Korean Journal of
Veterinary Service

Heat stress is a physiological condition to maintain the homeostasis of organism and occurs when the combined effects of external environmental heat and internal metabolic heat production exceed an animal’s capacity for heat dissipation (Hale et al., 2017; Dickson et al., 2018). In stressful conditions including heat stress, the normal functioning of the female reproductive system, which is dominantly regulated by the hypothalamic-pituitary-gonadal (HPG) axis, is disrupted by stress hormones secreted from the hypothalamic-pituitary-adrenal (HPA) axis, such as corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and glucocorticoids; these stress hormones exert negative feedback on the reproductive hormones within the HPG axis, leading to a decrease in the synthesis and release of gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone, thereby impairing ovarian function (Liang et al., 2013; Kim et al., 2023).

Pigs are intrinsically susceptible to heat stress caused by their lack of functional sweat glands, thick subcutaneous adipose tissue, and increased metabolic heat production in individuals due to breeding strategy for increased meat production (Dickson et al., 2018; Kim et al., 2023). Of note, the seasonal characteristics of Korea, including high air temperatures approaching more than 35℃ during summer, can be one of the factors that cause heat stress, which can lead to decreased productivity in the Korean pig farms during summer season. This has been commonly known as summer infertility, which is characterized by symptoms including reduced conception rate, spontaneous abortion, decreased litter size, an extended weaning-to-estrous intervals, and a delayed puberty onset (Bidne et al., 2019; Kim et al., 2023).

Granulosa cells are essential for ovarian function in regards to playing a critical role in the ovarian follicle development, steroidogenesis for estradiol and progesterone, and oocyte maturation through their interaction via gap junction (Hale et al., 2017). Therefore, apoptosis of granulosa cells or alteration of normal structure of granulosa cell layer under unexpected stimuli including stressors can also influence the fate of ovarian follicles and its function. Several studies have demonstrated heat-induced atresia of the ovarian follicle and granulosa cell apoptosis. In mice, heat stress increased number of atretic ovarian follicles and apoptosis of granulosa cells with decrease level of estradiol in serum (Li et al., 2016). In addition, elevated cortisol levels with accumulation of reactive oxygen species induced by heat stress could also trigger granulosa cell apoptosis (Chaudhary et al., 2019).

Caspases are a family of intracellular cysteine proteases that play a central role in both the initiation and execution of apoptosis in the cells (Johnson and Bridgham, 2002; Végran et al., 2011). Under non-stressed condition, they present in the cells as the inactive precursor enzyme (zymogen) (Khalil et al., 2014; Kaur and Kurokawa, 2023). When death signals activate the initiator caspases (caspase-2, -8, and -9), they subsequently activate executor caspases (caspase-3, -6, and -7) by proteolysis. Activated executioner caspases cleave majority of the substrates for the induction of apoptosis (Khalil et al., 2012; Khalil et al., 2014). Of note, the zymogen form of caspase-3 exists in the cytoplasm as a 32-kDa protein and generates the p17 and p12 subunits as its active form, upon cleavage during apoptosis (Li et al., 2016). Changes in the expression of caspase-3 were clearly reported in the apoptosis of granulosa cells; when atresia began in antral stage of follicles, the concentration of activated caspase-3 was increased in granulosa cell layer (Johnson et al., 2002).

Although several studies have investigated heat-induced ovarian injury (Li et al., 2016; Hale et al., 2017; Kim et al., 2023), few have focused on the effects of chronic heat stress on ovarian function in swine, particularly in relation to caspase-3. Therefore, this study attempted to examine the alteration of caspase-3 expression within the ovaries of domestic sows in Korea in response to heat stress during summer, for the purpose of understanding the mechanisms of caspase-3 by which summer infertility causes reduced reproductive performance.

Ethics

All sampling procedures for animal specimen were approved by the Institutional Animal Care and Use Committee at Kyungpook National University (approval number: 2021-0098).

