Estradiol-17β regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1

Journal Pre-proof Estradiol-17␤ regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1 Shi-Yu An, Xiao-Xiao Gao, Zhi-Bo Wang, Ya-Xu Liang, Shu-Ting Wang, Shen-Hua Xiao, Jiang-Tao Xia, Pei-Hua You, Feng Wang, Guo-Min Zhang

PII:

S0378-4320(19)31128-5

DOI:

https://doi.org/10.1016/j.anireprosci.2020.106328

Reference:

ANIREP 106328

To appear in:

Animal Reproduction Science

Received Date:

14 December 2019

Revised Date:

2 February 2020

Accepted Date:

20 February 2020

Please cite this article as: An S-Yu, Gao X-Xiao, Wang Z-Bo, Liang Y-Xu, Wang S-Ting, Xiao S-Hua, Xia J-Tao, You P-Hua, Wang F, Zhang G-Min, Estradiol-17␤ regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1, Animal Reproduction Science (2020), doi: https://doi.org/10.1016/j.anireprosci.2020.106328

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Estradiol-17β regulates proliferation and apoptosis of sheep endometrial epithelial cells by regulating the relative abundance of YAP1

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Running title: Estradiol-17β regulates proliferation and apoptosis in sheep EEC

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Shi-Yu An1, Xiao-Xiao Gao1, Zhi-Bo Wang1, Ya-Xu Liang1, Shu-Ting Wang1, Shen-Hua Xiao1, Jiang-Tao Xia1, Pei-Hua You3, Feng Wang1,

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Guo-Min Zhang1,2

Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China

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Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University,

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1

Nanjing 210095, China 3

Portal Agri-Industries Co., Ltd., Nanjing 211803, China

*Corresponding author: Feng Wang: Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing

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Agricultural University, No.1 Weigang, Nanjing, China; Tel.: +86-025-84395381; Fax: +86-025-84395314; E-mail address: caeet@ njau.edu.cn

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+86-025-84395314; E-mail address: [email protected].

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Guo-Min Zhang: College of veterinary medicine, Nanjing Agricultural University, No.1 Weigang, Nanjing, China; Tel.: +86-025-84395294; fax:

Highlights:

YAP1 suppression decreased the proliferation and promoted the apoptosis in sheep EEC

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Estradiol-17β could up-regulate the abundance of YAP1 in sheep EEC

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Estradiol-17β regulated the proliferation and apoptosis in sheep EEC through YAP1

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ABSTRACT

Yes-associated protein 1 (YAP1) transcription regulator of the Hippo protein kinase pathway, serves as a key regulator of tissue growth and

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organ size by regulating cell proliferation and apoptosis. Effects of YAP1 on proliferation and apoptosis of sheep endometrial epithelial cells

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(EEC) as a result of estradiol-17β (E2) treatment, however, remain unclear. In the present study, the abundance of YAP1 protein in the uterine horn was greater than that in the uterine body or cervix. The YAP1 protein was primarily localized in the endometrial luminal and glandular

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epithelial cells of the uterine horn of ewes on day 2 of the estrous cycle. Compared with control samples, there was a lesser abundance of YAP1

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mRNA transcript that was associated with a lesser proliferation and greater apoptosis of EEC. There were also lesser concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent culture medium when there was a lesser abundance of YAP1 mRNA in EEC compared

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with those in the control group. When there was a greater abundance of YAP1 mRNA transcript, there were greater concentrations of epidermal growth factor and insulin-like growth factor 1 in the spent media. Furthermore, with estradiol-17β treatment the abundance of YAP1 mRNA

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transcript was similar to that of the control samples. Taken together, estradiol-17β may function as an essential regulator of EEC proliferation and apoptosis by modulation of concentrations of YAP1 protein in the sheep uterus. These results indicate there are molecular mechanisms of estradiol-17β and YAP1 in EEC proliferation and apoptosis of ewes.

Keywords: Sheep; Endometrial epithelial cells; Estradiol-17β; YAP1

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1. Introduction

The uterus is an important tissue for mammalian reproduction because it contains the embryo/fetus throughout gestation (Arora et al., 2016).

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The physiological and cellular defects of the uterus result in the disruption of embryo implantation in mammals (Shukla et al., 2019).

