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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.34 No.1 pp.40-49

The Chronic and Unpredictable Stress Suppressed Kisspeptin Expression during Ovarian Cycle in Mice

Seung-Joon Kim*
College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
Correspondence Seung-Joon Kim College of Veterinary Medicine, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 41566, Korea Tel: +82-53-950-5971 Fax: +82-53-950-5994 E-mail:
25/01/2019 29/01/2019 29/01/2019


Chronic and unpredictable stress can disrupt the female reproductive system by suppression for secretion of gonadotrophin-releasing hormone (GnRH) and gonadotrophin, resulted in ovarian malfunction and infertility. In the recent days, kisspeptin has been highly highlighted as a hypothalamic peptide which directly stimulates synthesis and release for GnRH. However, in spite of the key role of kisspeptin in the female reproductive system, little information is still available on the changes of its expression during ovarian cycle under stressed condition. Therefore, we induced chronic and unpredictable stress series to the female mice to analyze kisspeptin expression in the brain and ovary. Stressed mice exhibited changes of behavior and body weight gain during the stress assessment, which suggested that the present stress model in mice was successfully established. In the brain level, kisspeptin expression was attenuated than control. In the ovary level, the stressed mice displayed irregularly shrunk oocytes with broken zona pellucida throughout the follicle stages, pyknotic granulosa cells, decreased number of developing follicles and increased number of atretic follicles than the control. In case of kisspeptin expression in the whole ovary tissue, the expression level was decreased in the stressed mice. In detail, the less intensity of kisspeptin expression in the antral follicles phase was observed in the stressed mice than control mice, indicating that local function of kisspeptin during ovary cycle is highly associated with development of ovarian follicles. We expect that the present study has important implications for the fields of reproductive biology.



    Stress is defined as a perceived threat by internal or external adverse events (stressors) to the homeostasis, and can take place to several neuropsychiatric disorders (e.g. anxiety, malnutrition and depression) and immune dysfunction (McEwen, 2004;Glaser and Kiecolt-Glaser, 2005). Of particular, a number of recent studies have shown that chronic stress disrupts the female reproductive system, ovarian function (ovarian cycle) and fertility, which are dependent on proper feedback of gonadotrophin synthesis and secretion. In the normal condition, reproduction is controlled by the brain where gonadotrophin-releasing hormone (GnRH) is secreted from the hypothalamus directly into the pituitary portal system and is transported to the anterior pituitary gland. Then GnRH stimulates the synthesis and secretion of the gonadotrophins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH). An appropriate pulsatile secretions of LH and FSH make sure of normal ovarian cycle with stimulations of the granulosa cells and theca cells during the follicular and luteal phase, and a surge secretion of GnRH at the end of the follicular phase induces LH surge and ovulation (Ralph et al., 2016). These mechanisms are called as hypothalamic-pituitary-gonadal axis (HPG axis). In contrast with aforementioned normal reproductive condition, glucocorticoid is released in response to stress from the adrenal gland and works in suppression of the HPG axis (Rivier and Rivest, 1991;Chrousos et al., 1998;Breen et al., 2007). In detail, stressors in animals inhibited GnRH secretion in the hypothalamus and diminished responsiveness to GnRH in the pituitary gland, followed by distruption of frequency of luteinizing hormone (LH) pulses; in addition, stressors could suppress LH surge, resulted in blocking or delaying ovulation (Battaglia et al., 1997;Dobson et al., 1999;Breen et al., 2007;Wagenmaker et al., 2009).

    In the recent days, the expression of kisspeptin has been identified in the brain, placenta, testis, ovary, pancreas and small intestine of animals and humans (Clarke et al., 2015;Cui et al., 2015). Especially, it has been well addressed that kisspeptin plays an important role in regulation of the onset of puberty, secretion of gonadotrophins and the control of fertility. Kisspeptin in the brain has been widely investigated as a hypothalamic peptide which directly stimulates GnRH synthesis and release (Roa et al., 2011;Terasaka et al., 2013). In the hypothalamus, kisspeptin is expressed in several regions with respect to the arcuate nucleus (ARC), anteroventral periventricular nucleus (AVPV), preoptic area (POA) and periventricular nucleus (PeN), and its neurons placed in close with GnRH neurons (d’Anglemont and Colledge, 2010;Clarkson and Herbison, 2006). The GnRH neurons which express kisspeptin receptor are activated by kisspeptin release from kisspeptin neurons, leading to GnRH release (Roa et al., 2011;Terasaka et al., 2013;Cui et al., 2015). The role of kisspeptin in the reproductive system could be identified when central and peripheral administrations of kisspeptin led to an elevation of circulating LH levels (Gottsch et al., 2004;Thompson et al., 2004).