Animals and treatment

Approximately 2-year-old three-way crossbred sows (Landrace×Yorkshire×Duroc) in the vicinity of Daegu city, Korea were employed; of note, after screening farms without air conditioning system, sows from those farms were only used in the present study. Ovaries and blood from sows were acquired from a local slaughterhouse approximately 30 minutes away from the laboratory. During sampling, the sows were divided into two experimental groups: a control group (CON, n=7), which consisted of sows exposed to the maximal atmospheric temperatures of the day ranging from 22 to 24.5℃ during the spring as suitable temperature for raising, and a heat-stressed group (HS, n=7), consisting of sows exposed to the maximal atmospheric temperatures of the day ranging from 30.5 to 37℃ during the summer in Korea; only ovaries and blood in the follicular phase (with growing ovarian follicles and regressing corpora lutea) were selected during sampling step in the slaughter house. One ovary was fixed in 4% paraformaldehyde (Duksan Chemical, Korea) for immunohistochemistry (IHC), and the cortex existing developing ovarian follicles was separated from the other ovary and snap-frozen in liquid nitrogen. The blood was allowed to clot and then centrifuged at 2,000×g for 15 min at 4℃. The serum, obtained from the supernatant, was immediately stored at -80℃ until further analysis.

Serum analysis for stress assessment

Serum levels of corticosterone were measured using enzyme-linked immunosorbent assay (ELISA) kits (Cayman Chemical Company, MI, USA). Upon thawing, the serum samples were processed for ELISA in accordance with the manufacturer’s instructions. Samples, enzyme immunoassay buffer, tracer, and antiserum were mixed in the antibody-coated 96-well plate and incubated for 90 min at room temperature (RT). Thereafter, the 96-well plate were reacted with detection reagents for 1 hr in incubator set at 37℃. The plate was then read at 405 nm using a microplate reader (Epoch, Biotek, VT, USA). Corticosterone concentrations in the serum were calculated using a 4-parameter logistic fit with free software (www.myassay.com).

Western blotting to assess the ovarian caspase-3 expression

The snap-frozen cortex from ovaries were homogenized in radioimmunoprecipitation assay buffer supplemented a proteinase inhibitor (Thermo Fisher Scientific, MA, USA) and centrifugated at 14,000×g for 15 min at 4℃ to obtain protein lysates. The supernatants then were gently collected and whole protein concentrations in the supernatant were measured using the Bicinchoninic Acid Assay kit in accordance with manufacturer’s instruction (Thermo Fisher Scientific). Equal amounts of protein (20 μg) from each group were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels. Separated protein transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA), followed by blocking of membrane with 3% bovine serum albumin (BSA) for 1 hr at room temperature (RT). Thereafter, the membranes were incubated with a mouse anti-human caspase-3 primary antibody (MA1-91637, 1:500 dilution with 1% BSA; Thermo Fisher Scientific) or a mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1,000 dilution with 1% BSA; Thermo Fisher Scientific) as a reference, for an overnight. The membranes were then washed with tris buffer saline containing 0.1% tween-20 (TBST), incubated with a horseradish peroxidase conjugated goat anti-mouse IgG (1:3,000 dilution with TBST; Thermo Fisher Scientific) for 1 hr at RT, and developed on X-ray films using an enhanced chemiluminescence kit (Thermo Fisher Scientific). Image J software (National Institutes of Health, USA) was involved for quantification of the band intensities at a 12 kDa in active form of caspase-3 and 35 kDa in GAPDH. The expression level of caspase-3 was relatively normalized against that of GAPDH.

Immunohistochemical staining to detect ovarian caspase-3

All fixed ovaries from each group, retrieved for IHC, were dehydrated, embedded in paraffin, and sectioned to a thickness of 5 µm using a microtome (Leica Microsystems, Germany). The paraffin-embedded sections on slides were deparaffinized through a series of alcohol washes (100% to 70%), exposed to antigen unmasking in 0.1 M citrate buffer (pH 6.0) by heating at 95℃ for 20 min, and then cooled to RT for 40 min. Peroxidase activity was quenched with 3% H₂O₂ for 30 min, followed by blocking with 2% normal horse serum (Vector Laboratories, CA, USA) for 1 hr at RT. The sections were then incubated overnight at 4℃ with a mouse anti-human caspase-3 primary antibody (1:100 dilution in 1% BSA). Subsequently, the slides were incubated with a biotinylated secondary antibody (1:200 dilution in phosphate-buffered saline), treated with an avidin-biotin complex solution (Vector Laboratories) at RT for 60 min, reacted with a 3,3’-diaminobenzidine (DAB) kit for 3 min (Vector Laboratories), and counterstained with hematoxylin solution (Vector Laboratories) for 3 min; the duration of DAB treatment and counterstaining was consistent across all slides. Negative control slides were processed in the same manner but without the primary antibody incubation. After staining, the slides were asessed under a light microscope. During the examination of caspase-3 localization around the ovarian follicles, we separately observed the follicular cells in immature follicles (primary and secondary follicles) and granulosa cell layers in mature follicles (tertiary and pre-ovulatory follicles). DAB-positive cells, indicated by a brownish color, were considered to represent the expression of both pro-caspase-3 and active caspase-3.

