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

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

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

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

Interactions Between Reproductive and Immune Systems

has not yet been determined for them (Shulz et al., 2009).

**cytokines and thymic peptides** 

During Ontogeny: Roles of GnRH, Sex Steroids, and Immunomediators 227

Estrogens have a protective effect on the progression of autoimmune diseases, in particular, multiple sclerosis and autoimmune encephalomyelitis as its model. In pregnant women with multiple sclerosis, clinical remission is observed during the last trimester, at a high level of estrogens and progesterone; after delivery, this level drops, and the disease is exacerbated. Exogenous estrogens at physiological concentrations suppress the progression of experimental encephalomyelitis, supposedly by inhibiting the synthesis of proinflammatory cytokines (Van den Broek et al., 2005). The protective effect of sex steroids is dependent on a number of factors, including their dose and the age, sex, and metabolic pattern of animals. Thus, in NZB/NZW mice, which spontaneously develop lethal glomerulonephritis by the age of 8–14 months, castration of 14-day-old females accompanied by testosterone injection significantly prolongs their life span. On the other hand, castration and estradiol injection in males has an opposite effect. Moreover, males castrated at the age of 14 days die of this disease earlier than do males castrated at the age of

5 weeks, while castration of 14- to 15-week-old males has no effect on their life span.

**2.3 Effect of immune system on the development of reproductive system: Role of** 

sensitization of the adult GnRH system to the inhibitory effect of stress.

**2.3.1 Regulation of the GnRH system in adult mammals** 

Numerous data are available on the effect of the neuroendocrine system, including its reproductive component, on the establishment and functioning of the immune system. The immune system, in turn, is not only the target for hormones but is itself involved in the regulation of neuroendocrine system functioning. Mediators produced by the immune system have a role in programming the development of reproductive system in the fetus (Igaz et al., 2006; Li et al., 2007; Goya et al., 2004). The thymus is the central organ of the immune system, and its absence in homozygous athymic or neonatally thymectomized mice leads to severe disturbances in immune–neuroendocrine regulation. These disturbances manifest themselves not only in the inhibited functions of the immune system but also in the impaired synthesis and secretion of neuropeptides and hormones of the hypothalamus, pituitary, and peripheral endocrine glands (Goya et al, 2004; Chapman et al., 2009; Zakharova, 2009). The impaired neuroendocrine functions can be modulated by thymic peptides (Goya et al., 2007). There is evidence that bacterial endotoxins and proinflammatory cytokines have influence on the GnRH system of newborns (Li et al., 2007). In particular, neonatal activation of the immune system by these factors results in long-term

As noted above, GnRH has a modulatory effect on the development and functions of the neuroendocrine and immune systems, which, in turn, control the functioning of the GnRH system. It is through GnRH neurons that various neurotransmitters and neuropeptides (monoamines, gamma-aminobutyric acid, neuropeptide Y, opioids, tec.) and also cytokines convey signals from external stimuli influencing the state of the reproductive system (Karsch et al., 2002; Ciechanowska et al., 2007; Pereira A et al., 2010). Sex steroids are the

It is noteworthy that the patterning of sexual behavior by sex steroids takes place not only during early ontogeny. As noted above, the brain retains its plasticity at later stages, and its responsiveness to sex steroids in males reaches a second peak during adolescence. Such a peak during the late postnatal period is also characteristic of females, but its exact timing

in the organ. The effects of androgens are realized via traditional receptor-mediated mechanisms (Olsen et al., 1996). Receptors for estrogens and androgens in the thymus are expressed as early as during embryonic development, with their level increasing by birth (Staples et al., 1999). Thymocytes carry the same numbers of functional androgen receptors as do target cells for these hormones in the reproductive system, with the least mature thymocyte subpopulations being the richest in such receptors. The thymic stroma also expresses androgen receptors. Estrogen receptors are expressed on mature peripheral T and B lymphocytes, which mediate the immunomodulatory effects of sex hormones (Tanriverdi et al., 2003.