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Endometrial epithelial cells (EEC) function at the site for embryo attachment (Kakar-Bhanot et al., 2019) and produce numerous cytokines, including epidermal growth factor (EGF) (Paiva et al., 2011), insulin-like growth factor 1 (IGF-1) (Wan et al., 2019), and transforming growth

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factor-β (TGF-β) (Yu et al., 2013). These cytokines are involved in cell proliferation, migration, and cell differentiation in the endometrium (Arai et al., 2014). Furthermore, EEC respond to the microenvironment of the uterus by maintaining uterine homeostasis in support of embryonic

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development. Disorders in EEC signaling are responsible for a wide spectrum of endometrial pathologies, ranging from infertility to cancer (Makieva et al., 2018). The regulatory mechanism involved in the proliferation and apoptosis of EEC, however, remain unclear, especially in small ruminants.

The Hippo transcription regulatory pathway is an evolutionarily conserved signaling cascade pathway that when active modulates tissue development, homeostasis, and regeneration (Mao et al., 2015) The Hippo transcription regulatory pathway is important in the development of

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the testes, epididymis, ductus deferens, and spermatogenesis in Hu sheep (Zhang et al., 2019). Furthermore, the Hippo transcription regulatory

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pathway is important in modulation of the process of steroidogenesis in fully developed granulosa cells (Fu et al., 2014) and sex-determination in mouse Sertoli cells (Levasseur et al., 2017). During early mouse embryo development, signaling as a result of the Hippo transcription

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regulatory pathway resulted from multiple cell biological functions, including cell polarization and cytoskeleton regulation to affect cell fate

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(Posfai et al., 2017). As a result of signals occurring as consequence of activation of the Hippo transcription regulatory pathway, yes-associated protein 1 (YAP1) was abundant in the placenta, prostate, testis, ovaries, and small intestine (Sudol et al., 1995). In addition, YAP1 protein was

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present in the human endometrium, and functioned to maintain homeostasis of the uterus by regulating cell proliferation and apoptosis (Song et al., 2016). Furthermore, the YAP1 protein was involved in regulating the functions of steroid hormones, and the abundance of this protein was

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regulated by gonadotropins and sex steroid hormones (Ji et al., 2017). The results of these studies indicate the Hippo transcription regulatory pathway may have important functions in male and female reproduction. The underlying mechanism of the proliferation and apoptosis effects of estradiol-17β (E2) on YAP1 in sheep EEC remain unknown. In the present study, the regulation of YAP1 production by E2 in the proliferation and apoptosis of sheep EEC was evaluated. There was investigation of the abundance and localization of YAP1 in the sheep uterus, and the effects of YAP1 on the proliferation and apoptosis of sheep

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EEC were examined using gene gain or loss-of-function procedures. Subsequently, the essential functions of E2 on the proliferation and

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apoptosis of sheep EEC were studied. 2. Materials and methods

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2.1. Ethics of animals and chemical reagents

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The study was approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (SYXK2011-0036) and National Institutes of Health Guide for Care and Use animals. The experiments were conducted in accordance with the approved guidelines. All

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antibodies were purchased from commercial suppliers, and the other chemicals were obtained commercially and of reagent grade. 2.2. Animal and sample collection

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Six clinically healthy Hu sheep (2.0 ± 0.2 years old) with good body conditions and clear pedigree from Taizhou Hailun Sheep Industry Co., Ltd were used for this study. The stage of the estrous cycle was synchronized among ewes using progestogen-impregnated pessaries that were inserted intravaginally for 12 days (Guo et al., 2017). The estrous behavior was assessed using vasectomized rams on the second day after pessary removal. The onset of the second estrous was denoted as day 0 of the estrous cycle. All ewes were slaughtered on day 2 of the estrous cycle. The sheep uteri were immediately collected and washed with 0.9% sodium chloride solution. The uterine body and the cervix were

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divided into two parts. One part was immersed in 4% paraformaldehyde for conducting immunohistochemistry (IHC) procedures, the other

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tissue was frozen at -80 °C for mRNA and protein extraction. The mid-section of the uterine horn was surgically removed and divided into three parts. The endometrial tissues were collected from one part of the uterine horn, stored at -80 °C until RNA and protein extraction. The second

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section of the uterine horn was fixed with 4% paraformaldehyde for IHC, and the third section was transported to the laboratory within 1 h of the

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time of tissue collection from the ewe carcass for EEC isolation.

