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Korean J. Vet. Serv. 2023; 46(3): 227-234

Published online September 30, 2023

https://doi.org/10.7853/kjvs.2023.46.3.227

© The Korean Socitety of Veterinary Service

Heat stress during summer reduced the ovarian aromatase expression of sows in Korea

Hwan-Deuk Kim 1,2†, Sung-Ho Kim 1†, Sang-Yup Lee 1, Tae-Gyun Kim 1, Seong-Eun Heo 3, Yong-Ryul Seo 2, Jae-Keun Cho 2, Min Jang 1, Sung-Ho Yun 1, Seung-Joon Kim 1, Won-Jae Lee 1*

1College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
2Department of Veterinary Research, Daegu Metropolitan City Institute of Health & Environment, Daegu 42183, Korea
3Gyeongsangbuk-do Livestock Research Institute, Yeongju 36052, Korea

Correspondence to : Won-Jae Lee
E-mail: iamcyshd@knu.ac.kr
https://orcid.org/0000-0003-1462-7798
These first two authors contributed equally to this work.

Received: September 1, 2023; Revised: September 6, 2023; Accepted: September 7, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0). which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

It has been addressed that heat stress due to high atmospheric temperature during summer in Korea induces impaired release of reproductive hormones, followed by occurring abnormal ovarian cyclicity, lower pregnancy ratio, and reduced litter size. Therefore, the present study attempted to compare seasonal change (spring versus summer) of the ovarian aromatase expression, an enzyme for converting testosterone into estrogen. While serum estrogen level in summer group was significantly lower than that of spring group, testosterone was not different between groups. Consistent with estrogen level, the ovarian aromatase expression in summer at follicular phase was significantly lower than the counterpart of spring. The ovarian aromatase expression was positively related with serum estrogen level significantly (r=0.689; P=0.008) and strongly negative correlation was identified (r=−0.533; P=0.078) with atmospheric temperature. The ovarian aromatase expression was not detected in immature ovarian follicles but specifically localized in the granulosa cell layers in both seasons. However, the aromatase intensity in the granulosa cell layers was stronger in spring than summer. Because testosterone level was not different between groups, it could be concluded that the lower level of estrogen during summer might be derived by not lack of substrate but lower expression of ovarian aromatase by heat stress.

Keywords Heat stress, Aromatase, Ovary, Sow, Summer

Domestic pigs are the most economically major livestock in the animal industry of Korea; at 2017, more than 10 million pigs were raised in approximately 4,400 farms (Kim et al, 2021). Since the reproductive performance of pig herd is complexly related with several factors in terms of nutrition, infectious diseases, genetics, husbandry, and stress control, appropriate breeding management of sows is important for increase of animal productivity and farm’s economic benefit (Kim et al, 2022). However, due to high atmospheric temperature during summer in Korea, heat stress has been a major concern in pig farms, known as summer infertility with presenting abnormal ovarian cyclicity, delayed puberty, less pregnancy rate, and reduced litter size (Shimizu et al, 2005; Kim et al, 2022).

Under normal circumstances, female reproductive system is governed by the hypothalamic-pituitary-gonadal (HPG) axis; pulsatile release of the hypothalamic gonadotropin-releasing hormone (GnRH) induces pulsatile secretion of gonadotrophins [GTHs; follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] from the pituitary gland, followed by developing ovarian follicles. Thereafter, increased ovarian estrogen positively feedbacks toward the hypothalamus and pituitary gland for LH surge and ovulation of mature follicles, followed by forming corpus luteum (CL) that can release progesterone to prepare pregnancy and negatively feedback to upstream reproductive hormones (Hwang et al, 2021; Kim et al, 2021; Kim et al, 2022). In contrast, the hypothalamic-pituitary-adrenal (HPA) axis secretes stress hormones such as corticotrophin-releasing hormone, adrenocorticotropic hormone, and glucocorticoid when animals are threatened by the stressed condition, which are negatively feedback to reproductive hormones in HPG axis. Then impaired GTHs due to stress hormones induce abnormal ovarian cyclicity, weakening ovarian follicle growth, and infertility (Li et al, 2016; Kim et al, 2022). Among several stressors, the effect of heat stress to animal’s reproductive system has been widely studied and known to change ovarian follicle dynamics, gonadal-steroidogenic ability, function of granulosa cell in the mature follicles, and oocyte maturation (Shimizu et al, 2005; Li et al, 2016).

Aromatase is the endoplasmic reticular enzyme, a member of the cytochrome P450 superfamily, and responsible for converting testosterone (C19 steroids) into estrogen (C18 steroids) (Miao et al, 2011; Kamal et al, 2022). Several organs such as the brain, adipose tissue, bone, prostate, and heart can express aromatase, but the main organs expressing aromatase are the placenta and gonads (Bell et al, 2014; Blakemore and Naftolin, 2016). Of note, aromatase expression in mature ovarian follicle is locally distributed in the granulosa cell layer where is under the control of endogenous FSH and LH level; testosterone derived from theca cell by LH stimulation is converted to estrogen in the granulosa cell by FSH stimulation (Britt et al, 2001; Shimizu et al, 2005).

