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Heat-Stress-Induced Metabolic Changes and Altered Male Reproductive Function Yuanlong Hou,†,⊥ Xiaoyan Wang,*,‡,⊥ Zhihai Lei,† Jihui Ping,† jiajian Liu,§ Zhiyu Ma,† Zheng Zhang,† Cuicui Jia,† Mengmeng Jin,† Xiang Li,† Xiaoliang Li,† Shaoqiu Chen,‡ Yingfang Lv,‡ Yingdong Gao,∥ Wei Jia,‡,§ and Juan Su*,† †

College of Veterinary Medicine, Nanjing Agriculture University, Nanjing 210095, China Ministry of Education Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China § Center for Translational Medicine, and Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth, People’s Hospital, Shanghai 200233, China ∥ Laboratory Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210095, China ‡

S Supporting Information *

ABSTRACT: Heat stress can cause systemic physiological and biochemical alterations in living organisms. In reproductive systems, heat stress induces germ cell loss and poor quality semen. However, until now, little has been known about such a complex regulation process, particularly in the perspective of metabolism. In this study, serum, hypothalamus, and epididymis samples derived from male SD (Sprague−Dawley) rats being exposed to high environmental temperature (40 °C) 2 h per day for 7 consecutive days were analyzed using metabonomics strategies based on GC/TOFMS. Differentially expressed metabolites reveal that the energy metabolism, amino acid neurotransmitters, and monoamine neurotransmitters pathways are associated with heat stress, in accordance with changes of the three upstream neuroendocrine system pathways in the SNS (sympathetic adrenergic system), hypothalamic pituitary adrenal axis (HPA), and hypothalamic pituitary testis axis (HPT) axis. Many of these metabolites, especially in the epididymis, were found to be up-regulated, presumably due to a self-preserving action to resist the environmental hot irritation to maintain normal functioning of the male reproductive system. KEYWORDS: metabonomics, heat stress, reproductive, serum, hypothalamus, epididymis, gas chromatography/mass spectrometry

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INTRODUCTION The body’s internal and external changes may result in stress reactions including a neuroendocrine and immune systemic response that will unbalance and bring disorder to the normally balanced metabolism of living organisms.1 Sustained high-load stress on the metabolism with a negative impact beyond the body’s compensatory capacity can lead to diseases such as diabetes, atherosclerosis, or secondary diseases.2,3 Heat stress is known to occur more commonly in hot weather, which often induces heat shock, tachypnea, cardiac failure, and inflammation, thus causing more death, particularly for old population.4 Heat stress has even more serious negative effects on farm animal growth performance and reproductive ability, as decreased feed intake, increased occurrence of disease frequency, and consumption of nutrition metabolism lead to poor growth of animals and great economic losses.5,6 Heat stress impacts most aspects of reproductive function in mammals. It is reported that male factor infertility is responsible in at least 50% of all infertile families.7 A main cause of male factor infertility, high scrotal temperature, due to occupational exposure, life style, or clinical disorders such as cryptorchidism © XXXX American Chemical Society

or varicocele, is negatively correlated with semen quality and induces male sterility.8,9 Compared with humans, livestock animals are thought to be more adversely affected by the detrimental effects of increasingly extreme climatic conditions. Heat stress has negative effect on male fertility, which has been proven in boars, bulls, and roosters, including decreased volume of semen, sperm concentration, sperm count, and vitality.10 Currently, heat stress research focuses primarily on two major areas: finding the molecular signaling pathway triggered by heat stress and seeking new molecules with antioxidant abilities, which may prevent germ cell apoptosis to improve sperm performance. However, few studies reported the systemic metabolic relationships between heat stress and male infertility because reproductive regulation itself is systemic. Learning the metabolic effects of heat stress on reproductive regulation system will certainly extend our existing knowledge on male infertility. An integrated NMR and GC−MS-based metabolite profiling method has been applied in conjunction Received: November 5, 2014