    There have been several researches to uncover the effect of stress to the reproductive system. The administration of glucocorticoid inhibited LH surge and ovulation in the pigs and rats (Baldwin, 1979;Turner et al., 1999). In addition, cortisol exhibited disruptive effects on preovulatory events in ewes (Breen et al., 2005). Furthermore, it was addressed that chronic and unpredictable stress suppressed reproductive function by inhibition of GnRH release, resulting that circulating level of gonadotrophin (FSH and LH) was declined (Choi et al., 2014). However, in spite of evidence that stress interferes with ovarian cycle in females, and changes GnRH and gonadotrophin (FSH and LH) secretion, little information is available on the changes of kisspeptin expression, the regulator of GnRH secretion, in the brain and ovary of stressed-female animal; in case of male, kisspeptin-mediated suppression in the reproductive system was demonstrated in stressinduced mice, with showing decrease of kisspeptin expression in the brain and testicular degeneration in the gonad (Hirano et al., 2014). Therefore, the purpose of the present study was to investigate the effect of chronic and unpredictable stress in the ovarian cycle, and kisspeptin expression in the ovary in the stress-induced female mice. We conducted the immunochemical analyses focusing on the expression of kisspeptin in the brain and ovary to understand the comprehensive effect of stress induction.


    Ethics, animals and chemicals

    All procedures, including induction of stress, animal care and animal sacrifice, were conducted in accordance with Kyungpook National University Guide for the Care and Use of Laboratory Animals. C57BL/6 mice, 7 week old females with 16-18 g body weight, were employed (Daehan Biolink, Cheongju, Korea) in this study. All chemicals and reagents were purchased from Sigma-Aldrich (MO, USA) and Thermo Scientific (IL, USA), unless otherwise specified.

    Stress-induced mice model

    The mice were housed in a room maintained for temperature at 23 ± 2ºC, humidity at 50-80% and approximately 12 h light/dark cycle. The standard feed (Jeil Feed Co., Ltd., Daejeon, Korea) and municipal water irradiated by ultraviolet light were provided to the animals. The mice were divided as two groups, non-stressed mice (control mice, n = 5) and stress-induced mice (stressed mice, n = 10). In case of stressed mice, we made them exposed to chronic and unpredictable stress to eliminate unwanted tolerance because tolerance of stressors into mice could elevate when they were repeatedly exposed to the predictable and same stressors (Choi et al., 2014). The protocol for stress induction was given to the mice in accordance with the previous study (Choi et al., 2014); in brief, the different stressors in terms of damp bedding for 12-14 h, 45ºC cage tilting for 14-18 h, continuous light on for overnight, water and food deprivation for 24 h, strong shaking for 10 min, confinement in a tube for 2 h, no bedding for overnight, cold water (8-12ºC) swimming for 3 min, cold environment (4-5ºC) for 1 h, lights off for 3 h during day time, social isolation (1/cage) for 24 h and hot environment (45ºC) for 5 min were randomly given to mice twice daily for 35 days. In contrast, control mice were kept in the same environment without stress induction during the experimental period.

    Stress assessment by changes of behavior and body weight

    The behavioral changes of stressed mice were compared with control mice by open-field test and marble-burying behavior test and tail suspension test, in accordance with the previous study (Choi et al., 2014). Briefly, in the openfield test, the mice were individually placed in a bright square box (26 cm × 26 cm, 250 lux) and monitored for the total movements, spending time in center, moving distance in center, number of entries into the center for 30 min using the TruScan Photo Beam Activity System (Coulbourn Instruments, PA,USA). For the marble-burying behavior test, 20 pieces of clean and light marbles (diameter: 1 cm) were equally spaced (each distance: 3-5 cm) on 5 cm deep sawdust in the plastic cages (26 cm × 20 cm), thereafter, the mice were put into cages without water and food. Then the number of marbles buried as more than two-thirds during 30 min was counted. The body weight (g) of all mice was measured every 5 days from Day 0 until Day 35.

