Chronic unpredictable stress (CUS) reduced phoenixin expression, induced abnormal sperm and testis morphology in male rats

Chronic stress caused by prolonged emotional pressure can lead to various physiological issues, including reproductive dysfunction. Although reproductive problems can also induce chronic stress, the impact of chronic stress-induced reproductive dysfunction remains contentious. This study investigates the effects of chronic unpredictable stress (CUS) on reproductive neuropeptides, sperm quality, and testicular morphology. Sixteen twelve-week-old Sprague Dawley rats were divided into two groups: a non-stress control group and a CUS-induced group. The CUS regimen involved various stressors over 28 days, with both groups undergoing behavioural assessments through sucrose-preference and forced-swim tests. Hypothalamic gene expression levels of CRH, PNX, GPR173, kisspeptin, GnRH, GnIH , and spexin neuropeptides were measured via qPCR, while plasma cortisol, luteinizing hormone (LH), and testosterone concentrations were quantified using ELISA. Seminal fluid and testis samples were collected for sperm analysis and histopathological evaluation, respectively. Results showed altered behaviours in CUS-induced rats, reflecting stress impacts. Hypothalamic corticotropin-releasing hormone ( CRH ) expression and plasma cortisol levels were significantly higher in CUS-induced rats compared to controls ( p < 0.05). Conversely, phoenixin ( PNX ) expression decreased in the CUS group (p < 0.05), while kisspeptin, spexin , and gonadotropin-inhibitory hormone (GnIH) levels showed no significant differences between groups. Despite a significant increase in GnRH expression ( p < 0.05), plasma LH and testosterone concentrations were significantly lower (p < 0.05) in CUS-induced rats. Histopathological analysis revealed abnormal testis morphology in CUS-induced rats, including disrupted architecture, visible interstitial spaces between seminiferous tubules, and absence of spermatogenesis. In conclusion, CUS affects reproductive function by modulating PNX and GnRH expression, influencing cortisol levels, and subsequently reducing plasma LH and testosterone concentrations. This study highlights the complex interplay between chronic stress and reproductive health, emphasizing the significant impact of stress on reproductive functions.