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Journal of Proteome Research Sample Pretreatment and GC/TOFMS Analysis

with modern multivariate statistical techniques to examine the effects of stress on the systemic and biochemical variations.11,12 Lately the emergence of novel technologies has prompted the hunting for new biomarkers of male infertility, and the metabolites related to infertile male have been paid increasing attention.13 Male fertility is a result of a complex and fine neuroendocrine control. Traditionally, the male reproductive function was considered to be controlled through a negative feedback signaling network that involves the hypothalamus, pituitary, and testis, known as HPT axis. Steroid level is tightly regulated by the HPT axis in males, which is needed to maintain normal male sexual function and fertility.14 Consequently, we performed a comprehensive metabolites analysis of hypothalamus, epididymis, and serum from SD rats exposed to heat stress to illustrate the biochemical responses and metabolic consequences as an aspect of reproduction, which makes for more information on the crucial biochemical changes in infertility induced by heat stress, an indispensable step for promoting progress of its clinical application.

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Serum samples were pretreated according to previous published methods.12 Briefly, 10 μL of 0.3 mg/mL L-2-chlorophenylalanine water solution and 10 μL of 1 mg/mL heptadecanoic acid methanol solution, as internal standards, were successively spiked into 200 μL serum samples of each rat. After a vortex mixing period of 30 s and 10 min of storage at −20 °C, the samples were put into centrifugal machine, centrifuged at 13 200 rpm for 10 min. We transferred 200 μL of the supernatant to a GC sampling vial by vacuum drying at 20 °C. The residue in vial was derived with 50 μL of 15 mg/mL methoxyamine pyridine solution at 30 °C for 90 min; then, 80 μL of BSTFA was added (contains TMCS at 1%) at 70 °C for 60 min. A 1 μL of solution of derivatives was then added to GC/TOFMS (Agilent 6890N gas chromatograph coupled to Leco Pegasus HT time-of-flight mass spectrometer) at 260 °C with a splitless mode. A DB-5 ms capillary column was applied to achieve the flow metabolite separation with helium as the carrier gas, and 1.0 mL/min was set as the constant flow speed. The temperature programming of GC part was set as follows: begin with a solvent delay for 5 min, then at 80 °C for 2 min, and ramped to 180 °C with the rate of 10 °C/ min, then to 230 °C with the rate of 6 °C/min and to 295 °C/min with the rate of 40 °C/min, and finally held for 8 min. The transfer interface was set to 270 °C with ion source at 220 °C. Data were acquired from 30 to 600 m/z at a 20/sec acquisition rate. The metabolites of organization extraction procedure followed our previous publication.15 Each hypothalamus and epididymis tissue (∼25 mg) was added to a 250 μL mixture of acetonitrile, chloroform, and water (2.5:1:1 v/v/v) and homogenized for 1 min. The samples were cooled to −20 °C for 20 min and then centrifuged at 12 000g for 10 min. The liquid layer was withdrawn and put into a new tube. The residue was extracted with 250 mL of methanol for the second extraction using the homogenizer for 10 min, followed by centrifugation at 12 000 rpm for another 10 min. The later supernatant was combined with the previous extraction and introduced to a new GC vial and then blow dried under a N2 gas stream. The follow-up derivatization and GC/TOF MS analysis process was in accordance with the above operation of serum samples.