    Tissue preparation

    Under ether anesthesia, the control and stressed mice were sacrificed by exsanguination at the 35th day of stress process. For the immunohistochemistry (IHC), the euthanized mice were subjected to cardiac perfusion fixation with flushing by a 4% paraformaldehyde through heart. The brains and ovaries were rapidly dissected, fixed in 10% neutral buffered formalin at room temperature (RT) for 48 h, dehydrated and embedded in paraffin. The paraffin-embedded tissues were cut into 5 mm thick in the ovary and 7 mm thick in the brain using a microtome (Leica Microsystems, Germany), and stained with hematoxylin and eosin (H&E) or used for immunohistochemical (IHC) analysis. In case of western blotting preparation, the brains and ovaries were collected, appropriately trimmed, snap-frozen with nitro nitrogen (LN2) and stored in deep freezer (-80ºC).

    Classification of ovarian follicles

    The H&E-stained ovarian follicles in the control and stressed mice were classified and counted (%) in accordance with the previous article with respect to primary follicles (a single layer of cuboidal granulosa cells), secondary follicles (surrounding of more than one layer of cuboidal granulosa cells without visible antrum), early antral follicles (multiple layer of granulosa cells and 1-2 small spaces of antrum), preovulatory follicles (the largest follicle with cumulus granulosa cell layer surrounding the oocyte), atretic follicles (more than 5% of pyknotic cells with showing oocyte shrinkage and breakdown of germinal vesicle) and corpus lutea (Myers et al., 2004).


    The slide sections of the brain and ovary from the control and stressed mice were deparaffinized, washed with distilled water (DW) twice for 5 min, treated with 0.01 M citrate buffer (pH 6.0), boiled in a microwave for 5 min and cooled at RT for 5 min. Then the slides were treated with 0.3% hydrogen peroxide in methyl alcohol for 20 min, washed with phosphate buffered saline with triton- X (PBS-T; pH 7.4), treated with serum blocking solution (Histostain-plus kit, Invitrogen, CA, USA) for 45 min and incubated with rabbit polyclonal anti-kisspeptin antibody (dilution: 1:200; Millipore, MA, USA) as primary antibody for overnight at 4ºC. Thereafter, the slides were incubated with biotinylated secondary antibody (Histostain-plus kit) for 45 min, incubated for 45 min with streptavidinperoxidase conjugate (Histostain-plus kit), reacted with a 3,3’-diaminobenzidine (DAB) kit (Vector Laboratories, CA, USA) and counterstained with H&E.

    Western blotting

    The snap-frozen tissues of the brains and ovaries from control and stressed mice were homogenized, lysed with a radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 14,000 rpm for 5 min at 4ºC. The supernatants were collected and quantified for the total amount of protein using a Bicinchoninic Acid Protein Assay Reagent Kit, in accordance with the manufacturer’s instruction. Equal amounts of protein (brains: 14 mg; ovaries: 7 mg) were separated by NuPAGE® 4-12% Bis-Tris Protein Gel via electrophoresis and transferred onto nitrocellulose membranes (Whatman GmbH, Germany). Then, the membranes were blocked through incubation with 3% skim milk in tris-buffered saline (TBS) for 3 h at 4ºC and incubated with rabbit polyclonal anti-kisspeptin antibody (dilution: 1:500; Abcam, UK) or anti-beta-actin (ACTB, dilution: 1:500; Abcam) for overnight at 4ºC. Thereafter, the membranes were washed with TBS containing 0.1% Tween-20 for 15 min, incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (dilution 1:10000; Santa Cruz biotechnology, CA, USA) for 2 h and developed using an enhanced chemiluminescence (ECL) kit (GE healthcare, IL, USA). Image J software (National Institutes of Health, USA) was used to quantitatively analyze the intensity of the western blotting bands and expression of kisspeptin was relatively normalized against the expression of beta-actin.

    Statistical analysis

    The Mann-Whitney U-test was conducted using PASW 18 (SPSS, IL, USA) to analyze significant differences between control and stressed mice groups. Data were presented as the mean ± standard errors (SEM). A p value of <0.05 was considered to be statistically significant.