Statistical analysis

All experimental assays were performed in triplicate. The raw data were statistically analyzed using Kruskal Wallis test with Bonferroni correction using SPSS 12.0 (SPSS Inc., IL, USA). In addition, Pearson’s correlation analysis was performed between several factors (maximal atmospheric temperature or stress hormone level) and caspase-3 expression level by western blotting. Differences were considered statistically significant at P<0.05.

Stress hormone level of heat-stressed domestic sows

In this study, the maximal atmospheric temperature on the sampling day for HS group was significantly higher (P<0.05) than that for CON group (Fig. 1A), indicating that HS group might be exposed to unavoidable chronic heat stress. Because the heat stress was associated with high atmospheric temperatures during the summer, we compared stress level (corticosterone) of CON and HS groups (Fig. 1B). Although there was no statistical significance (P=0.153) caused by high variations, a tendency towards higher corticosterone levels in HS group was identified, relative to CON group. These findings suggest that heat stress during summer might induce physiological changes in sows.

Figure 1. Characteristics of sows in the CON and HS groups. (A) The maximal atmospheric temperature on the day of sampling for the sow ovaries was significantly (P<0.05) different between groups, as 23.1±0.4℃ in the CON group and 34.4±0.8℃ in the HS group. (B) Corticosterone levels, used as a stress index, did not show a significant difference between the CON and HS groups (P=0.153) due to high variability, but the HS group showed a strongly increased tendency than CON group. *Superscript indicated a significant difference between groups (P<0.05). Graphs were presented as mean±SD in Fig. 1A and mean±SEM in Fig. 1B.
CON, control group sampled during spring; HS, heat-stressed group sampled during summer.

Caspase-3 expression in the ovaries of sows in different seasons

Caspase-3 activation was assessed by detecting cleaved (activated) caspase-3 via western blotting analysis of ovarian extracts (Fig. 2A). We observed a significant upregulation (P<0.05) of cleaved caspase-3 expression in HS group compared to CON group (Fig. 2B), indicating that apoptosis was increased in the ovarian follicles during summer. Furthermore, the expression of cleaved caspase-3 in the ovaries was positively correlated with degree of maximal atmospheric temperature (Fig. 3A; r=0.547, P=0.043) and serum levels of corticosterone (Fig. 3B; r=0.777, P=0.011). These findings suggested that stress induced by high atmospheric temperatures during summer might lead to increased apoptosis in the ovarian follicles.

Figure 2. Cleaved caspase-3 expression in the ovaries of sows in CON and HS group. (A) The expression of cleaved caspase-3 in the ovary was assessed by western blotting. (B) The ovarian cleaved caspase-3 expression in the HS group was significantly higher than in CON group. *Superscript indicated a significant difference between groups (P<0.05). The graph for cleaved caspase-3 expression levels was presented as mean±SEM.

Figure 3. Comparison of correlations between several factors and Caspase-3 expression. (A) A significantly (P=0.043) positive correlation was identified between maximal atmospheric temperature (℃) and Caspase-3 expression, with a correlation coefficient of r=0.547. (B) Positive correlation coefficient (r=0.777) between corticosterone levels and Caspase-3 expression was significantly (P=0.01) identified.

Immunohistochemistry of caspase-3 in the ovaries of sows in different seasons

The localization of caspase-3 in ovarian tissues from each group was examined using IHC. Caspase-3 was detected in cells surrounding follicles, with expression patterns varying depending on the follicle development stage. In immature ovarian follicles, low levels of caspase-3 expression were observed around follicular cells and there was no noticeable difference between two groups (Fig. 4A, 4B). However, caspase-3-positive cells were more prevalent in mature follicles with localization primarily in the granulosa cell layer and the intensities were stronger in HS group than CON group (Fig. 4D, 4E, 4G, 4H). These findings suggested that caspase-3 was prominently expressed in the granulosa cells of developing follicles in the ovary, and that its expression for apoptosis was influenced by heat stress, implying the lower function of mature ovarian follicle during summer.