MATERIALS AND METHODS

Animal Handling and Sampling

Fourteen male SD rats (8 weeks old) (200 ± 20 g) purchased from the Shanghai Laboratory Animal (SLAC, Shanghai, China) were randomly divided into two groups (the heat stress and control group). The rats in both groups were housed individually in cages provided with a certified standard rat chow and tap water ad libitum (room temperature 24 ± 1 °C, humidity 45 ± 15%), and the rats were maintained under a light cycle of 12 h (lights on at 8:00 a.m.). All experimental procedures were performed according to Chinese national legislation and local guidelines, and animal experimentations were conducted approved by Shanghai Jiao Tong University. The rats of H group were daily exposed to heat stress (40 °C) between time 11:00 and 13:00 for 7 days. The temperature of rat body and scrotal surface were recorded by InfraRed thermometer (DT-8861, CEM, Shenzhen, China) daily. The temperature of scrotal surface for each rat was compared with the average temperature of the whole testes surface, and the temperature of each body surface was compared with the average level of the animal’s torso, head, legs, and tail. Rats were fed in their individual feed bucket at 5 p.m., and 24 h later, the residual food and water bottle was taken out, weighed, and recorded, and each rat was weighed and recorded before and after the whole experiment. On the seventh day, all of the animals were decapitated immediately after heat stress, and blood, epididymis, and hypothalamus samples were collected and stored in −80 °C. The weight of each tissue (including testes, thymus, spleen, and adrenal gland) was recorded, and the individual ratio to bodyweight was worked out as its indices. The testicular tissue was fixed in Bouin for 3 days. After dehydration, the tissues were embedded into paraffin block; then, serial transverse sections at 5 μm were cut, mounted on glass slides, and stained with routine hematoxylin and eosin (HE) and Masson’s trichrome for histochemical and histometric analysis.

Statistical Analysis

The acquired GC−TOFMS mass chromatogram and spectrogram data were pretreated (involving denoising, smoothing, peak picking, alignment, and identification) using ChromaTOF software v4.22 (Leco), as described in a previous publication.15 The final data set included sample grouping information, peak retention time, and peak area (quant mass) of each metabolic compound. Artificial peaks including column bleed, noise peaks, N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) derivatization agents, and other signals known as interference peaks were all removed from the data set. PCA (principal component analysis) model and PLS-DA (partial least-squares discriminant analysis) were performed with SIMCA-p(13.0) software to identify the metabolites differentially. When VIP (the variable importance in the projection) values were greater than 1.0, they were considered to be differentiating variables. t test (P < 0.05) of all of the differentially expressed metabolites and fold-change values created from the arithmetic mean values of the ratio of the H group and control group, annotated by using our established standard reference library as described in our previous publications, were used for further differentiating variables selection and validation.

Radioimmunoassay Detection

Serum testosterone levels were assayed by double-antibody radioimmunoassay (RIA). The measurement was done by 125Itestosterone radioimmunoassay kits (purchased from Beijing North Institute of Biological Technology, Beijing, China), in accordance with the instruction. B

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Journal of Proteome Research Statistical comparisons included paired t test on temperature records and other classic pathophysiological indices in rats during the process of heat stress. GraphPad Prism version 5.0 software program (San Diego, CA) was used in statistical analysis. Thus, P values less than 0.05 are considered to be statistically significant.

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RESULTS

Temperature Assessment

After 2 h of heat exposure, scrotal and body surface temperatures of all rats were significantly elevated. The rectal temperature closed to 40 °C when rats suffered from heat stress, as indicated by accelerated respiratory rate, excessive drinking, and irritability. Food Consumption and Body Weight

Heat stress led to loss of body weight with increased food consumption. After 1 week of heat treatment, as compared with Control group (322 ± 4.17 g), we found that the body weight (304 ± 4.45 g) was significantly decreased in the H group (P < 0.05). However, food consumption showed no significant difference between the H group (23.53 ± 1.34 g) and the control group (20.98 ± 1.08 g). Histopathologic Evaluation

In control group, the seminiferous tubules were bounded together by loose intertubular connective tissue, which contained fibroblasts, and groups of germ cells (Figure 1A). Spermatogoniums, spermatocytes, and spermatids are arranged very closely to each other in the tubule with a high magnification image (Figure 1E). In the H group, there was a marked reduction in spermatogenesis. In the seminiferous tubules, there are separations between germ cells and the basement membrane (Figure 1B). Diameters of seminiferous tubules exposed to heat stress were significantly reduced, and the interstitial spaces were increased. The germinal epithelium and germ cells showed disorganization as well as marked degenerative changes with a high magnification image (Figure 1F). The numbers of fibroblasts as well as of collagen fibers were also increased (Figure 1C,D).

