeny (Morale et al., 1991; Zakharova et al., 2005). According to Marchetti et al. (1998), GnRH is one of the most important signal molecules involved in the neuroendocrine–immune interaction.

Sex steroids, in turn, control the development and functioning of the hypothalamo-pituitary and immune systems. In particular, they account for the programming of sexual dimorphism in the structure and functions of the hypothalamo-pituitary system of vertebrates and regulate GnRH production in the hypothalamus and expression of GnRH receptors in the pituitary, thereby modulating the response of gonadotrope cells to this hormone. Sex steroids also modulate the molecular processing of GnRH in the thymus and its concentrations in the thymus and spleen (Azad et al, 1998).

The effects of these hormones on the immune and endocrine systems apparently differ depending on the period of ontogeny, as is the case with many other factors. During the early period, they cause long-lasting or irreversible changes in the structure and functions of the above systems, whereas their effects in adult animals are short-term and reversible.

structures during early ontogeny but also activate sexual behavior in prepubertal and pubertal males and females. Thus, the brain retains its plasticity for programming at later stages of ontogeny, being most responsive to sex steroids in adolescence as well as in the perinatal period (Shulz et al., 2009). The formation of individual structural–functional elements of the reproductive and immune systems and the establishment of relationships between them are not strictly genetically controlled. These processes are characterized by high functional lability and sensitivity to various regulatory factors, which provides the

**2. Effect of the reproductive system on the immune system: Roles of GnRH** 

Distinctive features of GnRH-producing neurons include their small population size (800– 2000 cells), diffuse distribution in the septo-preoptic hypothalamic region, and extracerebral origin during ontogeny. Most these neurons have axons terminating in the median eminence, where GnRH is released in the portal circulation and regulates the synthesis and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by gonadotrope cells. GnRH isolated from the mammalian brain was initially regarded only as the hormone controlling the reproductive function, but subsequent studies have demonstrated that GnRH occurs in a variety of functionally different forms. To date, 23 such forms have been identified in vertebrates. Three of them—GnRH1, GnRH2, and GnRH3 are the most frequent, being expressed in groups of neurons differing in origin, location, and functional role. In adult animals, GnRH1 is expressed mainly in the hypothalamus; GnRH2, in the midbrain and tectum; and GnRH3, in the rostral regions of the forebrain. The population of hypothalamic neurons expressing GnRH1 is usually referred to as the GnRH1 system, and its basic function is to regulate the release of gonadotropins. GnRH2 and GnRH3 supposedly function as neuromodulators, the former being involved in the regulation of sexual behavior and the latter, in the integration of olfactory signals and other processes related to reproduction. Extracerebral synthesis is characteristic mainly of GnRH2, which has been revealed in the thymus, spleen, ovaries, testes, prostate, mammary gland, and placenta (Jacobson et al., 2000; Zakharova et al., 2005). Along with hypothalamic GnRH, extracerebral GnRH2 plays a role in the development and functioning of the immune system at different stages of ontogeny (Morale et al., 1991; Zakharova et al., 2005). According to Marchetti et al. (1998), GnRH is one of the most important signal molecules involved in the

Sex steroids, in turn, control the development and functioning of the hypothalamo-pituitary and immune systems. In particular, they account for the programming of sexual dimorphism in the structure and functions of the hypothalamo-pituitary system of vertebrates and regulate GnRH production in the hypothalamus and expression of GnRH receptors in the pituitary, thereby modulating the response of gonadotrope cells to this hormone. Sex steroids also modulate the molecular processing of GnRH in the thymus and

The effects of these hormones on the immune and endocrine systems apparently differ depending on the period of ontogeny, as is the case with many other factors. During the early period, they cause long-lasting or irreversible changes in the structure and functions of the above systems, whereas their effects in adult animals are short-term and reversible.

possibility of correcting disturbances in the reproductive process.

**and sex steroids** 

neuroendocrine–immune interaction.

its concentrations in the thymus and spleen (Azad et al, 1998).