Figure 4. Localization of caspase-3 expression in the sow ovary assessed using immunohistochemistry. (A and B) Low level of Caspase-3 expression in the follicular cells of immature follicles in both the CON and HS groups was observed. (D, E, G and H) In mature follicles, Caspase-3 was specifically expressed in the granulosa cell layer in both different groups and the intensities were stronger in HS group than CON group. (C, F, and I) DAB-positive cells were not detected in NC. *Asterisks in Fig. 4G∼4I indicated the granulosa cell layers. Magnification: ×400 in A∼C and G∼I; ×100 in D∼F.
NC, negative control; DAB, 3,3’-diaminobenzidine.

Heat stress in global animal agriculture industries has been shown to cause considerable economic damage, particularly through seasonal infertility (Pennarossa et al., 2012; Dickson et al., 2018). Due to the high atmospheric temperatures during summer and the inherent susceptibility of pigs to heat stress, pig farms in Korea also face significant reproductive challenges (Ross et al., 2017; Kim et al., 2023). Several studies have explained how heat stress negatively impacts reproductive system and leads to increased infertility in the animal. For instance, by compromising oocyte integrity and reducing the developmental competence of embryos, hyperthermia detrimentally impacted reproduction (Hale et al., 2017). Additionally, increased lipopolysaccharide levels induced by heat stress reduced the primordial follicle numbers, altered the endocrine system, and could lead to abortion (Dickson et al., 2018). Heat stress also triggered temporally inappropriate oxytocin and prostaglandin F2α production, which caused corpus luteum regression (Bidne et al., 2019). Moreover, mitochondrial dysfunctions, oxidative stress, and the cell apoptosis were found in both oocytes and granulosa cells of heat-stressed swine, which was followed by the poor quality of oocytes in cumulus-oocyte complexes (Yin et al., 2020). Therefore, we focused on heat stress-related apoptosis in the ovarian follicles of sow, particularly induced by caspase-3. Our findings demonstrated that the high expression of cleaved (activated) caspase-3 in the granulosa cells of mature ovarian follicles under heat stress during summer in Korea was likely associated with apoptosis-related alteration of ovarian follicle microenvironment, possibly resulted in decreased reproductive performance in sows.

Excessive levels of stress hormones such as cortisol and corticosterone could disrupt the immune system, repress the endocrine system, inhibit reproductive functions, and ultimately lead to reproductive failure (Wei et al., 2019). In the aspect of reproductive perspective, the activated HPA axis in response to stress for maintaining homeostasis in the body rather disrupts the HPG axis through stress hormones including CRH, ACTH, and glucocorticoids (Liang et al., 2013; Kim et al., 2023). In the present study, we measured corticosterone levels in the bloods to evaluate stress level of sows in different atmospheric temperatures during different seasons. Although the difference in seasonal temperatures between spring (23.1±0.4℃) and summer (34.4±0.8℃) did not result in a significant change (P=0.153) in corticosterone level in sows, a strong tendency to release higher level of stress hormone in HS group than that in CON group was identified (Fig. 1); this finding could be explained by a previous study that presented more variable level of corticosterone than that of cortisol in swine under stress (Rey-Salgueiro et al., 2018).