Figure 1. Light microscopy of testicular tissue in different groups stained with hematoxylin-eosin and Masson’s trichrome. (A) Control group. Normal morphology of seminiferous tubules (blue arrow) and inside lining smooth seminiferous epithelium (black arrow) (hematoxylineosin 50×). (B) H group. In the seminiferous tubules there are separations between germ cells and the basement (blue arrow) and gaps (black arrow) (hematoxylin-eosin 50×). (C) Control group. The normal structure of the tunica albuginea (red arrow) is observed (Masson’s trichrome 50×). (D) H group. The increase in connective tissue of the tunica albuginea (red arrow) is observed (Masson’s trichrome 50×). A high magnification image is shown in the black rectangle. (E) Control group. A high magnification image is shown in panel A with the black rectangle (hematoxylin-eosin 20×). Spermatogoniums, spermatocytes, and spermatids are arranged very closely to each other in the tubule. (F) H group. A high magnification image is shown in panel B with the black rectangle. (hematoxylin-eosin 20×). Germ cells showed disorganization changes as well as marked degenerative changes. Sg, spermatogonium; Sc, spermatocyte; Sd, spermatid.

Serum Concentrations of Testosterone

In the heat-treated group, serum testosterone concentration was increased significantly as compared with that of the control group (Figure 2). Testes, Adrenal Gland, Thymus, and Spleen Weights

The testes are the primary reproductive organs that maintain the health of the male reproductive system. They have the ability to produce significant amounts of testosterone and a regulated HPG axis.14 It is well accepted that short- or long-time testicular heat exposure induces a decrease in testes weight.16 In this study, we found that the testis index significantly decreased after 1 week of heat treatment. Adrenal, thymus, and spleen index, as indications of immune system, showed significant change in the 7th day compared with the Control group (Figure 3). In fact, heat stress can restrain the immune system and significantly influence the effect on the adrenal, thymus, and spleen indices. Heat-Stress-Induced Metabolic Changes

In this study, the PCA scores plot generated from GC/TOFMS data is depicted in Figure 4. There was a clear separation observed in the plot of serum samples (R2X = 0.597, Q2 = 0.157) between the heat stress and control groups (Figure 4A), suggesting that systemic metabolic variation might be induced by exposure to heat stress. Additionally, there were separations

Figure 2. Blood serum testosterone values in rats. The concentrations of testosterone in serum were measured. Each bar represents the mean ± SEM (n = 7). ** indicates p < 0.05 compared with the Control group. H: heat stress; C: control.

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DISCUSSION From previous literature, exposure to the high ambient temperature inhibits spermatogenesis and induces germ cell apoptosis, which leads to a reduction in testis size.16 In the present study, we found the reduced testes index (Figure 3) and increased testosterone concentration (Figure 2) after heat treatment on day 7, indicating that the heat stress disturbed the functions of testis, and testosterone level was elevated to prevent germ cells from heat-induced apoptosis.17 Sustained release of testosterone was affected by hypothalamic−pituitary− testis axis (HPT) in mammals. The results suggested that heat stress might have induced the male reproductive system dysfunction and affected the function of HPT as well. Metabonomic analysis of hypothalamus, serum, and epididymis indicated differentially expressed metabolites, including metabolites of the energy metabolism, organic acids, amino acid neurotransmitters, and monoamine neurotransmitters pathways (Figure 7), somehow reflecting the influence of three upstream neuroendocrine systems (the SNS (sympathetic adrenergic system), hypothalamic pituitary adrenal axis (HPA), and hypothalamic pituitary testis axis (HPT) axis).

Figure 3. Testis, thymus, spleen, and adrenal gland indices of rats during the period of heat stress. Values are expressed as mean ± SEM (n = 7).* indicates p < 0.05; **indicates p < 0.01 compared with the Control group. H, heat stress; C, control.

between the two groups found in the PCA scores plot of both hypothalamus (R2X = 0.698, Q2 = 0.326) (Figure 4B) and epididymis (R2X = 0.595, Q2 = 0.353) (Figure 4C). The PLS-DA scores plot in correlation with PCA model is given in Figure 5. There were 37 serum metabolites, 25 hypothalamic metabolites, and 19 epididymis metabolites identified in different levels between the control and heat stress rats (Figure 6A−C). Moreover, ANOVA and Kruskal−Wallis tests was used in this metabolite analysis, and the significance threshold was at p = 0.05, and the results of serum, hypothalamic, and epididymis are all shown in Tables S1−S3 in the Supporting Information. Fold changes came from the ratio of arithmetic mean values of these metabolites in H the Control group (Figure 6).