In the previous article, it was noted that heat stress in Korea affected to the reproductive system in sows with decreasing GnRH, GTHs, and estrogen and induced lower number of mature ovarian follicles (Kim et al, 2022). Even though it has been addressed that estrogen is produced by aromatase from granulosa cells, the relationship between heat stress in Korea during summer and aromatase expression in sows has not been identified yet. Therefore, the present study attempted to compare the seasonal change (spring versus summer) of the ovarian aromatase expression to reveal the effect of heat stress to reproductive system in Korean sows, by means of western blotting, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA).

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

All specimens were obtained during slaughtering domestic sows (three-way crossbred by Landrace×Yorkshire×Duroc, approximately 2-year-old, non-pregnant, and raised where the air temperature controller was not installed) at the local abbatoir. Since it was addressed that suitable temperature for raising pigs was ranged between 20∼22℃ in adults, the samples for control group were obtained from sows during spring when the atmospheric temperature was 18.4∼21.1℃, while heat-stressed group was derived from sows who were exposed to high atmospheric temperature as 29.5∼33.4℃ during summer in Korea (Kim et al, 2021; Kim et al, 2022). Whole bloods via jugular venipuncture were acquired before slaughtering, allowed to clot, and centrifugated at 2,000 g for 15 min in 4℃. The sera were collected from the supernatant and immediately stored a deep freezer at −80℃ until further analysis. During slaughtering, both ovaries per sow were collected; an ovary was instantly fixed in 4% paraformaldehyde (Duksan chemical, Korea) and the other was transported to the laboratory with storing at 4℃. Upon reaching the laboratory, the ovarian cortex was isolated from whole ovary and immediately snap-frozen into liquid nitrogen for western blotting. The fixed ovaries were cross-sectioned to morphologically determine the estrus cycle as the follicular or luteal phase when presenting growing mature follicles with degrading CL or mature CL with growing immature follicles were observed, respectively; estrus cycles of each subject were further rechecked by estrogen and progesterone level in the serum using ELISA. Thereafter, the cross-sectioned ovaries were dehydrated, embedded in paraffin, and sectioned into 5 mm thick using a microtome (Leica Microsystems, Germany) for immunocytochemical staining.

Western blotting to assess the ovarian aromatase expression in different seasons

The snap-frozen ovaries were homogenized with lysis buffer supplemented with a proteinase inhibitor (radioimmunoprecipitation assay buffer, Thermo Fisher Scientific, MA, USA), followed by centrifugation at 13,000 ×g for 10 min at 4℃. The supernatants then were carefully collected and quantified for the total amount of protein using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (25 μg) from ovaries were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). Thereafter, the membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature (RT) and incubated with a mouse anti-human cytochrome P450 aromatase primary antibody (1:500 dilution with 1% BSA; Bio-Rad, CA, USA) or a mouse polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2,000 dilution with 1% BSA; Abcam, UK) for overnight at 4℃. The membranes were further incubated with a horseradish peroxidase conjugated goat anti-mouse IgG (1:3,000 dilution with tris buffer saline; Thermo Fisher Scientific) for 1 h 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 used for quantification of the band intensities at a 58 kDa in aromatase and 37 kDa in GAPDH. The expression level of aromatase was relatively normalized against that of GAPDH.

Immunocytochemical staining to detect ovarian aromatase

The sectioned ovaries on slides were deparaffinized using alcohol series (100∼70%), treated with 0.01 M pH 6.0 citrate buffer at 95℃ for 60 min for antigen retrieval, cooled at RT for 30 min, treated with 3% H2O2 for 30 min to block endogenous peroxidase activity, blocked with 2% normal horse serum (Vector Laboratories, CA, USA) for 1 h at RT, and incubated with anti-human cytochrome P450 aromatase primary antibody (1:100 dilution with 1% BSA) at 4℃ for overnight. Then the slides were incubated with a biotinylated secondary antibody (1:200 dilution with PBS), treated with ABC solution at RT for 60 min (Vector Laboratories), reacted with a 3,3’- diaminobenzidine (DAB) kit (Vector Laboratories), and counterstained with hematoxylin (Vector Laboratories); the duration of DAB treatment was equal to all slides. Slides for negative control were treated in a same manner without incubation with a primary antibody. In accordance with previous articles, DAB positive cells as brownish color on the tissue in the granulosa cell layers of the mature follicles were determined as the positive expression of aromatase (Kamal et al, 2022; Lu et al, 2023).

ELISA to measure gonadal steroid hormones in different seasons

To assess estrus cycle as well as measure the level of gonadal steroid hormones, ELISA for estrogen, progesterone, and testosterone was performed (Cayman Chemical Company, MI, USA). A mixture of serum sample, enzyme-immunoassay buffer, tracer, and antiserum was incubated together for 120 min at RT, reacted with Ellman’s reagent for 60 min in the 35℃ incubator, and read at a wavelength of 414 nm using a microplate reader (Epoch, Biotek, VT, USA). The absorbance readings were calculated to the hormone concentrations using a four-parameter logistic fit with free software (www.myassay.com).

Statistical analysis

All 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 conducted between several factors and aromatase expression. Differences were considered statistically significant at P<0.05. The graphs are presented as mean±standard error (SEM).

