Effect of Heat Stress on Expression of Main Reproductive Hormone in Hypothalamic– Pituitary–Gonadal Axis of Wenchang Chicks

The hypothalamic–pituitary–gonadal (HPG) axis is a key neuroendocrine regulation system involved in the growth and reproduction of poultry. High-temperature conditions lead to the physiological dysfunction of target organs of the HPG axis of poultry, ultimately affecting the animals’ growth and development. In this study, we evaluated the effect of heat stress (HS) on the development of cells secreting major reproductive hormones of the HPG axis (i.e., hypothalamus, pituitary gland, ovary, and testis) of Wenchang chicks. Seventy-two one-day-old healthy Wenchang chicks were randomly divided into control (CK) and HS groups. The HS group was placed in a 40 ± 0.5°C artificial climate chamber for heat-stress treatment from 13:00 to 15:00 daily for six consecutive weeks. As development progressed, compared with the CK group, the gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the hypothalamus and testosterone (T) levers in the testes of male chicks in the HS group were significantly decreased at three weeks of age ( p <0.05). However, GnRH levels in the hypothalamus and estradiol (E 2 ) levels in the ovaries of female chicks in the HS group were variable and significantly lower than those of the CK group at four and five weeks of age ( p <0.05). In addition, the LH and FSH levels in the pituitary gland were significantly lower than those in the CK group at two and four weeks of age and at four and six weeks of age, respectively ( p <0.05). In brief, HS caused dysfunction of the corresponding target organs of the HPG axis in Wenchang chicks, and this affected the normal growth and development of the cells’ HPG axis.


INTRODUCTION
With global warming, high-temperature and high-humidity environments in tropical and subtropical regions are increasingly detrimental to the livestock industry. Poultry are highly susceptible to heat stress (HS), because they are covered with feathers and lack sweat glands. When the ambient temperature exceeds the upper limit of the thermoneutral zone of poultry, the body will be under stress, resulting in tissue and endocrine system disorders and suppressed organ development. These issues can result in stunted growth and reproductive performance and may even lead to shock or death of poultry in severe cases Zhu et al., 2017).
In animals, the endocrine and nervous systems work together to form a neuroendocrine-immune network. The hypothalamicpituitary-gonadal (HPG) axis is an important component of the endocrine system, regulating reproductive functions of the body. The GnRH secreted from the hypothalamus activates specific receptors on the pituitary gland to stimulate the synthesis and secretion of FSH and LH from gonadotropic cells of the pituitary gland. FSH and LH enter peripheral blood circulation to ultimately promote the secretion of sex hormones, and the sex hormones exert feedback inhibition to affect the synthesis of GnRH, FSH, and LH. This cycle is essential for studying the distributions of reproductive hormones (Chimento et al., 2014). High temperatures affect the synthesis and secretion of several relevant hormones such as FSH, LH, and E 2 (Zhang et al., 2012). Under such environmental circumstances, the body regulates the HPG axis by changing the secretion activities of these hormones. Studies have shown that high temperatures alter the secretion of GnRH in the hypothalamus as well as LH and FSH in the pituitary gland of poultry, leading to the dysregulation of the HPG axis (Elnagar et al., 2010;Chen et al., 2015;Tu et al., 2016). Existing studies on major reproductive hormones of the HPG axis have been mostly reported in mammals (Schams et al., 1980;Kim et al., 2013;Maurya et al., 2016), and related studies of Wenchang chicks, especially those evaluating the effects of high temperature on the synthesis of T and E 2 in the reproductive system and inhibiting the gonad development of poultry, have rarely been reported. Hence, in this study, we examined the developmental changes of related reproductive hormones in the hypothalamus, pituitary gland, ovary, and testis of chicks using immunohistochemistry. We evaluated the effect of HS on the growth and development of cells secreting major reproductive hormones in the HPG axis of Wenchang chicks. The results provide fundamental information concerning the growth and development of chicks in high-temperature environments.