Energy Metabolism

It is worth noting that there are several important intermediate products in TCA cycle, as the increased levels of succinate, fumarate, and malate in serum reflect an up-regulation of TCA cycle metabolism. Lower level of carbohydrates and higher level of glucogenic amino acids (GAAs) were detected in serum samples of animals undergoing heat stress, indicating negative energy balance. To begin with, down-regulation of fructose, galactose, rhamnose, mannose, D-ribose, and D-ribofuranose suggested that carbohydrate metabolism was accelerated to supply more energy. These findings in serum samples are in agreement with previous reports that the decrease in the

Figure 4. PCA scores plot of comparing control group and heat stress group in serum, hypothalamus, and epididymis. The blank dot is the Control group and the red dot is the Heat group. D

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Figure 5. PLS-DA score plots generated from the PLS-DA of the GC/TOFMS data derived from the serum (A), hypothalamus (B), and epididymis (C) samples of rats. The green dot is control group and the red dot is HS group (A) R2X = 0.41, R2Y = 0.963, Q2 = 0.808; (B) R2X = 0.534, R2Y = 0.848, Q2 = 0.478; and (C) R2X = 0.568, R2Y = 0.902, Q2 = 0.658.

epididymis barrier (BEB) has been considered to be closely connection between the epididymal epithelial cells, and thus it has a more restrictive environment to transport molecular than the blood−testis barrier (BTB).26 Therefore, this discrepancy might be related to a kind of self-preservation function of the epididymis; that is, when the reproductive system is damaged, certain metabolites with reproductive benefits would improve their concentrations to help the body resist the adverse stimulus to maintain normal reproductive functions. In addition, other studies have demonstrated a high concentration of fructose level in rams and boars when exposed to extreme environmental temperature.27 The up-regulated testosterone level during heat stress was another typical example of the self-preservation function. In addition, part of glucose can be converted into fructose associated with the stimulation of testosterone in the blood. As mentioned in our results, the elevated fructose level in epididymis and the depleted level in serum may be a result of the influence of testosterone. Stress is deleterious to fertility function because energy demands are crucial to the regulation of metabolic status. Heat exposure triggered significantly increased levels of fructose and amino acids identified, while citrate, malate, and nicotinamide abundances also increased in sperm. This result suggested that the alternation of these metabolites in energy pathways may be considered as a kind of compensatory mechanism to cope with the energy short. A similar pattern emerged in serum and hypothalamus as an abundance of energy significantly increased metabolism during heat stress. Some of these metabolites were confirmed by the plasma samples taken from young man displaying low, medium, and high sperm concentrations.28 Furthermore, on the basis of the seminal plasma, levels of citrate and lactate are altered in men with azoospermia, which were also in agreement with our results, suggesting a possible involvement of heat stress with infertility.13 As expected, we review the evidence that heat stress causes an increase in food consumption and a reduction of body weight. Taken together, these changes revealed that the disorder in the energy metabolism is a crucial