Characteristics of sows in the different seasons

By means of gross observation of ovaries for developing follicles and/or presence of CL as well as ELISA for estrogen/progesterone level in the serum, the subjects in the present study were divided into 4 groups as the follicular phase in spring (SP-fol; n=7), luteal phase in spring (SP-lut; n=7), follicular phase in summer (SU-fol; n=7), and luteal phase in summer (SU-lut; n=7) (Fig. 1A). As expected, serum concentration of estrogen or progesterone was significantly higher or lower in follicular phase than luteal phase during spring, respectively (Fig. 1B, 1C). However, the follicular phase exhibited significantly different estrogen level between spring (SP-fol) and summer (SU-fol) (Fig. 1B). In case of serum level of testosterone that implying a substrate of estrogen in the female, there was no difference among groups (Fig. 1C).

Fig. 1.Characteristics of sows in the different seasons. Representative images of the formalin-fixed ovaries (A) show follicular phase (growing follicles from small/medium follicles while degrading corpus luteum) and the luteal phase (mature corpus luteum) in the different seasons, serum estrogen (B), progesterone (C), and testosterone (D) are presented. Graphs are presented as mean±standard error (SEM). Significant differences (P<0.05) between groups are indicated by different letters at the top of bars.
SP-fol, follicular phase during spring; SP-lut, luteal phase during spring; SU-fol, follicular phase during summer; SU-lut, luteal phase during summer.

Aromatase expression in the ovaries of sows in different seasons

Because it was addressed that the stress under high atmospheric temperature during summer could alter HPG axis-related reproductive hormones (Kim et al, 2022) and the Fig. 1 in the present study presented lower level of estrogen in summer group than spring group, we further analyzed the ovarian aromatase expression level, which was known to be responsible for biosynthesis of estrogen from testosterone, in different seasons (Fig. 2). Consistent with estrogen level in different seasons in Fig. 1B, the ovarian aromatase expression in summer at follicular phase (SU-fol) was significantly lower than the counterpart of spring (SP-fol) (Fig. 2A, 2B). Notably, the ovarian aromatase expression in the follicular phase presented a positive correlation with serum estrogen level (r=0.689; P=0.008), which indicated that lower expression of the ovarian aromatase induced decreased level of estrogen. In addition, when the correlation was analyzed between the ovarian aromatase level and atmospheric temperature, it was not significant but strongly negative correlation was identified (r=−0.533; P=0.078), which implied heat stress could alter aromatase expression in the ovary.

Fig. 2.Aromatase expression in the ovaries of sows in different seasons. The ovarian aromatase expression in the follicular phase of summer is significantly lower than that of spring (A and B). The aromatase expression is positively correlated with serum estrogen level (C; r=0.689, P=0.008). The non-significant but highly negative correlation between aromatase expression and atmospheric temperature is identified (D; r=−0.533, P=0.078). Graph is presented as mean±standard error (SEM). Significant differences (P<0.05) between groups are indicated by different letters at the top of bars.

Localization of aromatase expression in the ovary

Fig. 3 showed the distribution of the ovarian aromatase in the follicular phase during different seasons. Compared with negative control (Fig. 3A, 3G), the immature ovarian follicles did not express aromatase around the follicular cells (red asterisks in Fig. 3B, 3H) in both different seasons (SP-fol and SU-fol). Positive DAB staining was specifically localized at the granulosa cells of the mature (preovulatory) follicles (blue asterisks in Fig. 3E, 3F of SP-fol, and yellow asterisks in Fig. 3K, 3L of SU-fol), compared to negative control (Fig. 3C, 3D, 3I, 3J). However, DAB-positive intensity in the granulosa cell layers during summer (SU-fol) was less abundant than those of spring (SP-fol) (blue asterisks versus yellow asterisks), which was thought to be highly related with lower estrogen concentration in Fig. 1B and decreased aromatase expression in Fig. 2B. In addition, because testosterone level was not different between groups in the follicular phase (SP-fol and SU-fol), it could be concluded that the lower level of estrogen during summer might be derived by not lack of substrate but lower expression of ovarian aromatase.

Fig. 3.Localization of aromatase expression in the follicular phase of ovary. The expression of aromatase is not detected in the follicular cells (red asterisks) of immature ovarian follicles in both spring and summer groups (A, B, G, and H). In mature ovarian follicles, aromatase is specifically expressed in the granulosa cell layer in both different seasons (C∼F and I∼L) but DAB-positive intensity is stronger in spring group (blue asterisks) than summer group (yellow asterisks). Magnification: ×100 in C, E, I, and K; ×400 in A, B, D, F, G, H, J, and L.
N/C, negative control; DAB, 3,3’-diaminobenzidine.

Because fluctuating breeding environment to farm pigs including temperature, humidity, wind, rain, and noise are stressors which can influence to their endocrine system and physiological balance, suitable establishment of breeding management strategy against several stressors is a main key in the industrial animals (Kim et al, 2021; Kim et al, 2022). Of note, it has been reported that chronic stress including heat stress affects to sow’s reproductive performance (summer infertility) with respect to pubertal delay, increased interval from weaning to estrus, higher pregnancy failure, and decreased litter size (Tummaruk et al, 2009; Lents, 2019). Therefore, we mainly focused on the heat stress-related changes in the ovarian aromatase expression during summer, so as to uncover the reason for lower estrogen level in this season. As a result, it was demonstrated that the lowered aromatase expression in the granulosa cells of mature ovarian follicle during summer was thought to be related to decreased estrogen level.