    Stress assessment by changes of behavior and body weight

    Since some of stressed mice were died during stress induction, further investigations were performed using remaining animals (n = 6). Both open-field test and marble- burying behavior test have been employed to assess anxiety and stress in rodents; stressed mice might present preference for staying in the edge of the field once placed in strange condition or bury more light-reflecting marbles during open-field test or marble-burying test, respectively (Deacon, 2006;Choi et al., 2014). In the present study, stressed mice significantly (p < 0.05) less moved toward the center area (Fig. 1A), spent shorter duration in the center area (Fig. 1B) and induced increased marble-burying behavior (Fig. 1D) in comparison with control mice. In addition, chronic and unpredictable stress affected on the body weight gain from Day 30 (Fig. 1E). These results indicated that the present protocols for induction chronic and unpredictable stress into mice were sufficient to change their physiology.

    Ovarian follicular morphology

    Whereas the control mice exhibited normal ovarian cycles with intact granulosa cells and oocytes in the secondary, antral and preovulatory follicles (Fig. 2A, 2C and 2E), the stressed mice demonstrated irregularly shaped and shrunk oocytes with broken zona pellucida throughout the follicle stages (white arrows in Fig 2B, 2D and 2F) as well as pyknotic granulosa cells (Fig. 2D and 2F). The zona pellucida remnants (ZPRs) which represent endstage atretic follicles were abundantly observed in the ovaries of stressed mice (arrowheads in Fig. 2H), indicating more increased atretic follicles in stressed mice in comparison with control mice. When the ovarian follicles were quantitatively classified, the rate of secondary and antral follicles, and corpus luteum in stressed mice significantly (p < 0.05) decreased but atretic follicles from them significantly (p < 0.05) increased in comparison with the control mice (Fig. 2I). These results indicated that chronic and unpredictable stress altered the ovarian follicular development.

    Immunohistochemistry and western blotting for kisspeptin expression in the brain

    The expressions of kisspeptin in the brain in the control and stressed mice were analyzed (Fig. 3). It has been well addressed that kisspeptin neurons in rodents places in various parts of the hypothalamus including ARC and AVPV, and kisspeptin neurons in the AVPV formed the major afferent neurons to GnRH neurons (Clarkson and Herbison, 2006; d’Anglemont and Colledge, 2010). Therefore, we focused on the expression of kisspeptin in AVPV in the control and stressed mice. A number of kisspeptinpositive cells were extensively observed in AVPV of the third ventricle (3V) in the control mice (white arrows in Fig. 3A), however, the abundance was apparently decreased in the stressed mice (Fig. 3B). In addition, the results by western blotting confirmed the significant (p < 0.05) down-regulation of kisspeptin in the hypothalamus of the stressed mice than the control mice (Fig. 3C and 3D).

    Immunohistochemistry and western blotting for kisspeptin expression in the ovary

    We further investigated kisspeptin expression in the ovaries of the control and stressed mice (Fig. 4); the expression of kisspeptin is associated with direct control of ovarian function in terms of follicular development, oocyte maturation, steroidogenesis and ovulation, in addition, dysregulation of kisspeptin in the ovary resulted in ovarian malfunction, followed by reproductive pathology and female infertility (Hu et al., 2018). Although both ovaries of the control and stressed mice expressed kisspeptin on the whole, the patterns were evidently different depending on stress induction; the number of kisspeptinpositive follicles was more prevalent in the ovary of control mice than stressed ones (Fig. 4A and 4B). The western blotting for investigation of the overall expression of kisspeptin in the ovary demonstrated significant (p < 0.05) decrease in kisspeptin expression in the stressed mice than the control ones (Fig. 4G and 4H). In detail, both groups exhibited little expression of kisspeptin (white arrow in Fig. 4C and 4D) in case of the secondary follicle phase. However, the expression level of kisspeptin in the antral follicle phase was became stronger in the granulosa cells in the antrum; however, the expression intensity in the stressed mice was generally weaker than the control mice (arrowheads in Fig. 4E and 4F). Collectively, these results suggested that chronic and unpredictable stress in the mice took place to less expression of kisspeptin in the granulosa cell in developing antrum, resulted in the hypocyclic ovary showing decreased number of secondary and antral follicles, and increased population of atretic follicles.