Experimental animals and heat-stress treatment
One hundred forty-four healthy one-day-old Wenchang chicks (1:1 male-to-female ratio) with no significant differences in body weights were purchased from Hainan YongJi Livestock (Hainan, China). The chicks were randomly divided into two groups: control (CK) and heat-stress (HS) groups, with 72 animals (36 males and 36 females) per group. The chicks were weighed, labeled, and fed ad libitum with free access to feed (purchased from Zhanjiang Yilong Feed Mill Co. Ltd., Zhanjiang, China) and sterile water, and were housed in a ventilated and lighted (14 h of light, 10 h of darkness) animal room. The Wenchang chicks in the HS group were placed in an artificial climate chamber (model LRH-800-GS, Tomorrow Environmental Protection Instrument, Shaoguan, China) daily for HS at 40 ± 0.5°C from 13:00 to 15:00, and the Wenchang chicks in the CK group were simultaneously placed in an unheated artificial climate chamber for 2 h of control treatment at 29.7 ± 2.3°C. After the HS or control treatment, all chicks were returned to their cages for routine feeding .

Sample collection and tissue sectioning
At the end of 1-6 weeks of age, six chicks were randomly selected from each group for dissection of the hypothalamus, pituitary gland, ovary, and testis. Excess fat and connective tissues were removed and placed on ice followed by removal of blood with saline solution, and the dissected organs were placed in Boiun's fixative solution. Fixed tissues were dehydrated, cleared, embedded in paraffin, and sectioned into 5-μm slices. The experiments were conducted with the approval of the Hainan Normal University Animal Experimentation Ethics Committee.
Six tissue sections stained with different antibodies were observed under an Olympus BX50F light microscope (Olympus Optical, Tokyo, Japan) by selecting 10 different fields of view to capture images using an Mshot MC50 digital camera (Guangzhou Mingmei Photoelectric Technology, Guangdong, China). The sections were analyzed using Image-pro Lu BB, Liang W, Liang C, Yu ZQ, Xie XZ, Chen Z

Effect of Heat Stress on Expression of Main Reproductive Hormone in Hypothalamic-Pituitary-Gonadal Axis of Wenchang Chicks
Plus 6.0 image analysis software (Media Cybernetics, Rockville, MD). We counted the numbers of cells with positive staining of the relevant hormones and recorded their integrated optical density (IOD) in different fields of view per tissue section.

Data processing and statistical analysis
The experimental results were presented as mean ± standard error mean (Mean ± SEM). Experimental data analysis was performed using Microsoft Excel (Version 12) and SPSS 16.0 (IBM SPSS, Chicago, IL) software. Analysis of variance (ANOVA) and Duncan's method were used for multiple comparisons. p<0.05 was considered a statistically significant difference.

Effect of HS on GnRH expression in hypothalamus of Wenchang chicks
Immunohistochemical analysis revealed that GnRHpositive staining was distributed in the cell bodies and around the nuclei of neurons in the hypothalamic tissues; this is illustrated by a light-brown color and irregular shape ( Figure 1). The average IOD of GnRH-positive cells in the hypothalamus was obtained by quantitative analysis of the positive expression (Table 1). As the chicks developed, the GnRH-positive staining in the hypothalamic tissues of the male Wenchang chicks of the CK and HS groups initially increased and then declined. The GnRH-positive staining of the female HS chicks showed high variability. Compared with the CK group, GnRH-positive staining in the hypothalamic tissues was significantly decreased in 3-6-week-old male chicks and 4-5-week-old female chicks of the HS group (Table 1; p<0.05).