Introduction
Reproductive dysfunction is defined as the abnormality and impairment of the reproductive system, and it does not arise from a singular issue.In fact, it is a combination of multiple problems comprised of hormonal imbalance produced by the reproductive system, abnormalities of gonadal organs (size and density), sexual dysfunction (low sexual drive and erectile dysfunction) and sperm production quality (count, motility, and morphology) (Almabhouh et al., 2020).Reproductive dysfunction arises from various factors, including psychological stress, metabolic stress, and other environmental influences, posing a global concern (Tournaye et al., 2017;Liu et al., 2022).In males, reproductive dysfunction is defined by the diminished quality of the male reproductive system, which can manifest as the inability to achieve satisfactory sexual performance or, in more severe cases, the inability to father a biological child (Anderson et al., 2022;Capogrosso et al., 2021).Male fertility issues are rarely discussed in society; however, awareness around this issue has gradually increased, leading to a reduction in the stigma associated with discussing it.Reproductive dysfunction in males is generally associated with infertility in the elderly; however, in recent years, the probability of younger men suffering from the same issue has not been excluded (Kaufman et al., 2019).According to the National Institute of Health (NIH), male factors can contribute to approximately 50% of all infertility cases around the world infertility cases worldwide (Kumar and Singh, 2015).Multiple factors may lead to reproductive dysfunction, such as obesity, sedentary lifestyle, genetic predispositions, and notably, chronic psychological stress (Norman and Clark, 1998;Foucaut et al., 2019;Kuroda et al., 2020).This psychological stress has recently emerged as a significant concern, particularly among infertile men aged between 19 and 33 years old (Jurewicz et al., 2010).
Random and prolonged exposure to numerous unpredictable stressful life events is a major risk factor for the development of chronic stress (Bondi et al., 2008).Cognitive and emotional bias due to chronic stress can take over rational thinking and cause the build-up of mental burden, which eventually may lead to mental disorders such as depression and anxiety (Fodor et al., 2020).Chronic stress also negatively affects various physiological functions, including dysregulation of the nervous system and the male reproductive system (Yaribeygi et al., 2017).Additionally, chronic stress may affect male reproductive function by altering the neuropeptides regulating reproductive system by altering the homeostasis of hypothalamus-pituitary-gonadal (HPG) axis by fluctuating the level of reproductive hormones (ref).In male, neuropeptides play a crucial role in regulating reproductive function involving each tier of the HPG axis.Research on hypothalamic neuropeptides and their effects on gonadal function has indeed progressed, yet gaps in understanding remain.For example, hypothalamic neuropeptides like GnRH and kisspeptin are pivotal in regulating the reproductive system by influencing the gonadotropins released by the pituitary gland, which in turn acts on gonadal organs to modulate their activity involving the release of sex hormones and fertilisation in mice (Selvage et al., 2004).Another recent study demonstrated that hypothalamic neuropeptide FF (NPFF) significantly inhibits FSH expression in the pituitary of medaka fish, resulting in a marked decrease in fertilisation rate and spermatogenesis in the testis (Tomihara et al., 2022).The findings from these studies follows the precise sequence of the HPG axis and serves as a reference for understanding the neural pathways from the hypothalamus to gonadal organs for other hypothalamic neuropeptides.Hence, the finding contributes an insight into the mechanisms of neuropeptides signalling in the control of testicular functions via HPG axis.
In relation of stress and reproductive system, few studies have demonstrated that stress affects the delicate balance of neuropeptides in the brain, including kisspeptin and gonadotropin-releasing hormone (GnRH) (Skorupskaite et al., 2014;Luo et al., 2016).Kisspeptin is a neuropeptide that act upstream of GnRH by directly activating GnRH neurons to regulate reproductive system (Xie et al., 2022).These two neuropeptides are the most studied under chronic stress (Hirano et al., 2014;Son et al., 2022).There are few other neuropeptides that are closely associated with reproduction and stress independently, namely PNX, gonadotropin inhibitory hormone (GnIH) and spexin (McIlwraith et al., 2022;Friedrich and Stengel, 2021).The neuropeptide PNX is recently discovered neuropeptide where it is cleaved from small integral membrane protein 20 (SMIM20) and the precursor is encoded by C4orf52 gene (Liang et al., 2022a;Lyu et al., 2013).Neuropeptide PNX consists of two active isoforms PNX-14, majorly play rule in peripheral region while PNX-20 commonly found in the central nervous system of vertebrates (Billert et al., 2020).Both isoforms are known potential ligand for G-protein-coupled-receptor 173 (GPCR173) that acts as receptor in both central and peripheral nervous system mainly gonadal area (Billert et al., 2020).Over the years, PNX has emerged as a notable neuropeptide, recognized for its pleiotropic effect on both reproduction and stress regulation (McIlwraith et al., 2022).Subsequently is GnIH, one of the known neuropeptides that acts as an inhibitory key hormone in reproduction and reproductive behaviours (Tsutsui and Ubuka, 2020).The inhibitory effect of GnIH in reproductive function always been an interest among the researchers to know further about the potential in relationship with other physiological function for instance stress regulation (Odetayo et al., 2023;Teo et al., 2021;Choi et al., 2017).Additionally, one of the most recent neuropeptides discovered is spexin, encoded by C12ORF39 gene where it was found to activate galanin receptor 2 (GALR2).The SPX expression has been documented in brain and peripheral tissue of humans, rats, mice and goldfish (Mirabeau et al., 2007;Wan et al., 2009;Liu et al., 2013;Porzionato et al., 2010).
In the 1970s and 1980s, a concept of prolonged stress and its effect on animals emerged and gave rise to "unpredictable chronic stress (CUS) paradigm" using experimental animal model (Matthews et al., 1980).The experiment was widely used to induce chronic stress, resulting in depression and anxiety-like behaviour (Matthews et al., 1980;Antoniuk et al., 2019;Seligman and Meyer, 1970;Baron and Brush, 1979).Subsequently, research has been conducted exploring the association between chronic stress and its impact on various physiological functions (Jiang et al., 2011;Karagiannides et al., 2014).However, the effects of CUS on male rat's reproductive neuropeptides and testis function remain unclear.Understanding the effect of chronic stress on male reproductive system is crucial to identify the potential therapeutic target for male reproductive dysfunction.Thus, this study aims to investigate into the impact of chronic unpredictable stress (CUS) on the male reproductive system, examining its potential influence on reproductive neuropeptides and its effects on sperm quality and testicular morphology in CUSinduced male rats.

Animals and experimental design
Experiments were conducted on young male specific-pathogen-free (SPF) Sprague-Dawley rats, weight 450 g-510 g (n = 16).Each rat was housed under a 12 h light/dark cycle (lights on at 12 am-12 pm) in a cage with ad libitum access to water and food.Initial acclimatisation took place for one week before the experiments.All experimental procedures were done humanely by trained personnel.The study was approved by Monash University Animal Ethics Committees (Ethics ID: 31768).

Chronic unpredictable stress (CUS) and behaviour tests
The animals were divided into two groups: (i) the non-stress control group (n = 8) (ii) CUS group (n = 8).The CUS protocol was adopted and modified to increase the intensity of the stressors from Alfarez, (Alfarez et al., 2003).The rats in the non-stress control group were single-caged and separated from the experimental group and received standard facility care with daily food and water support.The rats in the CUS-induced group were housed in a single cage and exposed to different types of stress (Table 1) for 28 consecutive days.Following the CUS procedure, the rats in non-stress control and CUS-induced groups were subjected to three behaviour tests.