Negative effects by heat stress on the ovarian aromatase activity and serum estrogen level commonly appear in every animal species. In the experimental animals, the induced heat stress to mice occurred the apoptosis of granulosa cells from the ovarian follicles and decreased serum estrogen concentration with lower abundance of aromatase in the ovarian follicles (Li et al, 2016). Intensive heat stress in rats suppressed the ovarian aromatase expression in granulosa cells, resulting in low capacity for estrogen production (Shimizu et al, 2005). In addition, the negative effect of naturally-induced heat stress during summer in farm animals is obvious. The estrogen levels in the ovarian follicles, derived from aromatase of granulosa cells, have been presented to decrease in heat-stressed goat herd (Ozawa et al, 2005). In case of another main livestock, the dominant follicles in cows exhibited decreased estradiol concentration and aromatase activity in the summer than in the autumn (Badinga et al, 1993). In chicken, a livestock with the hardest damage by heat stress, high temperature-exposed group presented reduced 17β-estradiol concentration with lower abundance of aromatase mRNA expression (Rozenboim et al, 2007). High temperature greatly induced a reduction in proliferation of both ovarian follicles and granulosa cells in pigs in vitro (Sirotkin, 2010). These reports demonstrated that heat stress affects the dynamics of ovarian granulosa cells and follicular estrogenic activity in livestock. Likewise, the present study also suggested that reproductive system of sows in Korea was affected by reduced expression of the ovarian aromatase, followed by reducing estrogen level.

Aromatase plays a critical role in developing body and maintaining homeostasis; a review article well consolidated for the role of ovarian aromatase/estrogen in the body in regards to cellular kinetics of several type of cells, metabolic effect, immune regulation, vascular homeostasis, brain development and homeostasis, bone homeostasis, and genital homeostasis (Blakemore and Naftolin, 2016). Especially, gonads of both sexes have known as the chief source of aromatase in adults. The Sertoli cells in testis in male, a corresponding tissue of the ovarian granulosa cell in female, expressed aromatase to convert androgen to estrogen for controlling spermatogenesis (Blakemore and Naftolin, 2016). In female, the theca cells around mature ovarian follicle produced androgens by LH stimulation, followed by aromatizing them by the granulosa cells via FSH stimulation; the aromatase knockout mouse showed a dysfunctional HPG axis, non-detectable serum estrogen level, infertility due to disruption of ovarian follicle development, and a failure to ovulate (Britt et al, 2001). Therefore, disturbance of GTHs release can induce abnormal synthesis of estrogen due to insufficient formation of theca cells and/or granulosa cells in female. Because several stressors activate HPA axis followed by inhibitory effects to HPG axis, stress finally induces negative effect on granulosa cells’ ability and synthesis of estrogen. The present study showed reduced serum estrogen level in summer than spring, while there was no difference in serum testosterone level. In accordance with previously published article, stress hormones selectively influence to FSH, responsible to develop the granulosa cells, followed by inhibitory effect on estrogen synthesis (Hsueh and Erickson, 1978). Likewise, experimental treatment of cortisol (stress hormone) to sheep suppressed the ovarian follicle development which was not up to estrogen-secreting preovulatory follicles, resulting in decreased serum estrogen level (Macfarlane et al, 2000). In addition, heat stress in Korea during summer decreased GnRH and GTHs, followed by more abundance of small ovarian follicles than large follicles and reduced serum estrogen level (Kim et al, 2022).

In conclusion, the present study demonstrated that heat stress during summer in Korea affected the ovarian aromatase expression and estrogen synthesis in sows. Based on the present results, it can be concluded that breeding management such as thermoregulation during summer is important to minimize the economic loss by summer infertility in sows. We hope that the present results have significant implications in the fields of reproductive biology and 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.

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Article

Original Article

Korean J. Vet. Serv. 2023; 46(3): 227-234

Published online September 30, 2023 https://doi.org/10.7853/kjvs.2023.46.3.227

Copyright © The Korean Socitety of Veterinary Service.

Heat stress during summer reduced the ovarian aromatase expression of sows in Korea

Hwan-Deuk Kim 1,2†, Sung-Ho Kim 1†, Sang-Yup Lee 1, Tae-Gyun Kim 1, Seong-Eun Heo 3, Yong-Ryul Seo 2, Jae-Keun Cho 2, Min Jang 1, Sung-Ho Yun 1, Seung-Joon Kim 1, Won-Jae Lee 1*

1College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
2Department of Veterinary Research, Daegu Metropolitan City Institute of Health & Environment, Daegu 42183, Korea
3Gyeongsangbuk-do Livestock Research Institute, Yeongju 36052, Korea

Correspondence to:Won-Jae Lee
E-mail: iamcyshd@knu.ac.kr
https://orcid.org/0000-0003-1462-7798
These first two authors contributed equally to this work.