Effect of HS on LH and FSH expression in pituitary tissues of Wenchang chicks
According to the LH and FSH immunohistochemistry, the LH-and FSH-positive staining was randomly distributed in the cytoplasm and membranes of the pituitary tissues ( Figure 2). Quantitative analysis of the positive staining was performed to obtain the average IODs of the FSH-and LH-positively stained cells in the pituitary gland ( Table 2). The LH-and FSHpositive cells in the pituitary tissues of the male chicks of the CK group initially increased and then decreased until the chicks were six weeks old. The LH-and FSH-positive cells in the pituitary tissues of the male chicks of the HS group initially increased and then decreased. Among the female chick pituitary tissues, the numbers of LH-positive cells initially decreased and then increased, and the FSH-positive staining displayed high variability. The numbers of LH-positive cells in male pituitary tissues of the HS group were significantly lower than in the CK group at 2-6 weeks of age. The numbers of FSH-positive cells of male pituitary tissues of the HS group were significantly higher than in the CK group at 1-2 weeks of age, but significantly lower than in the CK group at 3-6 weeks of age. Compared with the CK group, the LH-positive cells of female pituitary tissues of the HS group significantly decreased at two and four weeks of age, and the FSH-positive cells of female pituitary tissues of the HS group significantly decreased at four and six weeks of age (Table 2, p<0.05).

Effect of Heat Stress on Expression of Main Reproductive Hormone in Hypothalamic-Pituitary-Gonadal Axis of Wenchang Chicks
Effect of HS on respective E2 and T expression in ovaries and testis in Wenchang chicks Immunohistochemistry of E2 and T showed that E 2positive staining was distributed around the follicles in the ovary, and T-positive staining was distributed near the testicular stroma and the germ cells in the testis (Figure 3). The average IODs of E 2 in the ovary and T in the testis were obtained by quantitative analysis of the positive staining (Table 3). As the chicks developed, numbers of T-positive cells in male testicular tissues and E 2 -positive cells in the female ovary tissues of the CK group initially increased and then decreased.
Compared with the CK group, T-positive cells in male testicular tissues significantly declined at 3-6 weeks of age in male chicks, and E 2 -positive cells in the female ovary tissues significantly decreased at 4-5 weeks of age in female chicks of the HS group (Table 3, p<0.05).

DISCUSSION
The development and sexual maturation of animal reproductive organs is closely related to the regulatory function of the HPG axis. The hypothalamus promotes the synthesis and secretion of gonadotropin by secreting GnRH to act on the pituitary gland, and the peripheral blood circulation transports gonadotropin to act on

Effect of Heat Stress on Expression of Main Reproductive Hormone in Hypothalamic-Pituitary-Gonadal Axis of Wenchang Chicks
the gonads, thereby completing the regulation of the endocrine and exocrine systems (Qing et al., 2003).
The thermoregulatory system of young animals has not yet matured, and overly high ambient temperature easily affects the homeostasis of the body, leading to dysregulation of the HPG axis and ultimately affecting the growth and reproduction of the animals and even causing shock or death (Yu, 2009;Sun et al., 2015;Zhou et al., 2016). The hypothalamus is a gray-matter structure at the base of the forebrain that is involved in endocrine secretion and maintaining the balance of metabolic energy in the body (Blouet et al., 2010;Wilding, 2010). GnRH is one of the reproductive hormones synthesized by specific hypothalamic cells with bulging axons at their center, and GnRHs are released into the hypophyseal portal system in a pulsed manner. GnRH is important for the development of gonads and the maintenance of sexual maturity (Ye et al., 2003;Zhang, 2017). In this study, the GnRH-positive cells in the hypothalamus of male chicks of the CK group were significantly fewer than in male chicks of the HS group at the early stage of growth and development; this might be due to the high-temperature adaptability of the chicks, or to the high-temperature environment having no negative impact on growth and development of the chicks. After two weeks of age, the chicks entered a rapid growth period that was not conducive to the development of the hypothalamus and that initiated the blockage of neuronal development (Zhuang et al., 2004;Zhou et al., 2015;Lu et al., 2018). Studies have shown that hypothalamic tissue is susceptible to high temperature. Preoptic and anterior hypothalamic regions are thermoregulatory centers, and HS activates the neurons of the anterior and preoptic-anterior hypothalamic regions to resist stimulation (Jiang et al., 1994;Ishiwata et al., 2002). GnRH is secreted by GnRH neurons in the preoptic, anterior, and arcuate nucleus regions of the hypothalamus; therefore, HS can activate the preoptic area of the thalamus to affect GnRH neuronal development and inhibit GnRH secretion, which is consistent with the results of HS affecting the development of GnRH cells in the hypothalamus in this study. GnRH is a hypothalamicterminal output signal that plays a key role in regulating the downstream elements of the HPG axis. GnRH concentration in the hypothalamus is closely related to the development of the gonads and plays an important role in regulating the secretion of sex hormones (Asa et al., 2002;Pereyra, 2005;Liu, 2010). Therefore, GnRH was necessary to maintain the reductive functions of the chicks, and HS affected the normal growth and development of hypothalamic GnRH cells, resulting in insufficient secretion of GnRH in the hypothalamus, further causing neuroendocrine dysfunction of the HPG axis and affecting the reproductive system.
The pituitary gland is the most important endocrine gland in the body, and the pulsed release of GnRH from the hypothalamus enters the anterior pituitary gland through the hypophyseal portal system to promote gonadotrophin synthesis and secretion. LH and FSH are both gonadotrophins secreted by basophils in the pituitary gland that act synergistically on gonadal tissues (Mountford et al., 1989;Dahia et al., 2006). In this study, compared with the CK group, the numbers of LH-and FSH-positive cells of the male chicks in the HS group significantly decreased at 2-6 and 3-6 weeks of age, respectively. The LH-positive cells of the female chicks in the HS group were significantly fewer than in the CK group at two and four weeks of age, and the FSH-positive cells of the female chicks in the HS group were significantly fewer than in the CK group at four and six weeks of age. Our previous studies have shown that the development of basophils in the pituitary glands of chicks can be hindered by a hightemperature environment from three weeks of age Liang et al., 2018). Further, under HS, the development of GnRH cells in the hypothalamus is inhibited and the concentration of GnRH is decreased, leading to a decline of secretory function of the pituitary gland, further affecting the synthesis and secretion of LH and FSH. These results are consistent with the findings of Yu (2009); their study concluded that HS causes damage to the pituitary glands of rabbits,

