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. 2022 Apr 6;27(3):285–293. doi: 10.1007/s12192-022-01270-4

The effect of heat stress on the cellular behavior, intracellular signaling profile of porcine growth hormone (pGH) in swine testicular cells

Yan Zhang 1, Qingrong Zhao 1, Deyi Wu 1, Hainan Lan 1,✉
PMCID: PMC9106782  PMID: 35384615

Abstract

At present, heat stress caused by the thermal environment is the main factor that endangers the reproductive function of animals. Growth hormone (GH) is a polypeptide hormone, the biological function of reproductive organs has been reported, and it has many important physiological functions in the body. However, so far, the behavior and signal transduction of GH in testicular cells under heat stress are still unclear. To this end, in the current work, we use a swine testicular cell line (ST) as an in vitro model to explore the cell behavior and intracellular signaling profile of porcine growth hormone (pGH) under heat stress; the results showed that when cells were under heat stress, pGH and GHR were basically not internalized, and a large number of them accumulated on the cell membrane. In addition, we also studied the effect of pGH on the JAK2-STATs signaling pathway and IGF-1 expression under heat stress, we found that the ability of pGH to activate the JAK-STATs signaling pathway and IGF-1 under heat stress was greatly reduced (p < 0.05). In conclusion, our research shows that when cells undergo heat stress, the internalization of pGH and GHR were inhibited, and the activation of the JAK2-STATs signaling pathway and IGF-1 expression were reduced; this lays a solid foundation for further research on the effect of pGH on swine testicular tissue under thermal environment.

Keywords: Heat stress, Porcine growth hormone, Swine testicular cell, Cellular behavior, Intracellular signaling profile

Introduction

Livestock will be subjected to various pressures in the process of growth and development, which affects its production, reproduction, and health (Belhadj Slimen, et al., 2016). When the external environment temperature rises above the body temperature (37 °C) of the warm-blooded animal, the heat generated by the warm-blooded animal will exceed the heat lost to the environment, leading to the occurrence of heat stress, a large number of a highly conserved protein family, namely heat shock proteins (HSPs) will increase accordingly (Sun, et al., 2015; Al-Tamimi, 2007). This triggers the activation of heat stress response regulatory genes in the body, which control a variety of cellular activities, including protein folding, degradation, transportation, metabolism, DNA repair, and replication (Lindquist, et al., 1988; Feder, et al., 1999; Kregel, 2002). In short, heat stress basically disrupted normal homeostasis and severely led to a serious decline in livestock production and productivity (Bharati, et al., 2017).

Growth hormone (GH) is a single-chain amino acid containing 191 polypeptides with a molecular weight of approximately 22 kDa (Brooks, et al., 2010; Li, et al., 2013). GH participates in many biological activities in the animal body and assists the body’s growth, development, and metabolism (Waxman, et al., 2006). The key to GH’s function is the growth hormone receptor (GHR), which binds in the form of a dimer, then internalizes through the cell membrane, and finally locates in the nucleus (Waters, et al., 2006; Lan, et al., 2015a, b). The ligand-induced conformational change of the receptor is a crucial step in activating the signaling pathway; when GH binds to GHR, it can activate the endogenous tyrosine kinase janus2 (JAK2) and then initiate a variety of downstream signaling pathways, including signal transducer and activator of transcription 1, 3, 5 (STAT 1, 3, 5) and extracellular signal-regulated kinase 1, 2 (ERK 1, 2) (Brooks, et al., 2010; Zhu, et al., 2001). These activated signal molecules are finally transferred to the nucleus, where they regulate gene transcription and perform the biological functions of GH (Lan, et al., 2015a, b; Figueiredo, et al., 2016; Mertani, et al., 2003).

GH has a positive effect on the reproductive organs, but, as far as we know, there is no report on the cellular behavior and characteristics of GH in testicular cells under thermal environment. In the current work, we use a swine testicular cell line (ST) as an in vitro model. The experimental results show that when cells undergo heat stress, porcine growth hormone (pGH) and GHR are basically not internalized, and a large number of them accumulate on the cell membrane; at the same time, the activation of the JAK2-STATs signaling pathway is significantly reduced after pGH stimulation is added under heat stress. In summary, this study explored the behavior and intracellular signaling profile of pGH in swine testicular cells for the first time in a thermal environment, and laid a good foundation for the follow-up study of the effect of GH on testicular tissue under heat stress.