The EEC were obtained from sheep uteri and cultured as described previously (Zhang et al., 2018). Briefly, the uterine horn was washed

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with 75% alcohol for 1 min and then washed three times with Phosphate Buffered Saline (PBS). The epithelial layer was finely cut into small slices and cultured in low glucose Dulbecco's Modified Eagle Medium (DMEM)/F12 with 10% fetal bovine serum (FBS) and was subsequently

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washed with DMEM/F12. After 5 days of culture, the tissue sections were removed, and the remaining attached cells were digested using 0.25% trypsin. The EEC were subsequently centrifuged (500 g, 10 min), and re-suspended in DMEM/F12 containing 10% FBS. The purity of cultured EEC was confirmed using Cytokeratin rabbit monoclonal antibody (Nguyen et al., 2017). 2.3. Immunohistochemistry assay

The paraffin-embedded uterine tissues were cut into 6-µm-thick sections and mounted on glass slides. The sections were deparaffinized in

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xylene, followed by rehydration in a series of graded concentrations of ethanol, activation of the tissue antigen in citrate-buffered solution

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(100 °C, 10 min), and quenching of endogenous peroxidase by incubating the sections in methanol with 3% (v/v) H2O2 for 10 min. After being blocked with blocking buffer (Beyotime, Haimen, China) for 2 h, the sections were incubated with YAP1 Rabbit Polyclonal antibody (1:500

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dilution, Proteintech Group, IL, USA) at 4 °C for 14 h, followed by the addition of a horseradish peroxidase-labeled secondary antibody (diluted

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at 1:4000, Proteintech Group, IL, USA), and the visualization of specific protein immunoreactivity using the substrate chromogen 3, 3′diaminobenzidine (DAB) (Beyotime, Nantong, China). The stained sections were counterstained with hematoxylin and mounted with coverslips.

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Negative control samples were obtained by replacing the primary antibody with normal rabbit serum. 2.4. Cell transfection and E2 treatment

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When the EEC were 70% confluent, EEC were transfected with the siRNA-YAP1 oligo or pEX-4-YAP1 plasmid using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. The YAP1 (NCBI accession number: NM_001267881.2) overexpression vector (pEX-4-YAP1) was constructed by Genepharma Co., Ltd (Shanghai, China). The siRNAs for YAP1, and siRNA-control listed as Table S1 were synthesized and purified at Genepharma Co., Ltd, and the most effective siRNA of these three to make further assessments (Fig. S1). After 48 h of transfection, the cells and culture media were collected for further analysis.

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For E2 (Sigma, CA, USA) treatment, E2 was dissolved in alcohol and diluted to different concentrations (10-10 to 10-6 M). The siRNA-YAP1

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transfected EEC were incubated in a serum-free medium with or without E2 at 37 °C for 24 h, then the cells and culture media were collected for further analysis.

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2.5. EdU assay

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Cells were cultured in 24-well plates with 5×105 cells/well. Cell proliferation was evaluated using a kFluor647 Click-iT EdU kit (Keygene BioTECH, Nanjing, China). Briefly, EEC were fixed in 4% PFA for 20 min at room temperature (RT), and then incubated with 2 mg/mL glycine

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solution for 5 min. After being permeabilized using 0.5% (v/v) Triton X-100 for 20 min at RT, EEC were then washed twice with 0.3% BSA and incubated with Click-iT reaction mixture in the dark for 30 min at RT. Following counterstaining with 4ʹ, 6-diamidino-2-phenylindole (DAPI)

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for 3 min at RT to label cell nuclei, EEC were lightly compressed with a coverslip and observed using a microscope. 2.6. TUNEL assay

Cells were cultured in 24-well plates with 5 × 105 cells/well. Cell apoptosis was evaluated using a One-Step TUNEL Apoptosis Assay kit (Beyotime, Haimen, China). Briefly, EEC were fixed in 4% PFA for 30 min at RT, and then washed in PBS and permeabilized with 0.1% (v/v) Triton X-100 for 5 min at RT. The EEC were then washed with PBS twice and incubated with the TUNEL labeling medium in the dark for 1 h at

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37 °C. Following counterstaining with DAPI for 3 min at RT to label the nuclei, EEC were lightly compressed with a coverslip and observed

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using a microscope. 2.7. Measurement of Caspase activities

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Caspase 3 (CASP3) and caspase 9 (CASP9) activities were analyzed using a Caspase-3 Activity kit and Caspase-9 Activity kit (Beyotime,

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Haimen, China), with Ac-DEVD-pNA and Ac-LEHD-pNA as the colorimetry-specific substrate, respectively. Briefly, EEC were washed in PBS twice and then lysed in 100 µL of chilled lysis buffer (Beyotime, Haimen, China) at 4 °C for 15 min. The lysate was centrifuged at 20,000 × g at

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4 °C for 10 min, and the supernatant was incubated with 10 µL of caspase 3 or caspase 9 substrates for 8 h at 37 °C. Activities were quantified at an absorbance of 405 nm and expressed as the fold change in enzyme activity.