Received: September 1, 2023; Revised: September 6, 2023; Accepted: September 7, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0). which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

It has been addressed that heat stress due to high atmospheric temperature during summer in Korea induces impaired release of reproductive hormones, followed by occurring abnormal ovarian cyclicity, lower pregnancy ratio, and reduced litter size. Therefore, the present study attempted to compare seasonal change (spring versus summer) of the ovarian aromatase expression, an enzyme for converting testosterone into estrogen. While serum estrogen level in summer group was significantly lower than that of spring group, testosterone was not different between groups. Consistent with estrogen level, the ovarian aromatase expression in summer at follicular phase was significantly lower than the counterpart of spring. The ovarian aromatase expression was positively related with serum estrogen level significantly (r=0.689; P=0.008) and strongly negative correlation was identified (r=−0.533; P=0.078) with atmospheric temperature. The ovarian aromatase expression was not detected in immature ovarian follicles but specifically localized in the granulosa cell layers in both seasons. However, the aromatase intensity in the granulosa cell layers was stronger in spring than summer. Because testosterone level was not different between groups, it could be concluded that the lower level of estrogen during summer might be derived by not lack of substrate but lower expression of ovarian aromatase by heat stress.

Keywords: Heat stress, Aromatase, Ovary, Sow, Summer

INTRODUCTION

Domestic pigs are the most economically major livestock in the animal industry of Korea; at 2017, more than 10 million pigs were raised in approximately 4,400 farms (Kim et al, 2021). Since the reproductive performance of pig herd is complexly related with several factors in terms of nutrition, infectious diseases, genetics, husbandry, and stress control, appropriate breeding management of sows is important for increase of animal productivity and farm’s economic benefit (Kim et al, 2022). However, due to high atmospheric temperature during summer in Korea, heat stress has been a major concern in pig farms, known as summer infertility with presenting abnormal ovarian cyclicity, delayed puberty, less pregnancy rate, and reduced litter size (Shimizu et al, 2005; Kim et al, 2022).

Under normal circumstances, female reproductive system is governed by the hypothalamic-pituitary-gonadal (HPG) axis; pulsatile release of the hypothalamic gonadotropin-releasing hormone (GnRH) induces pulsatile secretion of gonadotrophins [GTHs; follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] from the pituitary gland, followed by developing ovarian follicles. Thereafter, increased ovarian estrogen positively feedbacks toward the hypothalamus and pituitary gland for LH surge and ovulation of mature follicles, followed by forming corpus luteum (CL) that can release progesterone to prepare pregnancy and negatively feedback to upstream reproductive hormones (Hwang et al, 2021; Kim et al, 2021; Kim et al, 2022). In contrast, the hypothalamic-pituitary-adrenal (HPA) axis secretes stress hormones such as corticotrophin-releasing hormone, adrenocorticotropic hormone, and glucocorticoid when animals are threatened by the stressed condition, which are negatively feedback to reproductive hormones in HPG axis. Then impaired GTHs due to stress hormones induce abnormal ovarian cyclicity, weakening ovarian follicle growth, and infertility (Li et al, 2016; Kim et al, 2022). Among several stressors, the effect of heat stress to animal’s reproductive system has been widely studied and known to change ovarian follicle dynamics, gonadal-steroidogenic ability, function of granulosa cell in the mature follicles, and oocyte maturation (Shimizu et al, 2005; Li et al, 2016).

Aromatase is the endoplasmic reticular enzyme, a member of the cytochrome P450 superfamily, and responsible for converting testosterone (C19 steroids) into estrogen (C18 steroids) (Miao et al, 2011; Kamal et al, 2022). Several organs such as the brain, adipose tissue, bone, prostate, and heart can express aromatase, but the main organs expressing aromatase are the placenta and gonads (Bell et al, 2014; Blakemore and Naftolin, 2016). Of note, aromatase expression in mature ovarian follicle is locally distributed in the granulosa cell layer where is under the control of endogenous FSH and LH level; testosterone derived from theca cell by LH stimulation is converted to estrogen in the granulosa cell by FSH stimulation (Britt et al, 2001; Shimizu et al, 2005).

In the previous article, it was noted that heat stress in Korea affected to the reproductive system in sows with decreasing GnRH, GTHs, and estrogen and induced lower number of mature ovarian follicles (Kim et al, 2022). Even though it has been addressed that estrogen is produced by aromatase from granulosa cells, the relationship between heat stress in Korea during summer and aromatase expression in sows has not been identified yet. Therefore, the present study attempted to compare the seasonal change (spring versus summer) of the ovarian aromatase expression to reveal the effect of heat stress to reproductive system in Korean sows, by means of western blotting, immunocytochemistry, and enzyme-linked immunosorbent assay (ELISA).