Materials and methods

Reagents and antibodies

Porcine growth hormone (pGH) (catalog no. 869008-M), FITC (catalog no. 46950), 4% paraformaldehyde (catalog no. P6148), Triton X-100 (catalog no. 93443), Hoechst 33,258 (catalog no. 94403), and DMSO (catalog no. D2650) were purchased from Sigma-Aldrich (USA). DME/F-12 medium (catalog no. 11330032), horse serum (HS) (catalog no. 26050088), fetal bovine serum (FBS) (catalog no. 10091148), penicillin–streptomycin (catalog no. 15140122), trypsin–EDTA (0.25%) (catalog no 25200072), enhanced chemiluminescence (ECL) (catalog no. WP20005), bovine serum albumin (BSA) (catalog no. 23210), and skimmed milk powder (catalog no. LP0033B) were purchased from Thermo Fisher (USA). RIPA kit (catalog no. P0013C) and BCA kit (catalog no. P0012S) were obtained from Beyotime Biotechnology (China). IGF-1 ELISA kit (catalog no. MM-038902) was obtained from MEIMIAN Biotechnology (China). GHR monoclonal antibody (catalog no. ab134078) was obtained from Abcam (USA). HRP-conjugated anti-rabbit IgG (catalog no. 7074), p-JAK2 (catalog no. 3771), p-STAT1 (catalog no. 9167), p-STAT3 (catalog no. 9145), p-STAT5 (catalog no. 9314), JAK2 (catalog no. 3230), STAT1 (catalog no. 14995), STAT3 (catalog no. 12640), and STAT5 monoclonal antibody (catalog no. 94205) were purchased from CST (USA). All other chemicals were obtained from Sigma-Aldrich (USA), unless otherwise stated.

Cell culture and heat treatment

The swine testicular cell line (ST) was purchased from Shanghai Cell Bank (China). The cells were cultured in DEME/F-12 medium with 5% horse serum (HS), 5% fetal bovine serum (FBS), 1% penicillin 10,000 units/ml, and streptomycin 10,000 μg/ml in an incubator with 5% CO2 at 37 °C. During the heat treatment, the control cells were kept at 37 °C, and the heat-treated group was incubated at 42 °C for 1 h and then returned to 37 °C for treatment.

Confocal laser scanning microscopy (CLSM)

The cells were starved for 6 h when the cell density reaches 30 ~ 50% confluence. Then, the cells were washed 3 times with PBS; FITC-pGH was added and incubated for the specified time point. After washing 3 times, the cell samples were fixed with 4% paraformaldehyde for 30 min. The cells were washed 3 times and incubated with Hoechst 33,258 in the dark for 15 min. The cell samples were observed with a laser confocal scanning microscope (Olympus FV3000).

Indirect immunofluorescence assay (IFA)

The cells were starved for 6 h when the cell density reached 30% confluence. After washing the cells, the cell specimen was fixed with 4% paraformaldehyde for 30 min. After washing, 0.1% Triton X-100 was added to permeate for 1 h. After washing, the cell specimen was blocked with 5% bovine serum albumin for 2 h. The cells were washed 3 times and incubated with the primary antibody overnight at 4 °C, followed by incubation with the fluorescently labeled secondary antibody at 37 °C for 2 h in the dark. The cells were washed 3 times and then incubated with Hoechst 33,258 in the dark for 15 min. The cells were observed with a confocal laser scanning microscope (CLSM; Olympus FV3000); FV10-ASW 1.7 Viewer software and ImageJ software were used for image analysis.

Western blot analysis

After the cells were treated with pGH, the cells were washed 3 times in PBS; the whole cell lysate was prepared with a RIPA kit. The protein concentration was determined with the BCA protein detection kit. The total protein specimen was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skimmed milk at 37 °C for 1 h, the PVDF membranes were incubated with specific primary antibodies at 4 °C overnight. After washing 3 times with TBST, the membranes were incubated with HRP-bound secondary antibodies for 1 h at 37 °C. After washing 3 times with TBST, the protein band was detected with ECL detection system. The grayscale analysis of immunoreactive protein bands was determined by ImageJ software for image analysis.

Cell viability analysis

MTT assays were performed to detect cell viability. The cells were cultured with a 96-well flat-bottom culture plate at a density of 5 × 104 cells/well. After adding pGH for 6 h (5 replicates in each group), the culture medium was removed, 100 μl of MTT solution (1 mg/ml) was added to each well and incubated for 4 h, the supernatant was then removed, and 100 μl of dimethyl sulfoxide (DMSO) was added and incubated for 15 min. Finally, a microplate reader (Thermo Scientific, Multiscan FC) was used to measure the optical density (OD) at 490 nm.