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2.8. EGF and IGF-1 secretion determination

The concentrations of EGF and IGF-1 in the spent culture media were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (Sheep EGF ELISA KIT and Sheep IGF-1 ELISA KIT; Jin Yibai, Nanjing, China) according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation for EGF and IGF-1 were 15% and 10.5%, respectively. 2.9. Quantitative real-time PCR

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Total RNA was extracted from the uterine tissues and cultured EEC by using TRIzol reagent (Takara, Dalian, China) according to the

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manufacturer's instructions. The RNA concentration was determined using an ND-2000 spectrophotometer (Thermo Fisher Scientific, DE, US). A reverse transcription kit (Takara, Dalian, China) was used to remove genomic DNA and reverse transcribe RNA samples. Quantitative

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real-time PCR (qRT-PCR) was performed using a Step-One Plus Real-Time PCR System (Bio Systems, CA, USA). Reactions were conducted

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using SYBR Green Master mix (Roche, Mannheim, Germany) in a reaction volume of 20 μL. Primer sequences are provided in Table 1. The relative abundances of mRNA transcripts were quantified using the 2−△△CT method. Sample quantities were normalized using the abundance of

times.

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2.10. Western blot analysis

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the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each experiment was independently repeated at least three

Proteins were extracted using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China), and protein abundances were determined using the bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). Protein samples were diluted in gel-loading buffer, and boiled for 10 min, followed by electrophoresis of 30 µg of total protein on 10% SDS polyacrylamide gel, and then electro-transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA). After blocking with 5% (v/v) bovine serum albumin, the membrane was incubated

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with a primary antibody (diluted according to Table 2) overnight at 4 °C, and then incubated with the secondary antibody (diluted according to

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Table 2) for 1 h. Protein signals were visualized using an ECL western blot detection system (Fijifilm, Tokyo, Japan). The chemiluminescent

estimated and normalized to abundance of GAPDH.

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2.11. Statistical analysis

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intensity for each protein band was quantified using ImageJ software (Wayne Rasband, MD, USA), and then the target protein abundances were

The distribution of all data was first confirmed to be of a normal distribution using the Kolmogorov-Smirnov goodness-of-fit test. Data

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were analyzed using SPSS 19.0 (SPSS Inc. Chicago, IL, USA) and are presented as mean values ± standard error of the mean (SEM) at least three independent experiments (n ≥ 3). Comparisons between two independent groups were determined by t-test; meanwhile, the differences

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among multiple groups were analyzed using a one-way ANOVA followed by Tukey’s test. Values of P < 0.05 were considered statistically significant.

3. Results

3.1. Abundances of YAP1 in uterus

To determine if YAP1 was present in the uterus of ewes on day 2 of the estrous cycle, the relative abundance of YAP1 mRNA transcript,

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protein and localization of the protein in EEC were examined. As depicted in Figure 1, the relative abundances of YAP1 mRNA transcript and

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protein in the uterine horn were larger than those in the uterine body or cervix, respectively (Fig. 1A and 1B, P < 0.05). Furthermore, YAP1 protein was localized in the endometrial luminal epithelium (LE), glandular epithelium (GE), micro-vessels, and myometrium of the uterine horn

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in ewes on day 2 of the estrous cycle (Fig. 1C a-c), while there was a lesser amounts of YAP1 in the endometrial stroma (ES). There was no

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positive signal for YAP1 in negative control samples (Fig. 1C d-f). 3.2. Effects of YAP1 on the proliferation of EEC

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To characterize the effects of YAP1 on the proliferation of EEC in vitro, EEC were labeled with EdU. As depicted in Figure S1, the abundances of YAP1 and Phospho-Ser127-YAP1 (p-YAP1) were greater as a result of transfection of EEC with the vector (pEX-4-YAP1) which

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resulted in a relatively greater expression of the YAP1 gene (P < 0.05), and there was transfection with siRNA3-YAP1 (siRNA-YAP1) which resulted in a marked reduction in relative abundance of YAP1 mRNA transcript and protein (P < 0.05). The EEC were examined 12 h after EdU incorporation. Compared with controls, overexpression of the YAP1 gene resulted in a larger number of EdU positive EEC, while suppression of expression of the YAP1 gene resulted in lesser numbers of EdU positive EEC (Fig. 2A). Consistently, the abundance of proliferating cell nuclear antigen (PCNA) was less in EEC when there was suppression of YAP1 gene expression as compared with the values for control samples, while

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there was a greater abundances of PCNA in EEC when there was overexpression of the YAP1 gene (Fig. 2B and 2C, P < 0.05). In addition, when

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there was a relatively greater expression of the YAP1 gene, there were greater secretions of EGF and IGF-1 for EEC (Fig. 2D, P < 0.05), while the concentrations of EGF and IGF-1 were relatively lesser when there was suppression of YAP1 gene expression (Fig. 2D, P < 0.05).