Sucrose-Preference Test (SPT)
The sucrose preference test was adopted from Serchov, 2016(Serchov et al., 2016).All rats from both groups were trained with two bottles of water on the last three days of CUS prior to the sucrosepreference test to familiarise themselves with the setting.Twenty-four hours after the CUS ended, the rats from both groups were given mL of 5% sucrose solution (Fisher Scientific, Loughborough, UK) and 150 mL of drinking water in two separate identical bottles for three consecutive days.The position of each bottle was changed daily to prevent biased consumption of any solution.The sucrose and water consumption were measured daily for the three consecutive days and the total consumption of both solutions was compared between the two groups.The water and sucrose preference percentages were calculated using the formula: Mohamed et al. water or sucrose consumption from one group ucrose consumption from one group × 100%

Force-swim Test (FST)
The force-swim test procedure was adopted from Yankelevitch-Yahav, 2015 (Yankelevitch-Yahav et al., 2015).The test was divided into two days: i) pre-test for familiarisation and ii) commencement day (n = 12).Prior to both days, all the rats were given food and water ad libitum.On the pre-test day, all the rats were exposed to a transparent cylindrical tank filled with water for 15 min to acclimate to the testing environment and reduce the risk of bias in analysis.Rats were monitored closely and transferred to a recovering cage for a blow-dry after the experiment was completed.On the second day, the same steps were repeated with the duration of 5 min for each rat and the whole experiment was recorded to analyse the mobility (active behaviour) and immobility (passive behaviour) of the rats in the water.

Copulatory test
The copulatory test procedure was adopted from Ågmo and Snoeren, 2015 (Ågmo and Snoeren, 2015).Sexually experienced female rats were used as mating stimuli (n = 10) for the male rats from both groups.The female rats were administered with a single subcutaneous dose of oestradiol benzoate (EB) (10 μg/100 g) (Sigma-Aldrich, Darmstadt, Germany) to initiate their oestrus cycle.Then, 72 h post EB injection, a subcutaneous dose of progesterone (0.5 mg/100 g) (Sigma-Aldrich, Darmstadt, Germany) was administered to the female rats to stop the oestrous cycle and were left for 6 h to induce the onset of heat for the copulatory test.Subsequently, male rats from both groups were introduced to female rats and were allowed to remain together for one hour.The whole session was recorded for sexual behaviour analysis.In this test, number of mounting frequencies, latency to first mounting (time to first pelvic thrusting), latency to first intromission (time to first pelvic thrusting with vaginal penetration) and latency to first ejaculation (time from first intromission to ejaculation) were recorded for statistical analysis.
Upon completion of the behaviour tests, the male rats were rested (ejaculatory abstinence) for 48 h before they were euthanised under ketamine (80 mg/kg)-xylazine (10 mg/kg) anaesthesia and were administered via intraperitoneal injection.During autopsy, the brain, blood, testis, and seminal fluid were collected from all the male rats for further analysis.

Quantitative PCR (qPCR)
Total RNA was isolated from hypothalamus of the male rat's brain (n = 16) using FavorPrep™ Tri-RNA Reagent (Favorgen BiotechCorp, Taiwan).The total RNA was converted to complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit using the manufacturer's recommended protocol (Applied Biosystems, Thermo Fisher Scientific, US).Seven genes (Table 2) involved in stress and reproductive function were selected, and GAPDH was used as a housekeeping gene for the quantitative polymerase chain reaction (qPCR).The primers for the genes were designed using the Primer3 online tool (https://primer3.ut.ee/).The qPCR was performed using the Sensifast SYBR Hi-Rox QPCR pre-mix (Bioline, Meridian Bioscience, UK) and run using the 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, US).The CT values for each gene were normalised against the housekeeping gene, GAPDH.The data obtained from this experiment were analysed using relative quantification 2^(-ΔΔCт) to determine the gene expression.

Hormones assay
Circulating hormones for stress (cortisol) and reproductive system (luteinising hormone (LH) and testosterone (n = 16) were measured using commercial enzyme-linked immunosorbent assay (ELISA) using the manufacturer's (Enzo Life Sciences, US) recommended protocols.The absorbance at 450 nm was obtained using the TECAN Infinite 200 PRO (Tecan, Switzerland).The sample concentration was calculated against the standard provided in the kit (cortisol and LH) to identify the hormone concentrations.

Seminal fluid analysis
Seminal fluid analysis including sperm count and sperm morphology was conducted.Male rats were rested (ejaculatory abstinence) for 48 h before sample collection to ensure optimal sperm production for seminal fluid analysis.