Effect of Heat Stress on Expression of Main Reproductive Hormone in Hypothalamic-Pituitary-Gonadal Axis of Wenchang Chicks
resulting in a significant decrease in the secreted hormone activities. Our previous study showed that changes in LH in the pituitary gland and serum of the male chicks of the HS group are basically consistent. However, serum FSH levels of the 1-6-week-old male chicks of the HS group were significantly higher than in the CK group (Zhang et al., 2014), which was contrary to the FSH levels in the pituitary glands of 3-6-weekold male chicks. Three weeks of age is a rapid growth period of chicks, and HS damages the pituitary gland, thereby lowering the pituitary and serum LH levels. Further studies are needed concerning the mechanism underlying the reduction of the pituitary and serum LH levels and how this affects the synthesis, processing, and secretion of FSH to cause the differences between the serum and pituitary FSH levels. For male chicks, FSH promotes the proliferation of spermatogonia and the development of spermatogenic epithelium in the testes (Zhang et al., 2014). Insufficient LH secretion leads to a decrease in the richness of testicular interstitial tissue, which further affects the secretion and synthesis of T. Spermatogenesis in the testes, which mainly depends on the interaction between FSH and T. If the secretion and synthesis of FSH and T are blocked, the reproductive function of the body will be seriously damaged (Mclachlan et al., 1996;França et al., 2001;Mclachlan et al., 2002). For female chicks, FSH promotes ovarian follicular and follicular granulosa cell development. Once FSH binds to its specific receptor, it not only induces LH receptor formation but also synergizes with LH to promote the synthesis and secretion of estrogen, which continuously synergizes with FSH to promote the growth and development of follicles. After two weeks of age, female chicks enter a period of rapid development, and HS begins to affect the development of the pituitary gland in females. In this study, FSH-and LH-positive cells in the pituitary tissues of female chicks in the HS group were significantly reduced, which interfered with the normal secretion of estrogen. However, at six weeks of age, LH-positive staining of the female chicks in the HS group was significantly higher than that of the CK group, which might be because of LH promoting the maturation and ovulation of follicles to further promote the luteinization of granular cells after ovulation in order to maintain secretion of progesterone from luteal cells. When the LH content is too low, the body regulates the LH content through a compensatory mechanism to maintain normal growth and development (Zhao et al., 2003). Therefore, HS affects the normal development of gonadotrophin cells in the pituitary glands of chicks, thereby preventing spermatogenesis in the testes and follicle development in the ovaries.
Testosterone (T) is the most active and highest content androgen in the testes. Its locally high concentration in the testes, significantly higher than in plasma, is essential for spermatogenesis. In this study, the changes of T in the testes were consistent with the FSH levels in the pituitary tissues of male chicks, suggesting that both T and FSH might synergistically regulate spermatogenesis. After three weeks of age, T-positive cells in the male testes of the HS group were significantly fewer than in the CK group, suggesting that the high-temperature environment began to suppress the normal development of the seminiferous tubules and had adverse effects on the spermatogenic cells of the male chicks . However, the high-temperature environment restricted the secretion of GnRH, FSH, and LH synthesis and secretion in the HPG axis of male chicks after three weeks of age, resulting in the absence of proper stimulation in the Leydig cells, thereby affecting their ability to secrete T. These findings are consistent with the previous report of significant reduction of T concentration in the testes of heat-stressed mice (Yang et al., 2013). Our previous study showed that serum T levels of 1-6-week-old male chicks in an HS group were significantly lower than in a CK group (Zhang et al., 2014). Thus, HS severely hinders the development of the testis germ cells, thereby hindering spermatogenesis and sperm maturation in male chicks.
Estrogens are the major hormones promoting the development of female reproductive organs and maintaining the metabolic processes that play a key role in the development of follicles and regulation of the estrous cycle. Estrogens secreted by the ovaries include E 2 and estrone, which are interconvertible. Results of this study showed that E 2 -positive staining in the ovaries in both groups was basically consistent; the numbers of positive cells initially increased and then decreased as the chicks developed. On one hand, FSH-positive staining in the pituitary glands of female chicks in the HS group was significantly stronger than that in the CK group before two weeks of age. The relatively high concentration of FSH promoted E 2 synthesis and secretion, resulting in high E 2 -positive staining. On the other hand, the high E 2 concentration led to positive feedback on the hypothalamus that promoted the synthesis and release of GnRH but also had a negative feedback on the pituitary that inhibited FSH synthesis. This effect on FSH ultimately led to the early termination of follicular development, Lu BB, Liang W, Liang C, Yu ZQ, Xie XZ, Chen Z