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3.3. Effects of YAP1 on the apoptosis of EEC

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To investigate whether YAP1 had functions in the apoptosis of EEC, the apoptosis related variables were analyzed in the EEC of the ewes by evaluation of the response when there was suppression or stimulation of expression of the YAP1gene as a result of the relevant transfections.

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The results from TUNEL staining indicated that suppression of YAP1 gene expression resulted in a decrease in percentage of cell apoptosis compared with the EEC of the control group, while stimulation of the expression of the YAP1 gene resulted in a decrease in percentage of EEC

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undergoing apoptosis (Fig. 3A, P < 0.05). As depicted in Figure 3B and 3C when there were comparisons with the EEC of the control group, suppression of YAP1 gene expression resulted in an increased ratio of Bcl-2-associated X protein/B-cell lymphoma 2 (BAX/BCL2) (P < 0.05), while stimulation of the expression of the YAP1 gene had the opposite effect. Furthermore, suppression of YAP1 resulted in greater relative abundances of mRNAs and activities of CASP3 and CASP9 compared with the values for the control samples (P < 0.05), while stimulation of the expression of the YAP1 gene with the relevant transfection resulted in lesser relative abundances of mRNA and activities of CASP3 and

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CASP9 (Fig. 3D, P < 0.05).

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3.4. Estradiol-17β effects on proliferation of EEC when there is suppression of YAP1 gene expression To further investigate the effects of E2 on the proliferation of EEC when there is YAP1 gene expression, there was evaluation of EEC when

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there was YAP1 gene expression combined with or without E2 treatment. The 10-7 M treatment of EEC with E2 was the optimal concentration for

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increasing EGF and IGF-1 secretions from EEC, as well as for enhancing the expression of the YAP1 gene in EEC (Fig. S2, P < 0.05). Treatment with 10-7 M of E2 inhibited the effects on the lesser abundance of YAP1 when there was the transfection conducted to suppress YAP1 gene

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expression in EEC. Furthermore, the treatment of EEC with10-7 M of E2 resulted in a lesser ratio of p-YAP1/YAP1 when there was suppression of YAP1-gene expression in the EEC (Fig. S2, P < 0.05).

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Results from conducting the EdU assay indicated that with E2 treatment there was an increase in numbers of EdU positive EEC when there was YAP1 gene suppression compared with values for the EEC in the control samples (Fig. 4A, P < 0.05). The abundance of PCNA in EEC was greater when there was treatment with E2 and with siRNA-YAP1 compared with the abundance of PCNA in the group treated with only siRNA-YAP1 (Fig. 4B and 4C, P < 0.05). Furthermore, treatment of EEC with E2 resulted in a suppression of the inhibition EGF and IGF-1 secretion from the EEC that resulted from suppression of YAP1 gene expression in the EEC (Fig. 4D, P < 0.05).

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3.5. Estradiol-17β effects on apoptosis of EEC when there is suppression of YAP1 gene expression

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There was assessment of E2 effects on apoptosis of EEC induced by suppression of YAP1 gene expression. Results from TUNEL staining indicated E2 treatment resulted in a decrease of apoptosis in EEC when there was suppression of YAP1 gene expression compared with number

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of cells that were undergoing apoptosis in EEC samples from the control group (Fig. 5A, P < 0.05). In addition, the ratio of BAX/BCL2 was less

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when there was treatment of EEC with E2 and transfection with siRNA-YAP1 compared with when there was transfection with only siRNA-YAP1 (Fig. 5B and 5C, P < 0.05). When there was transfection with siRNA-YAP1, the abundance of mRNA transcripts for and activities

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of CASP3 and CASP9 were less when there was treatment with E2 and transfection with siRNA-YAP1 as compared with only transfection with siRNA-YAP1 (Fig. 5D, P < 0.05).

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4. Discussion

Results of previous studies indicate YAP1 has important functions in the proliferation and apoptosis of the human endometrium (Song et al., 2016) and E2 promotion of proliferation of human EEC (Qi et al., 2018). Consistent with results of the present study that YAP1 is abundantly present in the LE and GE of the uterus of ewes on day 2 of the estrous cycle, and could enhance proliferation of sheep EEC. Furthermore, E2 could have functions in reduction of the incidence of apoptosis when there is a relatively lesser abundance of YAP1 in sheep EEC. These results

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indicated E2 has an important function in the proliferation and apoptosis of sheep EEC through modulation of abundance of YAP1 in these cells.