Total and abnormal sperm count
The total sperm count from the CUS-induced and non-stress control groups (n = 16) was counted and compared accordingly.Then, the total abnormal and normal sperm per mL were counted using an Improved Neubauer haemocytometer (>10 N's ring/fringes or iridescence lines) as described by Wang, 2002(Wang, 2002).The epididymal fluid was diluted in 1:5000 (sperm: normal saline) and 10 μL of the diluted seminal fluid was inserted into each space (counting chamber) between cover glass and haemocytometer.Both normal and abnormal sperm were counted separately on all 25 large squares at each counting chamber.The total sperm was calculated using the formula:

Sperm morphology
The diluted epididymal fluid collected as stated above was used, and 2 μL of the diluted sperm from all rats (n = 16) were placed on the glass slides respectively.Then, all of the samples were left overnight to dry.
After 24 h, all the samples were subjected to Haematoxylin and Eosin (H&E) staining (Leica, Manheim, Germany) and the standard protocols were used as perto manufacturer's protocol.The sperms were analysed by observing the morphology under an Olympus BX50 brightfield upright microscope (Olympus, Japan), and their shapes were compared according to Fig. 1.

Testes morphology
Testes were embedded in an Optimal Cutting Temperature (OCT) compound (Leica, Manheim, Germany) and were cryo-sectioned at 20 μM thickness.Haematoxylin and Eosin (H&E) staining (Leica, Manheim, Germany) was used to stain the sectioned using the standard manufacturer's protocol.The results were analysed by observing the morphology of the tissue under an Olympus BX50 brightfield upright microscope (Olympus, Japan).

Statistical analysis
Data for all experiments were analysed using GraphPad Prism 9, and statistical analysis for behaviour studies and sperm count were performed using one-way Anova followed by Tukey's Post-hoc test while the gene expression was performed using t-tests.The gene expression data between the CUS-induced rats and control rats were calculated by relative quantification and expressed as mean ± S.E.M to demonstrate the fold changes.The statistical significance data was set at p < 0.05.

CUS-induced anhedonia-like behaviour in male rats
Throughout the three days of the sucrose preference test, the nonstress control group demonstrated a significantly higher preference for sucrose consumption percentage at 33% more than the CUS-induced group (p < 0.05) (Fig. 2).The drinking water consumption percentage is significantly lower in the non-stress control group at 36% lower than in the CUS-induced group (p < 0.05) (Fig. 2).The non-stress control group demonstrated a higher preference for sucrose, 32% more than water.In contrast, CUS-induced rats groups showed significantly higher water preference and consumed 32% more water than sucrose solution (Fig. 2).

CUS induced despair-like behaviour in male rats
The FST result demonstrated that the non-stress control group showed significantly shorter immobility time (passive behaviour), at 136 s compared to CUS-induced rats, at 185 s (p < 0.05).Then, the nonstress control group demonstrated significantly longer mobility time at 49 s more than the CUS-induced rats.There is also significant difference between immobility and mobility time within each group (Fig. 3).

CUS does not affect the sexual motivation of the male rats
The copulatory test was done to evaluate the effect of CUS on the copulatory ability of the male rats.Three parameters, mounting,  intromission and ejaculation, were used during the test to analyse the sexual motivation and ability of the male rats for both groups.In this study, our data demonstrated a longer duration for mounting latency, intromission and ejaculation for the CUS-induced group compared to the non-stress control group; however, it was not statistically significant (Fig. 4a).Additionally, the frequency of mounting was recorded, and reduction of the mounting frequency of CUS-induced groups was observed; however, there is no statistical difference was demonstrated (Fig. 4b).

CUS-induced changes in stress and reproductive hypothalamus neuropeptide expression
The expression of the stress neuropeptide CRH in CUS-induced rats significantly increased by 0.6-fold compared to the control group (p < 0.05) (Fig. 5).The reproductive neuropeptides PNX, kisspeptin, spexin, GnIH and GnRH mRNA expression were also evaluated.Our results showed that CUS-induced rats demonstrated significantly low expression of PNX, which reduced by 0.5-fold compared to the non-stress control group (Fig. 6a).There were no significant differences in kisspeptin, spexin, and GnIH expressions between the non-stress control and CUS-induced groups (Fig. 6b-d).However, hypothalamic GnRH showed a significantly higher expression in CUS group compared to control (Fig. 6e).

CUS-induced changes in stress and reproductive hormones
CUS-induced male rats had a significantly higher cortisol concentration (2154.97pg/mL) compared to the non-stress control rats (1877.98 pg/mL) (p < 0.05) (Fig. 7).The concentration of two reproductive circulating hormones (LH and testosterone) was quantified in this study.There was a significant reduction (p < 0.05) of LH (22,683.2pg/mL) in the plasma from CUS-induced rats compared to control rats (24,610 pg/mL) (Fig. 8a).In addition, the plasma concentration of testosterone, a downstream reproductive hormone, was also significantly low (p < 0.05) in CUS-induced rats (1561.64pg/mL) compared to control rats (1892.42pg/mL) (Fig. 8b).