Effect of Heat Stress on Expression of Main Reproductive Hormone in Hypothalamic-Pituitary-Gonadal Axis of Wenchang Chicks
thereby reducing the secretion of E 2 (Chen, 2012). The difference in the E 2 -positive staining between the two groups of females was that the peak of E 2 -positive cells in the ovaries of the CK group was delayed by one week compared with the HS group, possibly due to heat accumulation in the ovarian tissues suppressing follicle development at all levels from three weeks of age and causing ovarian dysfunction . Previous studies have shown that when poultry are subjected to HS, a natural compensatory effect will alter the functional abnormality of the HPG axis to become normal (Cangar et al., 2008;Zhao et al., 2003). Since HS damages female reproductive function, the body must secrete large amounts of E 2 to maintain the growth and development of the reproductive system. At five weeks of age, E 2 -positive staining of the chicks in the HS group was significantly lower than that in the CK group, suggesting that the chicks tried to alleviate the injury caused by HS. Therefore, HS affected the growth of germ cells in ovaries and hindered the normal development of the reproductive system of female chicks.

CONCLUSIONS
In summary, HS caused the corresponding target organ dysfunction of the HPG axis in Wenchang chicks, affecting the normal development of the cells secreting the major reproductive hormones and the synthesis of steroid hormones. This process inhibited the development and sexual maturity of the reproductive system of chicks, and thus may reduce the breeding rate of Wenchang chickens. Therefore, further studies on reliable factors that improve the development of cells secreting the major reproductive hormones under HS may provide an empirical basis for improving the reproductive success rate of poultry in high-temperature environments.