Brazilian Journal of Poultry Science Revista Brasileira de Ciência Avícola Effects of Heat Stress on the Development of ISSN 1516-635X 2019 / v.21 / n.2 / 001-010 GABAergic Neurons in the HPG Axis of Wenchang http://dx.doi.org/10.1590/1806-9061-2018-0954 Chicks Original Article Author(s) ABSTRACT Liang WI https://orcid.org/0000-0003-4158-0104 This study conducted an in-depth investigation on the development Lu BBI https://orcid.org/0000-0003-0868-1333 of GABAergic neurons and their receptors in HPG axis-related target Liang CI https://orcid.org/0000-0002-0375-257X organs of Wenchang chicks under heat stress. One-day-old healthy I Yu ZQ https://orcid.org/0000-0003-1746-4058 Wenchang chicks were randomly divided into control (CK) and heat Xie XZI https://orcid.org/0000-0003-1161-6073 Chen ZI https://orcid.org/0000-0001-8156-8754 stress (HS) groups. Chicks in the HS group were placed in a 40±0.5°C climatic chamber for HS treatment from 13:00 to 15:00 daily. By I College of Life Science, Hainan Normal University, Haikou 571158, China. immunohistochemistry and Western blotting, GABA and GABAA receptor (GABAAR) expression in the hypothalamus of the HS group was significantly higher (p<0.05), but GABAB receptor (GABABR) expression was significantly lower than that of the CK group (p<0.05). Expression of GABA and its two receptors in the pituitary tissues of the HS group was significantly lower than in the CK group (p<0.05). Expression of GABA and GABABR in ovaries in the HS group was significantly higher, but expression of GABAAR in the testes of the HS group was lower than that of the CK group (p<0.05). In the male chicks, expression of GABA and its two receptors in the hypothalamus, pituitary, and testicular tissues of the HS group was significantly higher than that of the CK group (p<0.05). Western blotting showed that the GABAAR and GABABR expression of the HS group was significantly higher than that of the CK group at 3 and 5 weeks of age. Thus, HS caused GABAergic nervous system disorder in the HPG axis of Wenchang chicks and Mail Address seriously hindered the normal development of GABAergic neurons Corresponding author e-mail address in chicks, leading to the disorder of the expression of GABA and its Chen Z receptors in tissues. Hainan Normal University, Long Kun Nan Road, Haikou City, 571158, P.R. China. Phone: 0898-65883521 INTRODUCTION Email: [email protected] Hindered growth and reproductive performance of breeding animals caused by heat stress is one of the serious problems faced by animal husbandry. Given their coverage with feathers and lack of sweat glands, chicks are highly susceptible to heat stress, leading to Keywords regulatory disorders of the hypothalamic–pituitary–gonadal (HPG) Heat stress; GABAergic neurons; HPG axis; axis and ultimately resulting in an impaired reproductive system and Wenchang chick. decreased egg production (Xie et al., 2016). Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays an important role in the regulation of the HPG axis. It is widely present in the central nervous system and peripheral tissues and is also expressed in the reproductive system of animals. GABAergic neurons can cause gonadotropin- releasing hormone (GnRH) neurons to exhibit excitatory or inhibitory effects by acting on GABAA receptors (GABAAR) and GABAB receptors (GABABR) on the GnRH neuron surface and directly and indirectly impacting pituitary and ovarian functions by affecting the synthesis and release of GnRH in the hypothalamus (Tomaszewskaet al., 2003; Submitted: 07/December/2018 Zhu, 2016). Therefore, GABA can affect the HPG axis and its target Approved: 19/February/2019 organs by regulating the secretion of related hormones. For example, 1 eRBCA-2018-0954 Leme BB, Sakomura N, Viana GS, Effects of Heat Stress on the Development of Soares L, Melaré MC, Oliveira MJK, GABAergic Neurons in the HPG Axis of Wenchang Mansano CFM Chicks heat stress changes the amount of GnRH in the Heat stress treatment hypothalamus and the secretion of gonadotropin in the Chicks in the HS group were placed in a 40±0.5°C pituitary. These changes will further affect the growth climatic chamber with a relative humidity of 70–80% and spermatogenesis of male reproductive organs and for 2 h of heat treatment = from 13:00 to 15:00 daily. reduce the success rate of avian reproduction (Chen Simultaneously, chicks in the CK group were placed in et al., 2015; Türk et al., 2015). In addition, under heat a climatic chamber without being heated for 2 h. All stress conditions, developmental impairment of HPG axis-related target organs in chicks at different ages chicks were returned to the corresponding cages for routine feeding after heat stress (Chen et al., 2014). is accompanied by abnormal changes in GABAA and GABABR mRNA expression levels. This indicates that Sample collection and processing regulation of the HPG axis by GABAergic neurons is important for the homeostasis of the body under heat At the end