CUS-induced high abnormal sperm count
The total sperms were calculated and differentiated based on normal and abnormal morphology.There was no significant difference in the total number of sperms between the non-stress control and CUS-induced groups (Fig. 9a).However, there was a significant increase in the number of abnormal sperms (10,420 sperms/mL) (p < 0.05) in CUSinduced rats compared to the non-stress control group (2580 sperms/ mL) (Fig. 9b).
Fig. 3.The immobility time for the CUS-induced male rats was significantly longer than the control group (*p-value < 0.05; **p-value < 0.01).There is significant difference between mobility and immobility within each group and in between both groups.

CUS altered the sperm and testes morphology
Morphological analysis of sperm samples revealed a significant change in the CUS-induced rat's sperm morphology.For instance, the incidence of sperm with the head detached from the tails was commonly seen in the CUS-induced rats compared to the control (Fig. 10a).Morphological defects such as flat head, bent tail, double head, bent neck and flattened head were not commonly observed in the non-stress control group compared to the CUS-induced group (Fig. 10).The predominant type of abnormalities after the detached head was the flat head.The representative microphotographs of sperm morphology are shown in Fig. 10.
The microphotographs of the testes from the non-stress control group showed a consistent and round morphology of the seminiferous tubule (Fig. 11a-b) and CUS-induced male rats demonstrated an increased diameter of the seminiferous tubule as it appears elongated (Fig. 11e-f).Furthermore, the interstitial spaces were more visible in CUS-induced rats (Fig. 11f-g), as there were more spaces between the seminiferous tubules than in control groups (Fig. 11b-c).The microphotographs of the testes also showed that lesser to no spermatogenesis occurred in the CUS-induced rats (Fig. 11g-h).However, more spermatogenesis was Fig. 6.Relative quantification of reproductive system neuropeptides in male rat's hypothalamus.a) The PNX expression is lower by >0.5-fold in CUS group compared to control group (*p-value<0.05)b) Reproductive stimulator, kisspeptin showed no significant changes after the induction of CUS c-d) The reproductive system inhibitor, spexin and GnIH showed no significant changes after induction of CUS, however, expression of spexin in CUS-induced male rats showed an increase trend e) The expression of GnRH increased significantly in CUS-induced male rats (*p-value<0.05).Neuropeptide expressions were normalised against the housekeeping gene, GAPDH.visible in the non-stress control group (Fig. 11c-d) as observed by the lesser appearance of primary spermatids and elongated spermatids in the CUS-induced group compared to the non-stress control group (Fig. 11g-h).