Injection of testosterone, estrogen, or their derivatives to chick or quail embryos results in atrophy of the bursa of Fabricius, degeneration of lymphoid tissue in follicles and its substitution by fibrous tissue, and disturbances in the development of thymic stromal elements creating the microenvironment for lymphocyte maturation (Razia et al., 2006). In addition, excess sex steroids cause disturbances in mammal immune system. In particular, they suppress differentiation of regulatory and cytotoxic T cells in the thymus, with consequent increase in the numbers of immature lymphocytes in the circulation, and cause an impairment of negative selection mechanisms, which results in the formation of selfreactive T cells (Chapman et al., 2009). Estrogens also stimulate an increase in the numbers of self-reactive B lymphocytes and the level of circulating autoantibodies (Olsen & Kovacs, 1996; Tanriverdi et al., 2003). These data are in agreement with the observation that females, especially when pregnant, are more vulnerable to autoimmune diseases, compared to males. On the other hand, it has been noted that the level of testosterone in male mice genetically resistant to infectious diseases is maintained high after infection with bacterial (Salmonella enteritis) endotoxin, whereas this level in sensitive males dropped significantly on day 14 after infection (Zala et al., 2008).

Female mice kept at high density in the presence of only one male in the cage become aggressive, unresponsive to mating attempts and do not copulate. The aggressive behavior of females correlates with elevated levels of testosterone, corticosterone, and progesterone. Compared to female mice kept under standard conditions, the weight of their ovaries and adrenals is greater, while that of the thymus and uterus is smaller, and the lysis of corpora lutea in the ovaries is prolonged and incomplete. Supposedly, high corticosterone suppresses the activity of T lymphocytes normally involved in this process (Chapman et al., 2000).

Despite general similarity in the effects of male and female sex hormones on the thymus, the resultant changes in the composition of cell subpopulations in this organ are different: estrogens cause a decrease in the number of cortical T lymphocytes and an increase in the contents of more mature cell forms, whereas androgens have an opposite effect (Olsen and Kovacs, 1996).

Sex hormones also regulate the development of bone tissue and bone marrow and have an immunomodulatory effect on B lymphocytes in adults. Estrogens control differentiation of osteoclasts, mesenchymal stem cells, and myelopoiesis in the bone marrow (Carreras et al., 2008). Interacting with receptors on bone marrow stromal cells, sex hormones modulate Bcell differentiation. In either males or females, castration results in the increased numbers of pre-B cells in the bone marrow and mature B cells in peripheral organs, with the spleen growing in size (Olsen et al., 1996). In pregnancy, at a high estrogen background, the relative numbers of B lymphocytes in the bone marrow are decreased at almost all stages of differentiation (Tanriverdi et al., 2003).

in the organ. The effects of androgens are realized via traditional receptor-mediated mechanisms (Olsen et al., 1996). Receptors for estrogens and androgens in the thymus are expressed as early as during embryonic development, with their level increasing by birth (Staples et al., 1999). Thymocytes carry the same numbers of functional androgen receptors as do target cells for these hormones in the reproductive system, with the least mature thymocyte subpopulations being the richest in such receptors. The thymic stroma also expresses androgen receptors. Estrogen receptors are expressed on mature peripheral T and B lymphocytes, which mediate the immunomodulatory effects of sex hormones (Tanriverdi

Injection of testosterone, estrogen, or their derivatives to chick or quail embryos results in atrophy of the bursa of Fabricius, degeneration of lymphoid tissue in follicles and its substitution by fibrous tissue, and disturbances in the development of thymic stromal elements creating the microenvironment for lymphocyte maturation (Razia et al., 2006). In addition, excess sex steroids cause disturbances in mammal immune system. In particular, they suppress differentiation of regulatory and cytotoxic T cells in the thymus, with consequent increase in the numbers of immature lymphocytes in the circulation, and cause an impairment of negative selection mechanisms, which results in the formation of selfreactive T cells (Chapman et al., 2009). Estrogens also stimulate an increase in the numbers of self-reactive B lymphocytes and the level of circulating autoantibodies (Olsen & Kovacs, 1996; Tanriverdi et al., 2003). These data are in agreement with the observation that females, especially when pregnant, are more vulnerable to autoimmune diseases, compared to males. On the other hand, it has been noted that the level of testosterone in male mice genetically resistant to infectious diseases is maintained high after infection with bacterial (Salmonella enteritis) endotoxin, whereas this level in sensitive males dropped significantly on day 14