Apoptosis is a form of programmed cell death, and the caspase family plays a pivotal role at almost every stage of this apoptotic process (Johnson et al., 2002; Kaur et al., 2023). The zymogenic (non-activated) forms of caspases are activated through two main pathways: the extrinsic and intrinsic pathways. The extrinsic pathway is started by extracellular stress, leading to the formation of the death-inducing signaling complex, which activates caspase-8 and caspase-10 via death receptor activation. Conversely, the intrinsic pathway is initiated by intracellular stress, resulting in the release of mitochondrial cytochrome C into the cytoplasm. Cytochrome C then forms the apoptosome, which activates caspase-9. Finally, the executioner caspases, caspase-3 and caspase-7, are activated through proteolytic cleavage by caspase-8, -9, or caspase-10. The active forms of these executioner caspases then degrade the cell by cleaving specific cellular substrates (Kaur et al., 2023). Therefore, we mainly focused the alteration of an executioner caspases (caspase-3) in the heat-stressed ovary of the present study. Of note, caspase-3 is known as a key enzyme in the execution of apoptosis, but it can also play a role in other functions unrelated to apoptosis within cells, with respect to the differentiation of embryonic stem cells and erythroid cells, monocyte development, homeostasis of T and B cells, microglia activation, muscle function, and even long-term depression (Khalil et al., 2012). In addition, interestingly, there is evidences suggesting that caspase-3 is able to paradoxically preserve cells from death. For instance, under mild stress, healthy dividing cells may exhibit low level of activated caspase-3, and it might trigger pro-survival pathways. Upon closer examination, when cells are exposed to mild stress, caspase-3 is weakly activated, leading to partial cleavage of Ras GTPase-activating protein into fragments C and N. Fragment N inhibits further activation of caspase-3 by triggering an Akt-mediated anti-apoptotic response. However, when caspase-3 is strongly activated by severe stress, it inhibits the anti-apoptotic response by further cleaving fragment N, ultimately leading to cell death (Khalil et al., 2014). Based on these accumulated knowledges and our findings, the increased presence of activated caspase-3 in HS group than CON group suggested that chronic heat stress during summer in Korea was regarded as a more severe stress condition to the domestic sows, which caspase-3 in HS group was more activated to the apoptotic pathway rather than eliciting the pro-survival responses typically associated with mild stress as CON group.

Even under normal conditions, apoptosis can be occurred in the ovary, and apoptosis occurring in the ovarian follicle is called as follicular atresia. However, unexpected follicular atresia can be additionally initiated from increased apoptosis of ovarian germ or granulosa cells in response to extrafollicular physiological and pathological signals; of note, it was reviewed that the heat stress could also induce abnormally high rate of follicular atresia (Johnson et al., 2002). In addition, when the ovaries of mice were incubated for 24 hr at normal (37℃) or heat-stressed (41℃) temperature, level of activated caspase-3 significantly increased in granulosa cells at the heat-stressed ovaries, indicating that heat stress promoted apoptosis of ovarian follicle via conversion of inactive caspase-3 to its active form (Li et al., 2016). Likewise, as shown in Fig. 2B, it was general that active caspase-3 expression was existed in CON group who even raised suitable temperature. However, we observed a significant upregulation of activated (cleaved) caspase-3 expression in the HS group compared to the counterpart group, implying increased apoptosis of the ovarian follicle during summer (Fig. 2). Furthermore, the expression of activated caspase-3 in the ovaries was positively correlated with degree of maximal atmospheric temperature (Fig. 3A). These findings suggest that heat stress induced by high atmospheric temperatures during summer may lead to increased apoptosis in the ovary.

In the ovary, caspase-mediated apoptosis varies depending on the development stage of ovarian follicle. In primordial to preantral follicle, caspase-2 mediates apoptosis of oogonia and oocytes, which leads to follicle atresia. However, during antral to preovulatory stage of atretic follicle, caspase-3 initiates to apoptosis within the granulosa cell layer; when atresia begins in antral stage of follicles, the concentration of activated caspase-3 is increased in granulosa cell (Johnson et al., 2002). In this study, caspase-3-positive cells by IHC staining were predominantly observed in antral to preovulatory follicles, primarily localized in the granulosa cell layer. In contrast, only a few caspase-3 positive cells were found in immature follicles, such as primary and secondary follicles. Additionally, as consistent with western blot analysis result, the intensity of caspase-3 in HS group was higher than that in CON group (Fig. 4). These findings suggest that caspase-3 is prominently expressed in the granulosa cells of developing follicles during the antral and preovulatory stages, and that its expression is influenced by heat stress during the summer.

In conclusion, the present study demonstrated that chronic heat stress during summer in Korea increased activated caspase-3-related apoptosis in the granulosa cells in the mature ovarian follicles, with a corresponding rise in corticosterone levels, which might suggest that heat stress decreased reproductive performance in sows during summer. To mitigate summer infertility and economic losses, it is crucial to implement measures that reduce heat stress, such as providing optimal temperature- controlling housing conditions during summer. We hope that these findings will contribute to effective strategies for addressing heat stress and benefit the livestock industry.

This work was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the government of the Republic of Korea (RS-2023-00251171).

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