Cell cycle analysis

The cells were digested with trypsin (without EDTA) and then were centrifuged at 1200 r/min for 5 min. Cell specimen was collected to 1–5 × 106 cells, and the culture medium was discarded. After washing with PBS, ice-cold 70% ethanol was added for fixation overnight at – 20 °C. After centrifugation, the fixative was discarded, and the cells were resuspended in 3 ml PBS. The cells were centrifuged to remove PBS, RNase A enzyme (100 μg/ml) was added, after which 0.2% Triton X-100 was added and incubated at 37 °C for 30 min. After washing, 1 ml PI staining solution was added and incubated in the dark at 4 °C for 30 min (PI concentration 50 μg/ml). The cell specimen was detected by flow cytometry.

Cell apoptosis analysis

The cells were digested with trypsin (without EDTA) and then centrifuged at 1000 r/min for 5 min. Each specimen was collected to 1–5 × 106 cells, and the culture medium was discarded. The cell specimen was washed with PBS and centrifuged at 1000 r/min for 5 min. The cell specimen was resuspended with 1 ml Annexin V binding solution, and then, the cells were stained with FITC-Annexin V staining solution and PI staining solution according to the manufacturer’s instructions. Flow cytometry was used to detect the cell samples.

Data statistics

All results were expressed as mean ± standard deviation (SD). The statistical analysis system SPSS was used to analyze the results for variance analysis, and then, Student’s t test was performed. p < 0.05 was considered statistically significant.

Results

The expression pattern of GHR in ST cells

First, in order to verify whether GHR is an expression on ST cells, GHR monoclonal antibody was used to detect GHR. As shown in Fig. 1, ST cells expressed abundant GHR (green signal), which was mainly distributed on the cell membrane.

Fig. 1.

Fig. 1

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IFA detects GHR expression. The cells were washed 3 times with PBS, fixed, permeated, blocked, and incubated with GHR monoclonal antibody. After washing, the secondary antibody labeled with Alexa-Fluor 488 was incubated, and the nuclei were stained with Hoechst 33,258. Observe the cell specimen with a confocal laser scanning microscope. Bar = 10 μm. These images represent at least 3 independent experiments

The internalization kinetics of pGH in ST cells under heat stress

To explore the internalization kinetics of pGH under heat stress, we added 50 ng/ml FITC-pGH to the cells and incubated them at 37 °C or 42 °C for 3, 15, 30, 45, 60, and 75 min. As shown in Fig. 2, we found that pGH could internalize into the cell in a time-dependent manner when the cells were at 37 °C. When the cell was treated FITC-pGH with for 3 min, FITC-pGH began to enter the cells, the internalization of pGH was obviously increased with time increasing, the cytoplasmic fluorescence signal reached a peak at 30 min, and the nuclear fluorescence signal reached a peak at 45 min, after 60 min of pGH treatment, the fluorescence signal in the cytoplasm and nucleus began to decrease. But when the cells were at 42 °C, pGH was accumulated on the cell membrane in large amounts at the indicated time points, and there was basically no internalization.

Fig. 2.

Fig. 2

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CLSM analyses the internalization kinetics of pGH under heat stress. After the cells were starved, they were washed with PBS and incubated with FITC-pGH at different time points. Then, the cell sample was fixed with 4% paraformaldehyde, and the nucleus was stained with Hoechst 33,258. Observe the cell specimen with a confocal laser scanning microscope. Bar = 10 μm. These images represent at least 3 independent experiments

The internalization kinetics of GHR in ST cell under heat stress mediated by pGH

The above experiments have shown that pGH was basically not internalization under heat stress. After that, we further explored the internalization kinetics of pGH-induced GHR under heat stress. At 37 °C and 42 °C, the cells were treated with 50 ng/ml pGH for 3, 15, 30, 45, 60, and 75 min, and then detected with GHR monoclonal antibody. As shown in Fig. 3, we found that when the cells were at 37 °C, GHR was also internalized into the cell in a time-dependent manner. Three minutes after pGH treatment, GHR (green signal) began to enter the cell, the internalization of GHR increased with time, the cytoplasmic fluorescence signal reached a peak at 30 min, and the nuclear fluorescence signal reached a peak at 45 min; when pGH was treated for 60 min, the fluorescence signal of the cytoplasm and nucleus decreased slightly. Similar to pGH, when the cells are at 42 °C, GHR accumulated on the cell membrane at the indicated time points, and there was basically no internalization.

Fig. 3.