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This finding could improve the understanding of the mechanisms of proliferation or apoptosis in sheep EEC. Because YAP1 has modulating effects on the Hippo transcription regulatory pathway, YAP1 is an important protein in maintaining the

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homeostasis during the period when there is modulation of uterine morphology and functions that are important for optimal embryo development

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(Song et al., 2016; Yang et al., 2019). In the present study, relative abundance of YAP1 in the uterine horn was greater than in the uterine body and cervix. The functions of the tissues of the uterine horn are essential for development and implantation of the embryo and YAP1 appears to be

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an important protein in the regulation of embryo implantation (Sasaki, 2017; Yang et al., 2019). In addition, in the present study, YAP1 protein was present in the largest abundance in LE and GE of the ewe uterine horn on day 2 of the estrous cycle, and the LE and GE are known to

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produce numerous cytokines. Cytokines were recognized as principal components for the complex intercellular communication among cells in the uterus, and temporal release patterns of cytokines during both the menstrual and estrous cycles were regulated by sex hormones (Wira et al., 2005). These results indicate YAP1 has an important function in homeostasis maintenance and functional regulation of the uterine horn. The coordination and balance between cell proliferation and apoptosis is important for uterine homeostasis. Results from several studies indicate YAP1 is an important protein for organ size control and tissue regeneration by controlling cell proliferation and apoptosis (Fu et al.,

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2014; Zhu et al., 2015). These previous results are consistent with the findings in the present study that suppression of YAP1 gene expression is

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less during the period when there is relatively greater proliferation and relatively lesser apoptosis of EEC in ewes. Furthermore, the BAX/BCL2 ratio and activities of CASP3 and CASP9 were considered to be apoptosis biomarkers in a wide variety studies in which there were different

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approached to evaluation apoptosis (Jin et al., 2018; Zheng et al., 2019). In the present study, suppression of YAP1 gene expression resulted in an There was an increased BAX/BCL2 ratio and greater CASP3 and CASP9

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increased percentage of EEC of ewes undergoing apoptosis.

activities, consistent with previous results when there was assessment of different cell types such as vascular smooth muscle cells (Liu et al.,

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2017) and human prostate cancer DU145 cells (Jin et al., 2018). In addition, EGF is primarily secreted by LE and GE (Kaushic et al., 2000), and the IGF-1 protein was primarily localized in uterine epithelial cells, which were involved in regulating cell proliferation and apoptosis (Oner and

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Oner, 2007). Furthermore, YAP1 promotes cell proliferation in response to EGF and IGF-I signaling (Strassburger et al., 2012; Chen and Harris, 2016), and with these considerations combined with results from the present study YAP1 appears to regulate the production of EGF and IGF-1 in EEC of ewes. These results indicate that YAP1 functions as a local modulator of the endocrine activity in hormone-producing cells. The YAP1 protein, therefore, may affect homeostasis of the uterus by regulating the proliferation and apoptosis of sheep EEC. Estradiol-17β has wide ranging physiological and pathological functions in the development and maintenance of the female reproductive

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system (Deroo and Korach, 2006; Zhang et al., 2017). Results from previous studies indicate E2 is important for angiogenesis of uterine tissues

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(Albrecht et al., 2003) and development of the endometrial vasculature during the menstrual cycle. Furthermore, E2 promotes epithelial-mesenchymal cell transition (Thiery et al., 2009), which is recognized as an important mechanism in embryogenesis and the migration

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of EEC. In addition, E2 can specifically bind to nuclear receptors in the GE, and then induce the activities of a series of molecular signaling

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pathways that promote proliferation of EEC (Pavone and Bulun, 2012). In the present study, E2 appeared to inhibit apoptosis in the EEC when there was suppression of YAP1gene expression, and E2 also functioned to regulate the secretions of EGF and IGF-1 from EEC by regulation of

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the abundance of YAP1. These results indicate E2 could function as a local modulator of the endocrine activity in the EEC and the aberrant secretion of E2 could lead to the disruption of proliferation and apoptosis.

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There are functions of E2 and other synthetic estrogen ligands in activation of YAP1 gene expression and when there is treatment with these compounds there are important actions on proliferation of breast cancer cells (Zhou et al., 2015). The seven-transmembrane G protein-coupled estrogen receptor (GPER), as a G protein-coupled receptor (GPCR), can be activated by E2 to with the result being a reduction in the phosphorylation of YAP1, thereby promoting YAP1 transport into the nucleus and subsequent regulation of cell migration (Carmeci et al., 1997; Revankar et al., 2005). In addition, suppression of YAP1 gene expression leads to reduced cell proliferation, and decreased E2 synthesis in human

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ovarian granulosa cells (Fu et al., 2014). Results from many studies indicate GPER can transactivate the EGFR/ERK pathway through