MATERIALS AND METHODS

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

All specimens were obtained during slaughtering domestic sows (three-way crossbred by Landrace×Yorkshire×Duroc, approximately 2-year-old, non-pregnant, and raised where the air temperature controller was not installed) at the local abbatoir. Since it was addressed that suitable temperature for raising pigs was ranged between 20∼22℃ in adults, the samples for control group were obtained from sows during spring when the atmospheric temperature was 18.4∼21.1℃, while heat-stressed group was derived from sows who were exposed to high atmospheric temperature as 29.5∼33.4℃ during summer in Korea (Kim et al, 2021; Kim et al, 2022). Whole bloods via jugular venipuncture were acquired before slaughtering, allowed to clot, and centrifugated at 2,000 g for 15 min in 4℃. The sera were collected from the supernatant and immediately stored a deep freezer at −80℃ until further analysis. During slaughtering, both ovaries per sow were collected; an ovary was instantly fixed in 4% paraformaldehyde (Duksan chemical, Korea) and the other was transported to the laboratory with storing at 4℃. Upon reaching the laboratory, the ovarian cortex was isolated from whole ovary and immediately snap-frozen into liquid nitrogen for western blotting. The fixed ovaries were cross-sectioned to morphologically determine the estrus cycle as the follicular or luteal phase when presenting growing mature follicles with degrading CL or mature CL with growing immature follicles were observed, respectively; estrus cycles of each subject were further rechecked by estrogen and progesterone level in the serum using ELISA. Thereafter, the cross-sectioned ovaries were dehydrated, embedded in paraffin, and sectioned into 5 mm thick using a microtome (Leica Microsystems, Germany) for immunocytochemical staining.

Western blotting to assess the ovarian aromatase expression in different seasons

The snap-frozen ovaries were homogenized with lysis buffer supplemented with a proteinase inhibitor (radioimmunoprecipitation assay buffer, Thermo Fisher Scientific, MA, USA), followed by centrifugation at 13,000 ×g for 10 min at 4℃. The supernatants then were carefully collected and quantified for the total amount of protein using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (25 μg) from ovaries were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). Thereafter, the membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature (RT) and incubated with a mouse anti-human cytochrome P450 aromatase primary antibody (1:500 dilution with 1% BSA; Bio-Rad, CA, USA) or a mouse polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2,000 dilution with 1% BSA; Abcam, UK) for overnight at 4℃. The membranes were further incubated with a horseradish peroxidase conjugated goat anti-mouse IgG (1:3,000 dilution with tris buffer saline; Thermo Fisher Scientific) for 1 h 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 used for quantification of the band intensities at a 58 kDa in aromatase and 37 kDa in GAPDH. The expression level of aromatase was relatively normalized against that of GAPDH.

Immunocytochemical staining to detect ovarian aromatase

The sectioned ovaries on slides were deparaffinized using alcohol series (100∼70%), treated with 0.01 M pH 6.0 citrate buffer at 95℃ for 60 min for antigen retrieval, cooled at RT for 30 min, treated with 3% H2O2 for 30 min to block endogenous peroxidase activity, blocked with 2% normal horse serum (Vector Laboratories, CA, USA) for 1 h at RT, and incubated with anti-human cytochrome P450 aromatase primary antibody (1:100 dilution with 1% BSA) at 4℃ for overnight. Then the slides were incubated with a biotinylated secondary antibody (1:200 dilution with PBS), treated with ABC solution at RT for 60 min (Vector Laboratories), reacted with a 3,3’- diaminobenzidine (DAB) kit (Vector Laboratories), and counterstained with hematoxylin (Vector Laboratories); the duration of DAB treatment was equal to all slides. Slides for negative control were treated in a same manner without incubation with a primary antibody. In accordance with previous articles, DAB positive cells as brownish color on the tissue in the granulosa cell layers of the mature follicles were determined as the positive expression of aromatase (Kamal et al, 2022; Lu et al, 2023).

ELISA to measure gonadal steroid hormones in different seasons

To assess estrus cycle as well as measure the level of gonadal steroid hormones, ELISA for estrogen, progesterone, and testosterone was performed (Cayman Chemical Company, MI, USA). A mixture of serum sample, enzyme-immunoassay buffer, tracer, and antiserum was incubated together for 120 min at RT, reacted with Ellman’s reagent for 60 min in the 35℃ incubator, and read at a wavelength of 414 nm using a microplate reader (Epoch, Biotek, VT, USA). The absorbance readings were calculated to the hormone concentrations using a four-parameter logistic fit with free software (www.myassay.com).

Statistical analysis

All 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 conducted between several factors and aromatase expression. Differences were considered statistically significant at P<0.05. The graphs are presented as mean±standard error (SEM).

RESULTS

Characteristics of sows in the different seasons

By means of gross observation of ovaries for developing follicles and/or presence of CL as well as ELISA for estrogen/progesterone level in the serum, the subjects in the present study were divided into 4 groups as the follicular phase in spring (SP-fol; n=7), luteal phase in spring (SP-lut; n=7), follicular phase in summer (SU-fol; n=7), and luteal phase in summer (SU-lut; n=7) (Fig. 1A). As expected, serum concentration of estrogen or progesterone was significantly higher or lower in follicular phase than luteal phase during spring, respectively (Fig. 1B, 1C). However, the follicular phase exhibited significantly different estrogen level between spring (SP-fol) and summer (SU-fol) (Fig. 1B). In case of serum level of testosterone that implying a substrate of estrogen in the female, there was no difference among groups (Fig. 1C).

Figure 1. Characteristics of sows in the different seasons. Representative images of the formalin-fixed ovaries (A) show follicular phase (growing follicles from small/medium follicles while degrading corpus luteum) and the luteal phase (mature corpus luteum) in the different seasons, serum estrogen (B), progesterone (C), and testosterone (D) are presented. Graphs are presented as mean±standard error (SEM). Significant differences (P<0.05) between groups are indicated by different letters at the top of bars.
SP-fol, follicular phase during spring; SP-lut, luteal phase during spring; SU-fol, follicular phase during summer; SU-lut, luteal phase during summer.