Fig. 3

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IFA detects pGH-mediated GHR internalization kinetics under heat stress. After the cells were serum starved, the cells were washed 3 times with PBS. Then, the cells were fixed, permeated, blocked and incubated with GHR monoclonal antibody, and then incubated with secondary antibody labeled with Alexa-Fluor 488. Observe the cell specimen with a confocal laser scanning microscope. Bar = 10 μm. These images represent at least 3 independent experiments

Effect of pGH on JAK2-STATs signaling pathway under heat stress

In terms of regulating cell proliferation, differentiation, and immune regulation, the JAK2-STATs signaling pathway is one of the most important signaling pathways. In order to explore the effect of pGH on the JAK2-STATs signaling pathway under heat stress, we treated ST cells with pGH (50 ng/ml) for different times (3, 15, 30, 45, 60, 75) min, and then, we used Western blot to detect p-JAK2 and p-STAT1, 3, 5 expressions. As shown in Fig. 4a, at 37 °C, pGH could activate the JAK2-STATs signaling pathway in a time-dependent manner, but under heat stress (42 °C), the signal activation intensity was significantly weakened (p < 0.05).

Fig. 4.

Fig. 4

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The effect of pGH on JAK2-STATs signaling pathway and IGF-1 expression under heat stress. a Stimulate serum-starved cells with pGH (50 ng/ml) and incubate at different time points. The samples were evaluated by Western blot using anti-p-JAK2 and anti-p-STAT1, 3, 5, or anti-JAK2 and anti-STAT1, 3, 5, antibodies. b The effect of pGH on IGF-1 expression under heat stress. After stimulation of cells with pGH (50 ng/ml), the samples were evaluated by IGF-1 ELISA kit according to the instructions

In addition, GH signaling has multiple metabolic effects, primarily the expression of IGF-1. Therefore, we also explored the effect of pGH on IGF-1 expression under heat stress, we treated ST cells with pGH (50 ng/ml) for 40 min, and then, we used an ELISA kit to detect IGF-1 expression. As shown in Fig. 4b, the results showed that when the cells were under heat stress, the expression ability of IGF-1 was significantly reduced (p < 0.05).

The effect of pGH on cell viability under heat stress

In order to explore the effect of pGH on cell viability under heat stress, we performed an MTT assay. As shown in Fig. 5, we added pGH at a concentration of 50 ng/ml to ST cells at 37 °C and 42 °C and incubated them for 6 h. The results indicated that under the condition of pGH stimulation, when the cells were under heat stress, the cell viability was significantly reduced compared with the normal treatment group (p < 0.05).

Fig. 5.

Fig. 5

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The effect of pGH on cell viability under heat stress. Cell viability was measured by MTT assay. Add pGH at a concentration of 50 ng/ml to the serum-starved cells for 6 h. Then, add 100 μl MTT solution (0.1 mg/ml), incubate for 4 h, then add DMSO and shake for 15 min

The effect of pGH on cell cycle under heat stress

To study whether pGH on the cell cycle under heat stress, we used PI staining for detection. The results are shown in Fig. 6; compared with the normal treatment group, the proportion of the S phase was significantly reduced after adding pGH under heat stress (p < 0.05).

Fig. 6.

Fig. 6

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The effect of GH on the cell cycle. The PI staining method was used for processing, and the samples were detected by flow cytometry

The effect of pGH on cell apoptosis under heat stress

To investigate whether pGH on cell apoptosis under heat stress, we used the Annexin V-FITC/PI staining method and used flow cytometry to detect. The results are shown in Fig. 7; compared with the normal treatment group, the proportion of apoptosis rate was increased significantly after adding pGH under heat stress (p < 0.05).

Fig. 7.

Fig. 7

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The effect of pGH on cell apoptosis. The Annexin V-FITC/PI staining method was used for processing, and the data was detected by flow cytometry

Discussion

The metabolism of many species adapts to the temperature range of their surrounding environment; once the temperature of the surrounding environment rises, heat stress will occur (Sun, et al., 2015). Heat stress was defined as cytotoxic, as it alters biological molecules, disturbs cell functions, modulates metabolic reactions, induces oxidative cell damage, and activates both apoptosis and necrosis pathways (Du, et al., 2008). Furthermore, heat stress affects most aspects of both male and female reproduction function, such as the pregnancy rate, estrous activity, embryonic mortality, sperm viability, and spermatozoa mortality and abnormalities (Belhadj Slimen, et al., 2016). According to reports, in several mammalian species (including cattle, sheep, goats, pigs, and mice), the significant reduction in fertilization and pregnancy rates is directly related to heat stress (Kalo, et al., 2011; Roth, et al., 2005). However, until now, the behavior of GH in germ cells under heat stress is not very clear. In this study, we used a swine testicular cell line (ST) as an in vitro model to explore the cell behavior and intracellular signaling profile of pGH under heat stress. We found that pGH and GHR were basically not internalized, and a large number of them accumulated on the membrane; in addition, we also found that when cells undergo heat stress, the signal transduction ability was also significantly reduced.