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metalloproteinase actions and cleave heparin-binding EGF (Filardo et al., 2000; Maggiolini et al., 2004). Results from other studies indicate that activation of Hippo transcription regulatory pathway was regulated by specific hormones and the corresponding GPCR (Miller et al., 2012; Yu et

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al., 2012). In the present study, suppression of YAP1 gene expression resulted in a reduction in the production of EGF and IGF-1 and induction

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of cell apoptosis, whereas treatment of EEC with E2 inhibited those changes resulting from suppression of YAP1 gene expression in EEC of ewes. These results are consistent with those from a previous study where treatments with E2 and other GPER agonists led to the activation of Hippo

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YAP/TAZ transcription regulatory pathway to regulate breast cancer cell proliferation (Zhou et al., 2015). These observations raise the possibility that E2 regulated the proliferation and apoptosis of EEC of ewes by modulation of the abundance of the YAP1 protein.

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5. Conclusion

In conclusion, the results of the present study indicate E2 can regulate the proliferation and apoptosis of EEC of ewes through modulation of the abundance of YAP1. This finding extends understanding of the molecular mechanisms of the proliferation and apoptosis of EEC of ewes and provides a new perspective for genetic and molecular studies on EEC of ewes. Conflicts of interest

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All the authors of the manuscript declare that: we have no financial and personal relationships with other people or organizations that could

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inappropriately influence (bias) this work.

Author contribution statement

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Shi-Yu An, Guo-Min Zhang, and Feng Wang performed the experiments, analyzed the data, and prepared the manuscript. Xiao-Xiao Gao,

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Zhi-Bo Wang, Ya-Xu Liang, and Shu-Ting Wang participated in conducting the experiments. Shen-Hua Xiao and Jiang-Tao Xia contributed to

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data analysis. Pei-Hua You fed the animals. All authors critically reviewed the manuscript and figures.

Funding

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This study was supported by the National Nature Science Foundation of China (No.31802148), the China Agriculture Research System (CARS-38) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (No.280100745113).

Conflicts of interest

All the authors of the manuscript declare that: we have no financial and personal relationships with other people or organizations that could

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inappropriately influence (bias) this work.

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Acknowledgements

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The authors thank all the members of Feng Wang’s laboratory who contributed to sample collection.

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Figure legends

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Fig.1. Location of YAP1 and relative abundance of YAP1 mRNA transcript and protein in the uterus of ewes

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(A) Relative abundance of YAP1 mRNA transcript in the uterus was analyzed using qRT-PCR; Relative abundance was normalized based on abundance of GAPDH mRNA transcript; (B) Relative abundance of YAP1 protein in the uterus was analyzed using Western blot procedures;

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Relative mRNA abundance was normalized based on abundance of GAPDH protein (C) The localization of YAP1 protein in the uterine horn (a, b and c). LE: Luminal Epithelium; GE: Glandular Epithelial; ES: Endometrial Stroma; Negative control: d, e and f. a and d (400×); Scale bar =

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100 μm; b, c, e and f (400×); Scale bar = 50 μm; Corresponding data are represented as mean ± SEM from six individuals from three

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independent experiments (n = 3); a, b, cDifferent letters indicate differences (P < 0.05)

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Fig.2. Effects of YAP1 on the EEC proliferation in ewes (A) EEC were labeled with EdU and EdU positive cells were quantified; Red color indicates EdU positive cells, and blue color indicates cell

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nuclei with DAPI staining; Scale bars = 50 μm; (B) Relative abundance of PCNA mRNA transcript in EECs when there was inhibition or

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stimulation of YAP1 gene expression as determined using qRT-PCR; Relative abundance of YAP1 mRNA transcript was normalized to the abundance of GAPDH mRNA transcript; (C) Relative abundance of PCNA protein in EEC when there was inhibition or stimulation of YAP1

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gene expression as determined using Western blot procedures; Relative abundance of YAP1 protein was normalized based on abundance of

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GAPDH protein; (D) Concentrations of EGF and IGF-1 in the culture medium

when there was inhibition or stimulation YAP1 gene expression

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in EEC; Data are presented as mean ± SEM from four independent experiments (n = 4); a, bDifferent letters indicate differences (P < 0.05)

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Fig.3. Effects of YAP1 on the EEC apoptosis in ewes

(A) Apoptotic cells were labeled with TUNEL and the TUNEL positive cells were quantified; Green color indicates positive cells with TUNEL