## **2.1 Role of GnRH in immune system development and functioning at different stages of ontogeny**

The involvement of GnRH in the differentiation of lymphocytes and regulation of immune response is lifelong. Its neonatal administration in normal mice accelerates the development of immune reactions (Marchetti et al., 1989). In rats and monkeys, central or peripheral blockade of the GnRH receptor antagonists in the neonatal period leads to reduction of mature T- and B-lymphocyte counts in the thymus, spleen, and circulating blood and suppression of antibody production and mitogen-induced proliferative response of T cells, with the immune reactions returning to the norm only by the age of 3 months in rats and 5 years in monkeys (Morale et al., 1991). Thymic and splenic lymphocytes differ in sensitivity to GnRH. Neonatal administration of a GnRH antagonist in rats results in complete block of the mitogen-induced proliferative response of thymocytes, whereas this response of splenocytes is blocked only partially. GnRH and its agonists prevent age-related involution of the thymus and normalize the suppressed functional activity of thymocytes (Marchetti et al., 1989). In pregnancy, the functional activity of GnRH in controlling the numbers of thymocytes is suppressed due to the intensified synthesis of prohibitin, an antiproliferative protein; as a consequence, the maternal thymus undergoes involution. The suppression of T-lymphocyte development in pregnancy is an adaptation against allogeneic fetal rejection. Administration of an GnRH agonist results in normalization of thymocyte count (Dixit et al., 2003).

There is evidence that GnRH exacerbates progression of autoimmune diseases. In particular, this follows (by contradiction) from the data by Jacobson et al. (2000) that administration of an GnRH antagonist to New Zealand mice with systemic lupus erythematosus leads to a drop in the levels of both total IgG and anti-DNA antibodies, relief of disease symptoms, and extension of life span, with these effects being observed in both intact and castrated animals of both sexes. It should be noted that the diseases progresses more severely in females than males, which the authors attribute to sex-related differences in the expression of GnRH receptors or G protein (Jacobson et al., 2000). Although the available data on the involvement of GnRH in immune response modulation and exacerbation of autoimmune diseases in adults are fairly abundant, its role in these processes is not yet completely clear. However, since the functions of many hormones in postnatal life are aimed at the maintenance of immune system homeostasis in response to changes in ambient conditions (Dorshkind & Horseman, 2000), such a function cannot be excluded for GnRH.

According to our data (Zakharova et al., 2000), GnRH becomes involved in the regulation of T-cell immunity as early as during prenatal ontogeny. Surgical ablation of the hypothalamus (encephalectomy) *in utero* in 18-day rat fetuses results in 30–40% suppression of concanavalin A (Con A)-induced response in thymocytes isolated on day 22, but intraperitoneal injection of GnRH (0.2 μg per fetus) immediately after surgery restores this response to the norm. No such effect has been observed in experiments with sham-operated fetuses. Moreover, GnRH (10–9 and 10–7 M) added to a culture of thymocytes from encephalectomized fetuses has proved to enhance their Con A-induced proliferative response in a dose-dependent manner. The involvement of GnRH at early stages of immune system development is also confirmed by the results of experiments on central or peripheral blockade of the synthesis of GnRH or its receptors in rat fetuses (Zakharova et al., 2005). On day 20 of pregnancy, fetuses in one uterine horn were intraperitoneally injected with either the selective GnRH antagonist D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 or anti-GnRH antibodies, and fetuses in the other horn, with 0.9% NaCl solution or nonimmune rabbit serum. The GnRH antagonist (2 μg per fetus) caused 40–50%

Interactions Between Reproductive and Immune Systems

thereby regulating their numbers (Grasso et al., 1998).

Forhead, 2004; Wang et al., 2009; Wu et al., 2011).

development of infertility (Chapman et al., 2009).

During Ontogeny: Roles of GnRH, Sex Steroids, and Immunomediators 225

maturation, as well as directly interact with such receptors on lymphocytes. In addition, GnRH can induce both the expression of interleukin (IL)-2 receptors on lymphocytes and the synthesis of gamma-interferon (IFNγ), which, in turn, induces IL-2 production by T cells,

During early ontogeny, sex hormones (along with other hormones) participate in the development of the hypothalamo-pituitary and immune systems. It is known that exposure of the fetus to adverse factors can affect the structural and functional programming of these systems, with consequent disease susceptibility in adulthood (Langley-Evans, 2006). Alterations in a certain developing system usually entail alterations in other systems. External factors such as stress, treatment with pharmaceuticals, and mother's inadequate diet and behavior during pregnancy and breast feeding place the fetus (newborn) at risk for autoimmune, allergic, metabolic, nervous, and mental disorders in later life (Fowden &

A strong stimulus changing homeostasis of the fetus is exerted by sex steroids. The mechanisms of sexual differentiation of the brain were addressed even in the first studies on prenatal programming of the GnRH system. It has been shown long ago that the development of the brain in male mammals initially follows the female pattern, but specific brain regions in a certain period of ontogeny are influenced by testosterone aromatized into estradiol, which leads to masculinization of the brain and its subsequent development according to the male scenario. This period is critical for organization of the GnRH system, and its timing varies between species. In rodents, brain masculinization takes place during the late intrauterine–early postnatal period; in guinea pigs, during midpregnancy; in rhesus monkeys and humans, this process is accomplished by the second trimester of pregnancy;

whereas in sheep it continues from days 30--37 to 147 of intrauterine development.