Aromatase expression in the ovaries of sows in different seasons

Because it was addressed that the stress under high atmospheric temperature during summer could alter HPG axis-related reproductive hormones (Kim et al, 2022) and the Fig. 1 in the present study presented lower level of estrogen in summer group than spring group, we further analyzed the ovarian aromatase expression level, which was known to be responsible for biosynthesis of estrogen from testosterone, in different seasons (Fig. 2). Consistent with estrogen level in different seasons in Fig. 1B, the ovarian aromatase expression in summer at follicular phase (SU-fol) was significantly lower than the counterpart of spring (SP-fol) (Fig. 2A, 2B). Notably, the ovarian aromatase expression in the follicular phase presented a positive correlation with serum estrogen level (r=0.689; P=0.008), which indicated that lower expression of the ovarian aromatase induced decreased level of estrogen. In addition, when the correlation was analyzed between the ovarian aromatase level and atmospheric temperature, it was not significant but strongly negative correlation was identified (r=−0.533; P=0.078), which implied heat stress could alter aromatase expression in the ovary.

Figure 2. Aromatase expression in the ovaries of sows in different seasons. The ovarian aromatase expression in the follicular phase of summer is significantly lower than that of spring (A and B). The aromatase expression is positively correlated with serum estrogen level (C; r=0.689, P=0.008). The non-significant but highly negative correlation between aromatase expression and atmospheric temperature is identified (D; r=−0.533, P=0.078). Graph is presented as mean±standard error (SEM). Significant differences (P<0.05) between groups are indicated by different letters at the top of bars.

Localization of aromatase expression in the ovary

Fig. 3 showed the distribution of the ovarian aromatase in the follicular phase during different seasons. Compared with negative control (Fig. 3A, 3G), the immature ovarian follicles did not express aromatase around the follicular cells (red asterisks in Fig. 3B, 3H) in both different seasons (SP-fol and SU-fol). Positive DAB staining was specifically localized at the granulosa cells of the mature (preovulatory) follicles (blue asterisks in Fig. 3E, 3F of SP-fol, and yellow asterisks in Fig. 3K, 3L of SU-fol), compared to negative control (Fig. 3C, 3D, 3I, 3J). However, DAB-positive intensity in the granulosa cell layers during summer (SU-fol) was less abundant than those of spring (SP-fol) (blue asterisks versus yellow asterisks), which was thought to be highly related with lower estrogen concentration in Fig. 1B and decreased aromatase expression in Fig. 2B. In addition, because testosterone level was not different between groups in the follicular phase (SP-fol and SU-fol), it could be concluded that the lower level of estrogen during summer might be derived by not lack of substrate but lower expression of ovarian aromatase.

Figure 3. Localization of aromatase expression in the follicular phase of ovary. The expression of aromatase is not detected in the follicular cells (red asterisks) of immature ovarian follicles in both spring and summer groups (A, B, G, and H). In mature ovarian follicles, aromatase is specifically expressed in the granulosa cell layer in both different seasons (C∼F and I∼L) but DAB-positive intensity is stronger in spring group (blue asterisks) than summer group (yellow asterisks). Magnification: ×100 in C, E, I, and K; ×400 in A, B, D, F, G, H, J, and L.
N/C, negative control; DAB, 3,3’-diaminobenzidine.

DISCUSSION

Because fluctuating breeding environment to farm pigs including temperature, humidity, wind, rain, and noise are stressors which can influence to their endocrine system and physiological balance, suitable establishment of breeding management strategy against several stressors is a main key in the industrial animals (Kim et al, 2021; Kim et al, 2022). Of note, it has been reported that chronic stress including heat stress affects to sow’s reproductive performance (summer infertility) with respect to pubertal delay, increased interval from weaning to estrus, higher pregnancy failure, and decreased litter size (Tummaruk et al, 2009; Lents, 2019). Therefore, we mainly focused on the heat stress-related changes in the ovarian aromatase expression during summer, so as to uncover the reason for lower estrogen level in this season. As a result, it was demonstrated that the lowered aromatase expression in the granulosa cells of mature ovarian follicle during summer was thought to be related to decreased estrogen level.

Negative effects by heat stress on the ovarian aromatase activity and serum estrogen level commonly appear in every animal species. In the experimental animals, the induced heat stress to mice occurred the apoptosis of granulosa cells from the ovarian follicles and decreased serum estrogen concentration with lower abundance of aromatase in the ovarian follicles (Li et al, 2016). Intensive heat stress in rats suppressed the ovarian aromatase expression in granulosa cells, resulting in low capacity for estrogen production (Shimizu et al, 2005). In addition, the negative effect of naturally-induced heat stress during summer in farm animals is obvious. The estrogen levels in the ovarian follicles, derived from aromatase of granulosa cells, have been presented to decrease in heat-stressed goat herd (Ozawa et al, 2005). In case of another main livestock, the dominant follicles in cows exhibited decreased estradiol concentration and aromatase activity in the summer than in the autumn (Badinga et al, 1993). In chicken, a livestock with the hardest damage by heat stress, high temperature-exposed group presented reduced 17β-estradiol concentration with lower abundance of aromatase mRNA expression (Rozenboim et al, 2007). High temperature greatly induced a reduction in proliferation of both ovarian follicles and granulosa cells in pigs in vitro (Sirotkin, 2010). These reports demonstrated that heat stress affects the dynamics of ovarian granulosa cells and follicular estrogenic activity in livestock. Likewise, the present study also suggested that reproductive system of sows in Korea was affected by reduced expression of the ovarian aromatase, followed by reducing estrogen level.