    The series of stressors in the present study mimics the chronic and unpredictable stress of actual life events. It is well known that chronic stress impairs female reproduction by suppressing HPG axis (Vázquez, 1998). Likewise, the stressed mice in the present study exhibited attenuated expression of kisspeptin in AVPV region of the brain and in the ovary, especially antral follicle phase. It was found that kisspeptin expressed in the ovary but its local function and mechanism were not well understanding yet (Clarke et al., 2015;Cui et al., 2015;Hu et al., 2018). Therefore, our results may help to identify this mechanism and relation between stress and kisspeptin expression during ovarian cycles.

    The series of stressors in the present study is considered to be enough to induce behavioral changes as well as physiological alteration. In accordance with previous article, the activity of mice placed in the open-field is regarded as an indicator of its psychological state; animals stayed less in center and decrease of exploratory action during the open-field test indicate the anxiety-like activity and a loss of interest in novel (Castellano et al., 2006;Oakley et al., 2009). In addition, the marble-burying test is applied to test anxiety-related behaviors like compulsive and repetitive disorder (Deacon, 2006;Choi et al., 2014). Furthermore, attenuated body weight gain is also a key indicator for stress (Krajewski et al., 2005;Choi et al., 2014). The results from open-field, marble-burying test and body weight gain in the present study suggested that the stress model in mice was successfully established, with presenting significant (p < 0.05) behavioral disorder and attenuation of body weight gain during stress induction (Fig. 1).

    Several articles have revealed that impairment of reproductive function by various stressors with respect to psychological, metabolic and inflammatory stress is associated with suppression of gonadotrophin secretion in the brain level, due to enhanced presence of glucocorticoids derived from the adrenal cortex (Choi et al., 2014). Experimentally, when cortisol was applied to ovariectomized ewes, the pulsatile LH release was suppressed due to reduction of the sensitivity of the anterior pituitary gland to GnRH (Breen and Karsch, 2006a;Breen and Karsch, 2006b). In case of psychological stress, the series of stress consisting of social isolation, restraint, blindfolding and exposure to predatory cues could also induce a reliable elevation of plasma cortisol, followed by inhibition of LH secretion in ovariectomized ewes (Breen et al., 2007). In addition, the fasting, as one of stressor, presented decreased GnRH level in the brain, and reduced LH and FSH concentration in the plasma (Thomas et al., 1990;Luo et al., 2016).

    Moreover, the stressors induced the suppression in not only expression of GnRH, FSH and LH but also kisspeptin secretion, the upstream regulator of gonadotrophins; because kisspeptin is an important gatekeeper of reproductive control for the HPG axis by activating GnRH, suppression of kisspeptin can result in deterioration of the reproductive system. Since kisspeptin neurons in the ARC localize together with corticotrophin-releasing hormone receptor, the stress hormone is directly capable of disturbing the function of kisspeptin neurons (Takumi et al., 2012). For instance, kisspeptin expression was reduced in response to restraint, hypoglycaemia and lipopolysaccharide (LPS) (Kinsey-Jones et al., 2009). In the present study, kisspeptin expression in AVPV was suppressed by chronic and unpredictable stress, which could affect to HPG axis (Fig. 3). In the very recent article, the mechanism of stress-induced infertility was well reviewed (Iwasa et al., 2018). Under stress condition, several events concurrently occur in terms of decrease of kisspeptin expression, increase of gonadotrophin-inhibitory hormone (GnIH), activation of hypothalamic-pituitary-adrenal axis (HPA axis) and elevation of cytokines action. These events generate decrease of GnRH and LH secretion, followed by anovulation, decrease of reproductive behavior and infertility. However, these mechanisms are not sufficient to fully explain how stressors affect ovarian cycle with localization of kisspeptin expression on developing follicles.