An increase in the concentration of androgens in mice between intrauterine day 18 and postnatal day 14 causes changes in the development of the hypothalamo-pituitary system. Male transgenic hCGαβ+ mice overproducing human chorionic gonadotropin are characterized by an elevated GnRH level in the hypothalamus, a reduced FSH level in the pituitary and circulating blood, and inhibited expression of the mRNA of receptors for GnRH and estrogens in the pituitary (Gonzalez et al., 2011). The sexual behavior of a pregnant female has an effect on the functioning of the endocrine system in its offspring, which is mediated by epigenetic modifications at the promoter for oestrogen receptor alpha (ER) and subsequent effects on gene expression (Cameron et al., 2008). Estradiol and testosterone injected to female mice during the neonatal period induce the development of infertility, whereas their injection on postnatal day 7 causes no disturbances in the reproductive system. Hydrocortisone injected together with estradiol prevents the

Sex hormones also modulate the development of lymphoid organs, the thymus being their main target in the immune system. The drop in the level of sex hormones in male mice after pre- or postpubertal castration causes thymic hypertrophy (with increase in thymocyte count) and enhancement of graft rejection reaction. The phenomenon of twofold increase in thymus weight in castrated males was discovered more than a century ago, but its mechanism has not yet been elucidated in detail. Injection of androgens to castrated animals results in a rapid decrease in thymus weight, with signs of active apoptosis being observed

**2.2 Role of sex steroids in the development of immune and endocrine systems** 

suppression of Con A-induced proliferative response of thymocyte on day 22, compared to that in fetuses injected with saline (0.9% NaCl). It should be noted that this response did not differ between male and female fetuses, either in the norm or after the antagonist injection. When thymocytes from 22-day fetuses were cultured in the presence of the GnRH antagonist (10–5 or 10–6 М), no such decrease in the proliferative response was observed. In the case of injection with anti-GnRH antibodies, Con A-induced proliferative response of thymocytes was suppressed fivefold, compared to that in fetuses injected with either saline or nonimmune serum.

On the other hand, the injection of a long-acting GnRH agonist to prepubertal female mice has been found to suppress T- and B-cell maturation in the primary lymphoid organs. It appears that GnRH acts on lymphocyte precursors so that changes in its initial concentration lead to suppression of their differentiation in the central organs of the immune system (the thymus and bone marrow), which, in turn, accounts for the decreased numbers of differentiated T and B cells in spleen, a secondary lymphoid organ (Rao et al., 1995).

The synthesis of GnRH and its receptors in the thymus and spleen of adult animals has been confirmed experimentally. As shown by Azad et al. (1997), the *Jurkat* human leukemia T-cell line (phenotypically similar to normal human T lymphocytes) expresses GnRH mRNA and secretes this hormone and its precursor into the culture medium. The proliferative activity of these cells increases under the effect of either endogenous or exogenous GnRH, whereas its antagonist suppresses their proliferation. According to the same authors (Azad et al., 1998), the concentration of GnRH in the thymus significantly increases in castrated rats, but this increase is prevented by testosterone injection. It is considered that sex steroids modulate molecular processing of the GnRH precursor, with its processing in the thymus differing from that in the hypothalamus.

The results of our experiments exhibit that GnRH is also synthesized in the fetal thymus (Zakharova et al., 2005). Immunocytochemical analysis for GnRH in the thymus of 21-day rat fetuses revealed the presence of GnRH-positive cells morphologically identical to thymocytes. Quantitative assessment of GnRH in the thymus exhibited that its content was minimal in 18-day fetuses, increased by a factor of about 1.5 in 21-day fetuses, and further increased by postnatal day 3 (by 65 and 40%, compared to intrauterine days 18 and 21), reaching the level characteristic of the hypothalamus. The GnRH contents in the thymus were similar in males and females. A considerable GnRH level was also detected in the blood serum of rat fetuses. It reached a peak in 18-day fetuses and decreased by half in 21 day fetuses, remaining fairly high relative to those in the thymus and hypothalamus. After surgical ablation of the hypothalamus (encephalectomy) on intrauterine day 18, the concentrations of GnRH in the thymus and peripheral blood of 21-day fetuses, either male or female, was half as low as in sham-operated fetuses. The origin of circulating GnRH is as yet unclear. Since its level drops after encephalectomy, it appears that at least half its amount is of brain origin and, therefore, the level of this hormone at the periphery is controlled by the hypothalamus. It cannot be excluded, however, that GnRH found in the circulating blood of fetuses comes from other sources (e.g., the placenta).