production of carbohydrate level is a compensatory mechanism in rats during the acclimation to hyperthermia.18 Meanwhile, the elevation of the GAAs in heat stress rats, such as alanine, serine, valine, and methionine (Met), possibly stemmed from protein decomposition, which caused a negative nitrogen balance state in the body.19 During high-temperature exposure, there was an increased urea concentrations compared with thermoneutral controls.20 Therefore, the enhanced GAAs and urea indicated increased protein disintegration to supply the excess energy production in heat stress because they can be converted to pyruvate, which can then enter the TCA cycle and gluconeogenesis. Valine, leucine, and isoleucine, as named branched-chain amino acids (BCAAs), were also found to be up-regulated after heat stress. As an amino acid group of special function, BCAAs cannot only help cells uptake more energy but also prevent protein decomposition and reduce excessive free radicals induced by heat stress.21 Besides, a similar variation tendency of the 3-hydroxybutyric acid (3-HB) level was detected in the hypothalamus. 3-HB served as an indispensable source of energy for extrahepatic tissues, especially in the brain.22 Meanwhile, the diminished D-ribose further illustrated the lack of energy metabolism because it is a substrate help to regenerate 5phosphoribosyl-l-pyrophosphate (PRPP) contributing to ATP production. (Figure 7). There were the highest glycolytic rates and the lower Krebs cycle rates in spermatozoa of all germ cells for its single energy source of glucose or fructose.23 Recently published studies showed that the activities of the glycolytic pathway, HMP pathway, and TCA cycle enzymes were estimated in caput, corpus, and cauda segments of epididymis.24 So the significantly increased levels of citrate, malate, and nicotinamide in the epididymis indicated reproductive energy metabolic disorders upon exposure to heat stress. Furthermore, the increased levels of malate and aspartate (Asp) suggest there would be alteration of the malate-aspartate shuttle in ATP generation in spermatozoa due to the effect of heat stress.25 Under such conditions, we found the opposite tendency for fructose, ribose detected from epididymis, and serum. The anatomical location of the blood− E

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Figure 6. Fold changes of the arithmetic mean values (p < 0.05) of heat-induced changes in (A) serum, (B) hypothalamus, and (C) epididymis metabolites.

(Figure 7). Additionally, the elevated hydrocinnamic acid is an analogue of phe and a substrate of the enzyme oxidoreductases in the pathway of phe metabolism. These changes indicate that an increased SNS activity and an up-regulation of catecholamine metabolism followed. The taurine (Tau) level was found decreased in the hypothalamus of the H model. Corticotrophin secretion is positively regulated by Tau at the supraoptic level during times of stress in rats.31 In contrast with the change of Tau, hypotaurine was increased in the serum and epididymis of H rats (Figure 7). In the brain, hypotaurine is a product of cysteamine dioxygenase in Tau and hypotaurine metabolism pathway.32 In the male reproductive system, Tau and hypotaurine have been observed in semen of numerous species and are considered to have beneficial effects on sperm characteristics in mammals.33 The covariation of the two

part of a self-preserving mechanism in response to male reproductive system to deal with the heat stress (Figure 7). Amino Acid and Monoamine Neurotransmitters Metabolism

Consistent with the findings of different types of stress,29 it is thought that heat stress activated the SNS and HPA axis, providing two opposite influences: one is to regulate material and energy metabolism to maintain homeostasis in the organism, and the other is to restrain neuroendocrine and immune system, causing damage to the reproductive system, as evidenced by the improved adrenal index and reduction of testis, thymus, and spleen index in the heat stress group. In this study, phenylalanine (Phe) and phenylethanolamine, two precursors of catecholamines in the tyrosine pathway,30 were significantly increased F

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Figure 7. Changes in metabolite contents in the hypothalamus, serum, and epididymis with heat stress compared with those in the Control group revealed the underlying metabolic regulatory mechanism to maintain homeostasis and protect reproductive organs. HPA, hypothalamic pituitary adrenal axis; HPT, hypothalamic pituitary testis axis; SNS, sympathetic adrenergic system; BEB, blood−epididymis barrier; BTB, blood−testis barrier; SAM, S(5′-adenosyl)-L-methionine chloride.

pulsatile release of GnRH.41 Given our results, it could be inferred that the higher level of testosterone might be the result of increased synthesis of acetylcholine, which plays a positive role in the HPT function to fight against heat-induced damage (Figure 7).