Aromatase plays a critical role in developing body and maintaining homeostasis; a review article well consolidated for the role of ovarian aromatase/estrogen in the body in regards to cellular kinetics of several type of cells, metabolic effect, immune regulation, vascular homeostasis, brain development and homeostasis, bone homeostasis, and genital homeostasis (Blakemore and Naftolin, 2016). Especially, gonads of both sexes have known as the chief source of aromatase in adults. The Sertoli cells in testis in male, a corresponding tissue of the ovarian granulosa cell in female, expressed aromatase to convert androgen to estrogen for controlling spermatogenesis (Blakemore and Naftolin, 2016). In female, the theca cells around mature ovarian follicle produced androgens by LH stimulation, followed by aromatizing them by the granulosa cells via FSH stimulation; the aromatase knockout mouse showed a dysfunctional HPG axis, non-detectable serum estrogen level, infertility due to disruption of ovarian follicle development, and a failure to ovulate (Britt et al, 2001). Therefore, disturbance of GTHs release can induce abnormal synthesis of estrogen due to insufficient formation of theca cells and/or granulosa cells in female. Because several stressors activate HPA axis followed by inhibitory effects to HPG axis, stress finally induces negative effect on granulosa cells’ ability and synthesis of estrogen. The present study showed reduced serum estrogen level in summer than spring, while there was no difference in serum testosterone level. In accordance with previously published article, stress hormones selectively influence to FSH, responsible to develop the granulosa cells, followed by inhibitory effect on estrogen synthesis (Hsueh and Erickson, 1978). Likewise, experimental treatment of cortisol (stress hormone) to sheep suppressed the ovarian follicle development which was not up to estrogen-secreting preovulatory follicles, resulting in decreased serum estrogen level (Macfarlane et al, 2000). In addition, heat stress in Korea during summer decreased GnRH and GTHs, followed by more abundance of small ovarian follicles than large follicles and reduced serum estrogen level (Kim et al, 2022).

In conclusion, the present study demonstrated that heat stress during summer in Korea affected the ovarian aromatase expression and estrogen synthesis in sows. Based on the present results, it can be concluded that breeding management such as thermoregulation during summer is important to minimize the economic loss by summer infertility in sows. We hope that the present results have significant implications in the fields of reproductive biology and the livestock industry.

ACKNOWLEDGEMENTS

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).

CONFLICT OF INTEREST

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

Fig 1.

Figure 1.Characteristics of sows in the different seasons. Representative images of the formalin-fixed ovaries (A) show follicular phase (growing follicles from small/medium follicles while degrading corpus luteum) and the luteal phase (mature corpus luteum) in the different seasons, serum estrogen (B), progesterone (C), and testosterone (D) are presented. Graphs are presented as mean±standard error (SEM). Significant differences (P<0.05) between groups are indicated by different letters at the top of bars.
SP-fol, follicular phase during spring; SP-lut, luteal phase during spring; SU-fol, follicular phase during summer; SU-lut, luteal phase during summer.
Korean Journal of Veterinary Service 2023; 46: 227-234https://doi.org/10.7853/kjvs.2023.46.3.227

Fig 2.

Figure 2.Aromatase expression in the ovaries of sows in different seasons. The ovarian aromatase expression in the follicular phase of summer is significantly lower than that of spring (A and B). The aromatase expression is positively correlated with serum estrogen level (C; r=0.689, P=0.008). The non-significant but highly negative correlation between aromatase expression and atmospheric temperature is identified (D; r=−0.533, P=0.078). Graph is presented as mean±standard error (SEM). Significant differences (P<0.05) between groups are indicated by different letters at the top of bars.
Korean Journal of Veterinary Service 2023; 46: 227-234https://doi.org/10.7853/kjvs.2023.46.3.227

Fig 3.

Figure 3.Localization of aromatase expression in the follicular phase of ovary. The expression of aromatase is not detected in the follicular cells (red asterisks) of immature ovarian follicles in both spring and summer groups (A, B, G, and H). In mature ovarian follicles, aromatase is specifically expressed in the granulosa cell layer in both different seasons (C∼F and I∼L) but DAB-positive intensity is stronger in spring group (blue asterisks) than summer group (yellow asterisks). Magnification: ×100 in C, E, I, and K; ×400 in A, B, D, F, G, H, J, and L.
N/C, negative control; DAB, 3,3’-diaminobenzidine.
Korean Journal of Veterinary Service 2023; 46: 227-234https://doi.org/10.7853/kjvs.2023.46.3.227

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KJVS
Dec 30, 2024 Vol.47 No.4, pp. 193~317

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