First, we detected the expression of GHR in ST through IFA, according to our previous reports, liver, muscle, and express abundant GHR (Lan, et al., 2017), and we also observed the existence of abundant GHR in ST, and a large number of them are distributed on the cell membrane (Fig. 1). Studies have shown that GH not only plays an active role in the cell membrane, but also in the cytoplasm and nucleus (van Kerkhof, et al., 2000; Conway-Campbell, et al., 2007, 2008). When GH binding to GHR, the internalization process starts immediately (Lobie, et al., 1994). Theoretically, the internalization process of GH-GHR could be divided into 3 stages, GH-GHR passes through the cell membrane, GH-GHR is transported in the cytoplasm, and GH-GHR crosses the nuclear membrane (Lan, et al., 2018). In this work, we found that when the cells were at 37 °C, both pGH (Fig. 2) and GHR (Fig. 3) were internalized in a time-dependent manner; however, when the cells were at 42 °C, pGH and GHR were concentrated on the membrane, and there was basically no internalization. In fact, the internalized pGH and GHR may have important biological functions, which indicates that heat stress may attenuate the biological effects of GH-GHR by preventing the internalization of GH-GHR.

JAK/STAT signaling pathway is essential to promote cell proliferation and differentiation, and maintains the body’s internal homeostasis (Ihle, et al., 1997). It is generally believed that after pGH and GHR form a pGH-GHR complex on the cell membrane, JAK2 and STAT1, 3, 5 were subsequently phosphorylated by tyrosine, and downstream signals were activated, and IGF-1 began to expression (Brooks, et al., 2010; Zhu, et al., 2001). Through the results, we found that pGH activated the JAK2-STATs signaling pathway in a time-dependent manner (Fig. 4a), but when the cells undergo heat stress, the signal activation intensity was significantly reduced (p < 0.05); at the same time, we also found the IGF-1 expression (Fig. 4b) was also significantly reduced (p < 0.05), which indicates that heat stress may reduce signal transduction ability to weaken the biological effects of cells.

When cells encounter mild heat stress, they will trigger positive responses, such as increasing signaling pathways and reprogramming gene expression to re-regulate their internal environment (Park, et al., 2005). However, when cells are exposed to severe heat stress, the cell cycle will stop, and more severe heat stress will lead to programmed cell death (Kühl, et al., 2000; Rowley, et al., 1993; Fuse, et al., 1996; Nitta, et al., 1997; Li, et al., 1999; Punyiczki, et al., 1998). At the same time, heat stress can also cause mitochondrial protein denaturation, which is also an obvious manifestation of mitochondrial damage (Ahmad, et al., 2007). In the current study, we found that the cell viability (Fig. 5) and the proportion of the S phase (Fig. 6) at 42 °C were significantly reduced compared with the 37 °C (p < 0.05), and the rate of apoptosis (Fig. 7) was significantly higher (p < 0.05). The above results indicate that heat stress has already caused damage to cells and reduced the positive and positive effects of pGH. In conclusion, in this work, we explored the cellular behavior and signaling profiles of pGH in swine testicular cells under heat stress; the results showed that when the cells were under heat stress, pGH was basically not internalized into the cell, and at the same time, JAK2-STATs signaling induced by pGH was also downregulated; these findings lay the foundation for further research on the function of pGH in swine testicular tissue under heat stress.

Acknowledgements

The first author thanks Hainan Lan and all colleagues for their assistance with this study.

Author contribution

Yan Zhang and Hainan Lan conceived and designed the experiments. Yan Zhang, Qingrong Zhao, and Deyi Wu performed the experiments. Yan Zhang, Qingrong Zhao, and Deyi Wu analyzed the data. Hainan Lan contributed reagents/materials/analysis tools. Yan Zhang and Hainan Lan contributed to the writing of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 30162022).

Data availability statement

All data and materials are available for publication. Data related to the paper can be obtained from the corresponding author, based on reasonable requirements.

Declarations

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Consent for publication

All authors read and approved the final manuscript.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Yan Zhang, Email: 1085607173@qq.com.

Qingrong Zhao, Email: zqr19950408@163.com.

Deyi Wu, Email: 528827001@qq.com.

Hainan Lan, Email: tougao@jlau.edu.cn.

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