Discussion
In this study, four weeks of CUS significantly induced chronic stress in the rats demonstrated by the behavioural test findings.The CUS  significantly reduced sucrose preference by >30% among the experimental rats (Fig. 2) which indicated that CUS-induced rats were not able to experience pleasure from the sweet taste of the sucrose.This behaviour deviated from the typical response of healthy rats which are generally drawn to the sweet taste from sucrose solution as observed in non-stress control group (Scheggi et al., 2018;Gregory et al., 2012).The inability to experience pleasure, also known as anhedonia, is one of the behaviours that can be found in individuals who suffer from chronic stress and known as one of the depressive-like behaviours (Stanton et al., 2019).Simultaneously, the non-stress control group showed a higher sucrose preference at 66% which indicates that the non-stress control group experienced joy or hedonic behaviour.Hence, it is possible to postulate that the daily stress inflicted on these CUS-induced rats reflects a decreased sensitivity to pleasure, further reinforcing the CUS paradigm model.In addition, this study supports the previous finding which reported a low sucrose preference percentage value observed in CUS-induced and socially isolated rats compared to control rats (Hong et al., 2012;Muscat and Willner, 1992).
Subsequently, the forced swimming test (FST) was done to assess both "behavioural despair" and the slowing down of psychomotor abilities in the presence of chronic stress conditions (Unal and Canbeyli, 2019).Psychomotor retardation was defined as slowing down of a movement (torso or head) in extreme conditions as part of the coping mechanism in animal models (Buyukdura et al., 2011).Furthermore, helplessness or passive behaviour in extreme stress conditions has been reported in previous studies (Unal and Canbeyli, 2019;Kraeuter et al., 2019).In our study, CUS-induced rats demonstrated longer immobility time, indicating passive behaviour compared to the control group.These findings imply that the CUS approach applied in this study elicited a state resembling depression in the rats, as evidenced by their display of helplessness and lack of motivation to engage in survival behaviours.Our results are in agreement with previous study where longer  e-h).Non-stress control group showed round morphology of seminiferous tubule (a-b) while CUS-induced rats showed elongated and enlarged seminiferous tubules (e-h).There is expansion of interstitial spaces (black arrow) in CUS-induced rats (f-g) while less to no space visible in control rat's testes.There is obvious present of primary spermatids (black arrow) (c) and elongated spermatids (black arrow) (d) while less primary spermatids and absent of elongated spermatids demonstrated in CUS-induced group (g-h).
immobility time in FST indicates that the rodents demonstrate the core symptom of chronic stress.Based on these data, suggesting that the CUS paradigm model in our present study successfully induced chronic stress in the experimental group (He et al., 2020).
Generally, chronic stress may reduce male sexual activity, and the effect of prolonged stress may cause significant changes in locomotor ability (Mancini et al., 2021).In a previous study, 14 and 28 days of chronic mild stress (CMS) significantly reduced the latency to intromission, ejaculation, and frequency of mounting (Grønli et al., 2005).However, our findings appear to contradict with the data since there were no significant differences observed in the sexual activities of rats from the CUS-induced or control groups.Based on these data, it is possible that the rats from the CUS-induced group could require longer exposure time with female rats to experience first mounting, first intromission and first ejaculation.Nevertheless.the variability in the duration of each parameter was high among the CUS-induced male rats and this could be due to the difference in the adaptation of male to female rats and the reception pattern of the female to male rats.Hence, it is possible that the effect of CUS on sexual behaviour may be more prominent if the experimental rats were exposed for longer than 28 days to CUS.The effect of longer duration of CUS was observed in a previous study where the rats in the experimental group were exposed to CUS for 35 days and demonstrated significantly lower sexual activity compared to normal rats (Grønli et al., 2005).
To further determine the effect of CUS exposure on reproductive dysfunction, gene expression study measuring stress and reproductive hypothalamic neuropeptides was performed, followed by measuring the stress and reproductive plasma circulatory hormones levels.Neuropeptides can act as neurotransmitters by directly regulating signals between nerve cells, and modulate existing neurotransmission by acting as autocrine and paracrine regulators within a close cellular environment, and as hormones in long-range signalling (Burbach, 2011).Therefore, neuropeptides can affect various physiological processes, for instance, the reproduction and stress response (Zup et al., 2022).Neuropeptides such as CRH, pituitary adenylyl cyclase-activating polypeptide (PACAP) and orexin have been associated with stress response and involved in the pathophysiology of psychiatric disorders including chronic stress, depression, and anxiety (Kormos and Gaszner, 2013;Jiang et al., 2023;Grafe and Bhatnagar, 2018).In this present study, CRH expression was measured due to its pivotal function in integrating neuroendocrine and behavioural responses to stress (Sukhareva, 2021).
In our study, continuous and longer duration of stress events were inflicted on the male rats to stimulate the CRH expression and cortisol level in HPA axis.Thus, the expression of the CRH gene has been found to be highly expressed in the brain, promoting the release of peptides from the hypothalamus to the pituitary to stimulate the release of adrenocorticotropic hormone (ACTH) (Zhou et al., 2022).The ACTH, in turn, will be released from pituitary and stimulate the adrenal cortex of the kidney to release cortisol in the event of stress (Chai et al., 2022).The increase of cortisol level in stress response was demonstrated our current finding where the cortisol concentration was significantly increased in the CUS-induced group (Fig. 3).Subsequently, the level of stress hormones will be restored to normal once the stressors are undetected in the central nervous system (Zhou et al., 2022;Chai et al., 2022).Interestingly, in our study, the expression of the CRH gene and cortisol concentration remained high in the CUS-induced rats even after two weeks of CUS and behavioural test were completed.These findings could prove that the CUS model efficiently induced chronic stress and showcased depressive-like behaviour in the male rats.Our findings aligned with previous studies that reported exposure to CUS increased the density of CRH neurons in the paraventricular nucleus (PVN) of CUSinduced rats model (Wang et al., 2010;Gao et al., 2017).