    The expression of kisspeptin has been identified in the brain, placenta, testis, ovary, pancreas and small intestine of animals and humans (Clarke et al., 2015;Cui et al., 2015). In the brain, kisspeptin secretion from the hypothalamus stimulates GnRH neurons to release GnRH into the pituitary gland for controlling HPG axis, as the upstream regulator for pulsatile and surge secretion of GnRH and gonadotrophins (Roa et al., 2011;Terasaka et al., 2013;Cui et al., 2015). After first reporting that kisspeptin is highly expressed in the rat ovary, the extrahypothalamic expression of kisspeptin has been recently highlighted to uncover its local function during the reproductive biology (Uenoyama et al., 2016). Kisspeptin knockout mice exhibited small ovarian size, less weight of ovary, absence of the large follicles and increased population of the atretic follicles in comparison with the wildtype counterparts (Lapatto et al., 2007;d’Anglemont et al., 2007;Hu et al., 2018). In addition, whereas kisspeptin infusion in the rats demonstrated lower number of antral follicles but increased population of preovulatory follicles and corpora lutea, administration kisspeptin antagonist produced exactly the contrary effect (Fernandois et al., 2016). Given that chronic and unpredictable stress (e.g. cold stress) in the rodent attenuated the number of secondary and antral follicles, and induced atresia of growing follicles (Dorfman et al., 2003;Wu et al., 2012;Choi et al., 2014), the present findings for normal cyclic ovary in the control group and less cyclic ovary (small population of the large follicles and increased number of the atretic follicles) in the stressed group implied that chronic and unpredictable stress mediated insufficient expression of kisspeptin in the ovary and failure of follicle development (Fig. 2). Furthermore, it was addressed that kisspeptin expression was higher in the granulosa cell than other ovarian cells, was gradually elevated in the developing follicles and was highest in the pre-ovulatory follicle phase (Shahed and Young, 2009;Ricu et al., 2012;Mondal et al., 2015). The control group in the present study displayed high expression of kisspeptin from the antral follicle phase (Fig. 4E). However, the same follicles in the stressed group exhibited weaker expression of kisspeptin than the control mice (Fig. 4F). In addition, overall expression of kisspeptin in ovarian tissue of stressed group demonstrated significant (p < 0.05) decrease than the control one (Fig. 4G and 4H). Collectively, these results suggested that local function of kisspeptin during ovarian cycle was associated with initial follicle recruitment as well as maintenance of developing follicle; stress-induced kisspeptin suppression could inhibit the local function of kisspeptin in the ovary.

    Because reproduction system in animal is a highly orchestrated and is precisely controlled by the HPG axis, comprehensive and multifactorial studies are required. Especially, in the recent days, stress-mediated infertility has become an issue in humans as well as animals such as the densely populated livestock and isolated animal in the zoo. Here, we demonstrated the chronic and unpredictable stress-induced suppression of kisspeptin expression in the ovary. The local function of kisspeptin expression in the present study may have important implications for the fields of reproductive biology, livestock industry and animal welfare.


    No potential conflict of interest relevant to this article was reported.



    Stress assessment by changes of behavior and body weight. The behavioral changes of chronic and unpredictable stress in mice by open-field test (A-C) and marble-burying test (D). The effect of chronic and unpredictable stress on the body weight gain from Day 0 until Day 35 (E). Graphs were presented as mean ± SEM. *p < 0.05 indicated a significant difference between the control and stressed mice.


    Ovarian follicular morphology in the control and stressed mice. The representative images of H&E staining of follicles in terms of primary follicles, secondary follicles, early antral follicles, preovulatory follicles and atretic follicles in control and stressed mice (A-H). White arrows or arrowheads indicated irregularly shaped and shrunk oocytes with broken zona pellucida or zona pellucida remnants (ZPRs) in the ovary of stressed mice, respectively. Quantitative classification of the ratio (%) for each type of follicles in the control and stressed mice (I). Graphs were presented as mean ± SEM. *p < 0.05 indicated a significant differe nce between the control and stressed mice. Magnifications: × 200 in A-H and × 100 in G and H.


    The effect of chronic and unpredictable stress on the kisspeptin expression in anteroventral periventricular nucleus (AVPV) of hypothalamus. Representative images of immunohistochemistry for kisspeptin expression in AVPV in control and stressed mice (A and B). Representative images of the western blotting for kisspeptin and beta-actin (ACTB) expression of control and stressed mice (C and D). Graph was presented as mean ± SEM. *p < 0.05 indicated a significant difference between the control and stressed mice. 3V, the third ventricle. Magnifications: × 100.


    The effect of chronic and unpredictable stress on the kisspeptin expression in the ovary. Representative images of immunohistochemistry for kisspeptin expression in ovaries in control and stressed mice (A-F). White arrows or arrowheads indicated the secondary follicle or kisspeptin expression in the granulosa cells in the antrum. Representative images of the western blotting for kisspeptin and ACTB expression in ovaries of control and stressed mice. Graph was presented as mean ± SEM. * p < 0.05 indicated a significant difference between the control and stressed mice. Magnifications: × 40 in A-B and × 200 in C-F.



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