All the above data suggest that GnRH can control the development and functioning of the immune system via the hypothalamo-pituitary axis and is involved in an autocrine or paracrine regulation of the immune response during postnatal life. There are several possible mechanisms of GnRH action on the immune system: it can interact with specific receptors on thymic epithelial cells that synthesize peptides participating in T-cell

suppression of Con A-induced proliferative response of thymocyte on day 22, compared to that in fetuses injected with saline (0.9% NaCl). It should be noted that this response did not differ between male and female fetuses, either in the norm or after the antagonist injection. When thymocytes from 22-day fetuses were cultured in the presence of the GnRH antagonist (10–5 or 10–6 М), no such decrease in the proliferative response was observed. In the case of injection with anti-GnRH antibodies, Con A-induced proliferative response of thymocytes was suppressed fivefold, compared to that in fetuses injected with either saline

On the other hand, the injection of a long-acting GnRH agonist to prepubertal female mice has been found to suppress T- and B-cell maturation in the primary lymphoid organs. It appears that GnRH acts on lymphocyte precursors so that changes in its initial concentration lead to suppression of their differentiation in the central organs of the immune system (the thymus and bone marrow), which, in turn, accounts for the decreased numbers of

The synthesis of GnRH and its receptors in the thymus and spleen of adult animals has been confirmed experimentally. As shown by Azad et al. (1997), the *Jurkat* human leukemia T-cell line (phenotypically similar to normal human T lymphocytes) expresses GnRH mRNA and secretes this hormone and its precursor into the culture medium. The proliferative activity of these cells increases under the effect of either endogenous or exogenous GnRH, whereas its antagonist suppresses their proliferation. According to the same authors (Azad et al., 1998), the concentration of GnRH in the thymus significantly increases in castrated rats, but this increase is prevented by testosterone injection. It is considered that sex steroids modulate molecular processing of the GnRH precursor, with its processing in the thymus

The results of our experiments exhibit that GnRH is also synthesized in the fetal thymus (Zakharova et al., 2005). Immunocytochemical analysis for GnRH in the thymus of 21-day rat fetuses revealed the presence of GnRH-positive cells morphologically identical to thymocytes. Quantitative assessment of GnRH in the thymus exhibited that its content was minimal in 18-day fetuses, increased by a factor of about 1.5 in 21-day fetuses, and further increased by postnatal day 3 (by 65 and 40%, compared to intrauterine days 18 and 21), reaching the level characteristic of the hypothalamus. The GnRH contents in the thymus were similar in males and females. A considerable GnRH level was also detected in the blood serum of rat fetuses. It reached a peak in 18-day fetuses and decreased by half in 21 day fetuses, remaining fairly high relative to those in the thymus and hypothalamus. After surgical ablation of the hypothalamus (encephalectomy) on intrauterine day 18, the concentrations of GnRH in the thymus and peripheral blood of 21-day fetuses, either male or female, was half as low as in sham-operated fetuses. The origin of circulating GnRH is as yet unclear. Since its level drops after encephalectomy, it appears that at least half its amount is of brain origin and, therefore, the level of this hormone at the periphery is controlled by the hypothalamus. It cannot be excluded, however, that GnRH found in the

All the above data suggest that GnRH can control the development and functioning of the immune system via the hypothalamo-pituitary axis and is involved in an autocrine or paracrine regulation of the immune response during postnatal life. There are several possible mechanisms of GnRH action on the immune system: it can interact with specific receptors on thymic epithelial cells that synthesize peptides participating in T-cell

circulating blood of fetuses comes from other sources (e.g., the placenta).

differentiated T and B cells in spleen, a secondary lymphoid organ (Rao et al., 1995).

or nonimmune serum.

differing from that in the hypothalamus.

maturation, as well as directly interact with such receptors on lymphocytes. In addition, GnRH can induce both the expression of interleukin (IL)-2 receptors on lymphocytes and the synthesis of gamma-interferon (IFNγ), which, in turn, induces IL-2 production by T cells, thereby regulating their numbers (Grasso et al., 1998).