Neuropeptides exhibit pleiotropic effects on physiology, development, reproduction, and behaviour, functioning both as hormones and neuromodulators within the nervous system (Souza-Moreira et al., 2011;Friedrich and Stengel, 2023).In this study, the effect of CUS on the reproductive system was evaluated by identifying the effect on reproductive neuropeptides PNX, kisspeptin, spexin, GnIH and GnRH expressions.Our findings revealed that CUS significantly reduced PNX expression in male rats.PNX neuropeptide is acknowledged as a pivotal regulator of the reproductive system by promoting gonadotropinreleasing hormone (GnRH) secretion and/or directly stimulating luteinising hormone (LH) and follicle-stimulating hormone (FSH) release from the pituitary gland (Liang et al., 2022b).Additionally, PNX was identified to be associated with stress response, where the reduction of PNX expression was found in restraint stress involving male rats (Schalla et al., 2020).This corresponds with our present study, where PNX expression was reduced in the CUS-induced rats.The similar findings was demonstrated in another study where PNX was reduced in response to metabolic stress of anorexia patients with high cortisol levels (Pałasz et al., 2021).Our present data also consistent with a study involving an unexpected noise disturbance stress, a recent report examined how phoenixin treatment affects the excitability of NTS (Nucleus Tractus Solitarius) neurons and unexpectedly found that stress greatly influences these effects (Grover et al., 2020).Initially, they observed that phoenixin treatment caused the neurons to increase or decrease their spike frequency (Grover et al., 2020).However, this effect disappeared when construction noise began in their animal care facility suggesting that stress altering phoenixin's effects on NTS neurons (Grover et al., 2020).These studies further support the role of PNX in the stress response and its importance in regulating reproductive system.Despite of all the findings, there is no study have been done to identify the exact mechanism on how stress affecting the phoenixin signalling.More study needs to be accomplished as phoenixin has pleiotropic effects hence it may work synergistically with other molecules in central nervous system in stress response.Interestingly, PNX is closely associated with the regulation of kisspeptin and GnRH in hypothalamus where is has stimulatory effect on both gene expressions and secretion (McIlwraith and Belsham, 2018).
In hypothalamus, kisspeptin is a positive regulator for GnRH secretion and responsible for regulation of reproductive function (Novaira et al., 2009).Recent research found that PNX can stimulate both GnRH and kisspeptin expression via two distinct pathways: the cAMP/protein kinase A pathway and the C/EBP-β pathway (Treen et al., 2016).However, our present study demonstrated no significant changes in the kisspeptin expression in both CUS-induced and the non-stress control groups.While the GnRH significantly increased in the CUS-induced group.Our findings are rather contradictory to most studies related to the interlinking of these neuropeptides.For instance, a study demonstrated that PNX treatment stimulates the kisspeptin expression in immortalised cells (Treen et al., 2016).Hence, this suggests level of PNX in chronic stress may not affect the kisspeptin and GnRH signalling, in fact, it may reduce the pituitary responsiveness towards hypothalamic GnRH by altering the GnRH receptor expression in the pituitary gland.This inference is supported by a previous study where the finding demonstrated that phoenixin regulates gonadotropins released from the pituitary by enhancing the expression of GnRH receptor (Liang et al., 2022a;Yosten et al., 2013).The previous study also suggested that in the absence of GnRH, phoenixin signalling leads to an increase in GnRH receptor expression in the pituitary (Yosten et al., 2013).
The reproductive function inhibitor genes, spexin and GnIH expression demonstrated no significant changes in CUS-induced and non-stress control groups.Spexin expression in our study aligned with the previous study which reported no changes in spexin expression in the hypothalamus of the CUS-induced male mice and the control group (Zhuang et al., 2020).In contrast, additional study by Kirby et al and Clark et al demonstrated high expression of GnIH in chronic stress-induced infertility in female rats (Kirby et al., 2009;Clarke et al., 2016).These suggest that the GnIH and spexin were not affected by the high CRH expression in the CUS-induced rats.Our present findings suggest that in prolonged chronic stress, the reproductive system inhibitors, Spexin and GnIH might not be responsible in regulating the HPG axis; and PNX expression might not be involved in regulating the HPG axis inhibitors.
Although the GnRH is significantly high in our study, the plasma LH level was significantly low in CUS-induced group which indicates that the GnRH did not stimulate the LH in regular physiological level.The present finding supported the inference that high cortisol levels in CUSinduced rats might reduce the responsiveness of the pituitary to hypothalamic GnRH and, therefore, affect the secretion of LH (Karsch and Breen, 2009).Besides, the significantly high of GnRH expression in the hypothalamus in chronic stress may affect the pulsatility of GnRH release tom the pituitary causing low plasma LH concentration.Previous studies demonstrated continuous administration of GnRH stimulator decreased the gonadotropin and sex hormone due to the phenomenon of "desensitization" of GnRH pulsatile released whereas pulsatile administration of GnRH/GnRH agonists improve the gonadotropins released (Kaiser et al., 1997;Belchetz et al., 1978).
The significant elevation of cortisol was demonstrated in the circulating testosterone level, where testosterone concentration is significantly low in the CUS-induced group.Testosterone is mainly produced by Leydig cells in the testis, and it was demonstrated that high cortisol inhibits testosterone production by acting directly on the Leydig cells (Bambino and Hsueh, 1981).Therefore, our findings suggest that cortisol is essential in regulating the reproductive system, which provides insight that stress response and reproductive system might interact via cortisol.Additionally, our results also suggested that high cortisol levels negatively affected PNX expression.Although there is no direct evidence of how cortisol inhibits PNX, it was demonstrated that in chronic hypocaloric stress, cortisol level was elevated significantly and circulating PNX level was significantly decreased (Friedrich et al., 2020).This strengthens the suggestion that cortisol levels might be involved in regulating PNX or vice versa.
Impaired male reproductive health often identified using the quality of sperm, in which seminal fluid is used to measure whole aspects including sperm count, motility and morphology.Numerous studies have shown that various type of stress could lower sperm count and decreased sperm motility, nevertheless the mechanism on how stress affects the quality of seminal fluid is not fully understood (Jurewicz et al., 2010;Ilacqua et al., 2018).Hence in our present study, the effect of CUS on seminal fluid quality was determined by the sperm count, where it revealed no significant difference in total sperm count.However, there is a significant difference in the percentage of healthy and abnormal sperm count in both control and CUS-induced groups.These findings indicate that spermatogenesis still occurred; however, there might be impairment in its process as represented by the high abnormal sperm count (Fig. 9b).The present finding suggests that stress response does not inhibit spermatogenesis; however, affecting the circulating testosterone level; where this hormone is needed to support the completion of meiosis and the elongation of primary spermatids to Sertoli cells (Franchimont, 1983).To support the inference, the testes morphology was observed, and the CUS-indued group demonstrated fewer primary spermatids and elongated spermatids than the control group (Fig. 11).The impaired spermatids detected in the seminiferous tubules of CUS-induced rats in this study are in agreement with the previous study where the CUS significantly induced cell cycle arrest of spermatogonia and showed a considerably high number of apoptotic spermatids in the seminiferous tubules of CUS-induced rats (Zou et al., 2019).The testes morphology of CUS-induced rats revealed elongated and increased interstitial space between the seminiferous tubules, contradicting the typical structure of healthy testes.This condition diminished the discrete layer of germ cells that are used to aid the development of healthy sperms, thereby influencing the pathological processes occurring in this organ.This condition was demonstrated in the previous study where the discrete layer of germ cells are disrupted hence affecting the testicular morphology and the sperms quality produced in the mice (Souza et al., 2021).