Female mice kept at high density in the presence of only one male in the cage become aggressive, unresponsive to mating attempts and do not copulate. The aggressive behavior of females correlates with elevated levels of testosterone, corticosterone, and progesterone. Compared to female mice kept under standard conditions, the weight of their ovaries and adrenals is greater, while that of the thymus and uterus is smaller, and the lysis of corpora lutea in the ovaries is prolonged and incomplete. Supposedly, high corticosterone suppresses

Despite general similarity in the effects of male and female sex hormones on the thymus, the resultant changes in the composition of cell subpopulations in this organ are different: estrogens cause a decrease in the number of cortical T lymphocytes and an increase in the contents of more mature cell forms, whereas androgens have an opposite effect (Olsen and

Sex hormones also regulate the development of bone tissue and bone marrow and have an immunomodulatory effect on B lymphocytes in adults. Estrogens control differentiation of osteoclasts, mesenchymal stem cells, and myelopoiesis in the bone marrow (Carreras et al., 2008). Interacting with receptors on bone marrow stromal cells, sex hormones modulate Bcell differentiation. In either males or females, castration results in the increased numbers of pre-B cells in the bone marrow and mature B cells in peripheral organs, with the spleen growing in size (Olsen et al., 1996). In pregnancy, at a high estrogen background, the relative numbers of B lymphocytes in the bone marrow are decreased at almost all stages of

the activity of T lymphocytes normally involved in this process (Chapman et al., 2000).

et al., 2003.

after infection (Zala et al., 2008).

differentiation (Tanriverdi et al., 2003).

Kovacs, 1996).

Estrogens have a protective effect on the progression of autoimmune diseases, in particular, multiple sclerosis and autoimmune encephalomyelitis as its model. In pregnant women with multiple sclerosis, clinical remission is observed during the last trimester, at a high level of estrogens and progesterone; after delivery, this level drops, and the disease is exacerbated. Exogenous estrogens at physiological concentrations suppress the progression of experimental encephalomyelitis, supposedly by inhibiting the synthesis of proinflammatory cytokines (Van den Broek et al., 2005). The protective effect of sex steroids is dependent on a number of factors, including their dose and the age, sex, and metabolic pattern of animals. Thus, in NZB/NZW mice, which spontaneously develop lethal glomerulonephritis by the age of 8–14 months, castration of 14-day-old females accompanied by testosterone injection significantly prolongs their life span. On the other hand, castration and estradiol injection in males has an opposite effect. Moreover, males castrated at the age of 14 days die of this disease earlier than do males castrated at the age of 5 weeks, while castration of 14- to 15-week-old males has no effect on their life span.

It is noteworthy that the patterning of sexual behavior by sex steroids takes place not only during early ontogeny. As noted above, the brain retains its plasticity at later stages, and its responsiveness to sex steroids in males reaches a second peak during adolescence. Such a peak during the late postnatal period is also characteristic of females, but its exact timing has not yet been determined for them (Shulz et al., 2009).

#### **2.3 Effect of immune system on the development of reproductive system: Role of cytokines and thymic peptides**

Numerous data are available on the effect of the neuroendocrine system, including its reproductive component, on the establishment and functioning of the immune system. The immune system, in turn, is not only the target for hormones but is itself involved in the regulation of neuroendocrine system functioning. Mediators produced by the immune system have a role in programming the development of reproductive system in the fetus (Igaz et al., 2006; Li et al., 2007; Goya et al., 2004). The thymus is the central organ of the immune system, and its absence in homozygous athymic or neonatally thymectomized mice leads to severe disturbances in immune–neuroendocrine regulation. These disturbances manifest themselves not only in the inhibited functions of the immune system but also in the impaired synthesis and secretion of neuropeptides and hormones of the hypothalamus, pituitary, and peripheral endocrine glands (Goya et al, 2004; Chapman et al., 2009; Zakharova, 2009). The impaired neuroendocrine functions can be modulated by thymic peptides (Goya et al., 2007). There is evidence that bacterial endotoxins and proinflammatory cytokines have influence on the GnRH system of newborns (Li et al., 2007). In particular, neonatal activation of the immune system by these factors results in long-term sensitization of the adult GnRH system to the inhibitory effect of stress.