compounds suggested that the pathway of hypotaurine and Tau might have been changed when exposed to heat stress. Collectively, according to the above results, we believe that heat stress is a strong activator of the SNS and HPA axis, which mobilizes systemic power to deal with the heat stress. Tryptophan (Trp), an essential monoamine neurotransmitter and an important precursor of serotonin, was increased in the hypothalamus and epididymis samples (Figure 7). It is vital in the regulation of a healthy nervous system because it helps the production of serotonin that supports mental health and helps to reduce stress, insomnia, anxiety, and depression.34,35 The alteration in brain Trp level depends largely on the serotonin synthesis. There was a close physiological relationship between them: when Trp levels in brain vary in view of insulin secretion or dietary intake, the rate of serotonin production alters as a result.36 The NMR study showed that the Trp acts as an attractant to spermatozoa.37 Thus, the significant changes of Trp levels are considered to play a key role in the heat-stress-induced reproduction dysfunction. There was also considerable evidence suggesting that the regulation of body temperature and fluid equilibrium is controlled by the cholinergic system, which induced the expression of Fos protein in some hypothalamus areas.38 The up-regulated SAM (S-(5′-adenosyl)-L-methionine chloride) and glycine (Gly), two important precursors of acetylcholine, were found in the heat stress animals, hinting that the synthesis of acetylcholine might be increased as well.39,40 Moreover, a previous report has provided direct neuromorphological evidence of the cholinergic system in the afferent neuronalregulation of GnRH (gonadotropin-releasing hormone neuron), while the testosterone secretion is regulated by the

Other Amino Acids Metabolism

Meanwhile, some other amino acids were changed by heat stress. High lysine levels in all three samples were detected. It was found that chronic ethanol consumption caused testicular failure along with significantly lower contents of lysine.42 Many studies have reported that lysine has been shown to increase testosterone and sperm production.43 Moreover, it is known that testosterone has an effect on epithelial cell proliferation in the rat epididymis.44 Given our results, it could be inferred that the higher level of testosterone might be connected to the lysine in epididymis. Similarity was seen in the level of Met. Studies show that Met in protein acts as an endogenous antioxidant in cells. Furthermore, Met was able to reverse ROS-induced injury, possibly through the activation of antioxidant like GSH or GPx, which was observed in animals being exposed to high ambient temperature and fed a Met supplementation diet.45 Herein, the elevated lysine and Met levels might play a protective self-preservation function in response to high temperature. In summary, this study offers a novel insight into the influence of heat-induced reproductive and metabolic disorder via the combination of pathophysiological indices and metabonomic outcomes, highlighting the role of underlying metabolic regulatory mechanism against heat. We have identified various metabolites that were implicated in a multitude of functions associated with heat stress in the hypothalamus, serum, and G

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Journal of Proteome Research

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epididymis, revealing a probable endogenous metabolic selfpreservation function of several critical metabolites.

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ASSOCIATED CONTENT

S Supporting Information *

Table S1. Differential expressed metabolites in serum contributing to the separation between H group and Control group. Table S2. Differential expressed metabolites in hypothalamus contributing to the separation between H group and Control group. Table S3. Differential expressed metabolites in epididymis contributing to the separation between H group and Control group. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*J.S.: E-mail: [email protected]. Phone:86- 25-84395294. Fax: +86 25 84398669. Address: 6 Tongwei Road, Nanjing 210095, China. *X.W.: E-mail: [email protected]. Phone: 86-21-34207343. Fax: 86-21-34206059. Address: 800 Dongchuan Road, Shanghai 200240, China. Author Contributions ⊥

Y.H. and X.W. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) (280100745113) and the National Nature Science Foundation of China (31372388, 30901997).

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ABBREVIATIONS GAA, glucogenic amino acid; BCAA, branched-chain amino acid; Trp, tryptophan; Met, methionine; Asp, aspartate; Cit, citrulline; Orn, ornithine; Arg, arginine; Tau, taurine; Phe, phenylalanine; Gly, glycine; SAM, S-(5′-sdenosyl)-L-methionine chloride; 3HB, 3-hydroxybutyric acid; HPA, hypothalamic pituitary adrenal axis; HPT, hypothalamic pituitary testis axis; SNS, sympathetic adrenergic system; BEB, blood−epididymis barrier; BTB, blood−testis barrier; GnRH neuron, gonadotropin-releasing hormone neuron; 5-HT neuron, serotonin neuron; ACH neuron, cholinergic neuron

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REFERENCES

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