Conclusion
In conclusion, our findings demonstrated that CUS influenced reproductive function by modulating PNX and GnRH expression.The stress response increases cortisol concentration, affecting the circulating plasma LH and testosterone concentration.Furthermore, CUS can impact sperm and testicular morphology, emphasizing its significant role in compromising reproductive health.The morphological findings in this study suggest that CUS affects not only the number of abnormal sperms produced but also the testes architecture of the male rats.This is aligned with our low testosterone concentration finding in the CUSinduced rats, as testosterone is primarily known to be involved in the development of male reproductive tissues including the testes and prostate.The present study is the first to uncover the impact of CUS on various levels of the reproductive system, elucidating the relationship between stress-related molecules and the effect on testes.

Fig. 1 .
Fig. 1.The abnormal and normal rats sperm morphology.a) normal sperm b) bent tail c) double head d) bent neck e) flat head f) detached head g) detached tail h) coiled tail.

Fig. 2 .
Fig. 2. Sucrose preference test was done to identify the anhedonia effect on the CUS-induced rats.The drinking water and sucrose consumption presented in percentage.

Fig. 4 .
Fig. 4. The duration and frequency for sexual behavioural test.a) There is no significant difference for latency to first mounting, first intromission and first ejaculation for both control and CUS-induced rats.b) No significant difference in the mounting frequency for both non-stress control and CUS-induced groups.

Fig. 5 .
Fig. 5.The relative quantification of CRH in the male rat's hypothalamus.The CRH expression increased significantly by more than half fold from the control group (*p < 0.05).The expression values were normalised against the housekeeping gene, GAPDH.

Fig. 8 .
Fig. 8.The plasma circulating hormones for the reproductive axis were measured by ELISA.Both plasma LH (a) and testosterone (b) concentration demonstrated a significant decrease in CUS-induced rats (*p-value<0.05).

Fig. 9 .
Fig. 9.The total of normal and abnormal sperm counts in both control and CUS-induced male rats.a) No changes demonstrated in the total sperm count in both groups b) The abnormal sperm was demonstrated higher in CUS-induced rats and the normal was demonstrated lower by 7840 sperms/mL (*p-value<0.05).

Fig. 10 .
Fig. 10.Microphotographs illustrating morphologically normal sperm and various sperm impairments.a) The overview of CUS-induced rats sperm sample, multiple defects can be seen under 10× magnification b) Detached head (black arrow) bent neck (red arrow) c) Flat head d) Double heads e) Bent tail f) Normal sperm (H&E staining; b-f: 40× magnification).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 11 .
Fig. 11.Morphological evaluation of H&E staining showing testes of the non-stress control group (a-d) and CUS-induced group (e-h).Non-stress control group showed round morphology of seminiferous tubule (a-b) while CUS-induced rats showed elongated and enlarged seminiferous tubules (e-h).There is expansion of interstitial spaces (black arrow) in CUS-induced rats (f-g) while less to no space visible in control rat's testes.There is obvious present of primary spermatids (black arrow) (c) and elongated spermatids (black arrow) (d) while less primary spermatids and absent of elongated spermatids demonstrated in CUS-induced group (g-h).

Table 1
Schedule of CUS stressors.Stressors were randomly shuffled for the three subsequent weeks.

Table 2
List of primers and genes.