of each week from 1 to 6 weeks of age stress conditions (Xie et al., 2016). and after the 2-h heat stress treatment, 6 chicks from The pharmacological properties of GABA have each group were randomly selected to immediately been found to be calming, anxiety-reducing, and dissect the hypothalamus, pituitary, ovary, and testis, antidepressant. GABA can be used as feed additive to followed by weighing. Part of the tissues was rapidly regulate immune and reproductive functions and to placed in Bouin’s solution for 12 h fixation, and alleviate the harm of heat stress to animal husbandry another part of the tissues was immediately placed in (Chen et al., 2002; Leventhal et al., 2003; Chen et al., a cryotube and immersed in liquid nitrogen, followed 2014). For example, one study showed that GABA by preserving in a −80°C freezer after 24 h. Tissues alleviated the adverse effects of high temperature in the fixative were paraffin-embedded and sectioned on the reproductive system of poultry by increasing for immunohistochemistry (IHC). Cryopreserved tissues the serum reproductive hormones, such as follicle- were used for protein analysis in Western blotting. The experiments were conducted with the approval from stimulating hormone (FSH) and estradiol (E2), thereby ensuring the egg laying performance and egg quality of the Hainan Normal University Animal Experimentation hens in a high-temperature environment (Zhang, 2012) Ethics Committee. and providing basic information for improving the Measurement of the expression of anti-stress ability of poultry. However, the relationship GABAergic neurons in the HPG axis between heat stress-induced HPG axis dysfunction and GABAergic neurons in chicks remains unclear. This Primary anti-GABA antibody (orb6065), anti- study used immunohistochemistry (IHC) and Western GABAAR antibody (orb10677), and anti-GABABR blotting to explore the development of GABA, GABAAR, antibody (orb10679) were purchased from Biorbyt Ltd. and GABABR in the hypothalamus, pituitary, ovary, and (Cambridge, United Kingdom). Rabbit IHC detection kits testicular tissues of HPG axis-related target organs in (36312ES75) were purchased from Shanghai Yeasen chicks to reveal the mechanism of heat stress on the Biotechnology Co., Ltd. (Shanghai, China) and used in development of HPG axis GABAergic neurons in chicks. strict accordance with the manufacturer’s instructions. After paraffin embedding, the tissues were routinely MATERIALS AND METHODS prepared into 5-μm serial sections, followed by routine immunohistochemical staining,3,3′-diaminobenzidine Experimental animals and feeding (DAB) color development, and hematoxylin nuclear management counterstaining. Tissue sections were observed under One hundred forty-four healthy one-day-old light microscopy, and positive IHC staining presented Wenchang chicks (1:1 male-to-female ratio) purchased as yellowish brown granules. Three tissue sections of from Hainan YongJi Live Stock Co., Ltd. (Hainan each sample were randomly selected to observe the Province, China), with no significant differences in positive IHC staining in 10 fields of view each section body weight, were randomly divided into control (CK) under Olympus CX31 microscope (Olympus, Japan). and heat stress (HS) groups (n = 72 chicks, including Mshot Image Analysis System v1.0 (Mshot, Guangdong 36 males and 36 females, per group). All chicks were Province, China) was used for photographing and weighed and numbered, followed by housing in a image storage. Image-Pro Plus 6.0 software (Media ventilated and regularly disinfected animal room (L:D Cybernetics Corporate, Rockville, MD) was used to = 14:10) with free access to feed (purchased from measure the integrated optical density (OD) values of Zhanjiang Yilong Feed Factory, Guangdong Province, GABA-, GABAAR-, and GABABR-positive cells in the China) and sterile water. fields of view. 2 eRBCA-2018-0924 Leme BB, Sakomura N, Viana GS, Effects of Heat Stress on the Development of Soares L, Melaré MC, Oliveira MJK, GABAergic Neurons in the HPG Axis of Wenchang Mansano CFM Chicks Detection of GABA receptor protein integrated OD value of the target protein band to the expression in ovary and testis image-integrated OD value of the reference protein band (Cheng et al., 2013). Ovary and testicular tissues were rinsed with PBS, followed by removing blood using filter paper and Data processing and statistical analysis adding in RIPA lysate (containing PMSF) at a ratio of 0.2 mg/μL for homogenization with a glass pestle on All data were processed by Microsoft Excel software an ice bath to ensure low-temperature grinding until (Microsoft Corporation, Redmond, WA) and analyzed the tissue cells were completely ruptured. After 30-min by ANOVA with SPSS 20.0 software (IBM SPSS, Chicago, lysis on the ice bath, the lysate was transferred into a IL). Multiple comparisons were performed by Duncan’s 1.5-ml centrifuge tube and centrifuged at 12,000 rpm multiple range test.