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staining and blue color indicates the cell nuclei with DAPI staining; Scale bars = 50 μm; (B) Relative abundances of BAX and BCL2 mRNA transcripts, and the ratio of BAX/BCL2 mRNA transcripts in EEC when there was inhibition or stimulation of YAP1 gene expression were analyzed; The relative abundances were normalized based on the abundance of GAPDH mRNA transcript; (C) Relative abundance of BAX and BCL2, and the ratio of BAX/BCL2 proteins in EEC when there was inhibition or stimulation YAP1 gene expression; Relative abundances were normalized based on abundance of GAPDH protein; (D) Relative abundances of mRNA transcripts, and proteins, and activities of CASP3 and

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CASP9 when there was inhibition or stimulation of YAP1 gene expression as determined using qRT-PCR and a caspase activity kit, respectively;

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Relative abundances of proteins were normalized based on the abundance of GAPDH protein, and activities of CASP3 and CASP9 were expressed as the fold change normalized to the activity of the control group; Corresponding data are represented as mean ± SEM from four

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independent experiments (n = 4); a, bDifferent letters indicate differences (P < 0.05)

Fig.4. Estradiol-17β effects on EEC proliferation when there was inhibition of YAP1 gene expression (A) EEC were labeled with EdU and EdU positive cells were quantified; Red color indicates EdU positive cells, and blue color indicates the cell

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nuclei with DAPI staining; Scale bars = 50 μm; (B) Treatment with estradiol-17β inhibited the effects of suppression of YAP1 gene expression

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on relative abundance of PCNA mRNA in EEC as detected using qRT-PCR; Relative abundance was normalized based on the abundance of GAPDH mRNA transcript; (C) Treatment with estradiol-17β inhibited the suppressive effects on abundance of PCNA protein when there was

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inhibition of YAP1 gene expression in EEC as detected using Western blot procedures; Relative abundance was normalized based on the

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abundance of GAPDH protein; (D) Treatment with estradiol-17β resulted in an inhibition of the suppressive effects of YAP1 on EGF and IGF-1 in EEC; Corresponding data represent the mean ± SEM from four independent experiments (n = 4); a, bDifferent letters indicate differences (P <

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0.05)

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Fig.5. Estradiol-17β effects on EEC apoptosis in ewes when there is suppression YAP1 gene expression

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(A) Apoptotic cells were labeled with TUNEL and TUNEL positive cells were quantified; Green color indicates positive cells with TUNEL staining, and blue color indicates the cell nuclei with DAPI staining; Scale bars = 50 μm; (B) Relative abundances of BAX, BCL2 and the ratio of BAX/BCL2 mRNA transcripts in EEC when there was siRNA-YAP1 transfection with or without E2 treatment as detected using qRT-PCR; Relative abundances were normalized based on abundances of GAPDH mRNA transcript; (C) Relative abundances of BAX, BCL2 and the ratio of BAX/BCL2 proteins in EEC when there was siRNA-YAP1 transfection with or without E2 treatment were detected using Western blot

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procedures; Relative abundances were normalized to abundance of GAPDH protein; (D) Relative abundances and activities of CASP3 and

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CASP9 in EECs when there was siRNA-YAP1 transfection with or without treatment with E2 were detected using qRT-PCR and caspase activity kit, respectively; Relative abundances were normalized to the abundance of GAPDH protein, and activities of CASP3 and CASP9 were

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expressed as the fold change normalized to activities of the control group; Corresponding data are represented as mean ± SEM from four

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independent experiments (n = 4); a, bDifferent letters indicate differences (P < 0.05)

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Table 1

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Details of primer sequences, expected product sizes and Genbank accession numbers of genes used for qRT-PCR Primer sequence (5′- 3′)

Target gene

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F:5’-CTGCTTCGGCAGGAATTAGC-3’ YAP1

Product size (bp)

Accession number

166

NM_001267881.2

123

XM_004015363.3

176

XM_012103831.2

274

XM_015104560.1

236

XM_012187488.2

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R:5’-CGTCGCGAGAGTGATAGGTG-3’ F:5’-GTGTCTGAAGCGCATTGGAG-3’ BAX

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R:5’-TCGGAAAACATTTCAGCCGC-3’ F:5’- ATGACTTCTCTCGGCGCTAC-3’

BCL2

R:5’- CTCCACACACATGACCCCTC-3’

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F:5’-TCAGGGAAACCTTCACGAGC-3’

CASP3

R:5’-CCTCGGCAGGCCTGAATAAT-3’ F:5’-GCCAAGCCAAGGAAAACTCG-3’

CASP9

R:5’-CACGGCAGAAGTTCACGTTG-3’

f PCNA

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R:5’-GCCAAGGTGTCCGCATTATC-3’

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F:5’-AGTGGCGTGAACCTACAGAG-3’

F:5’-CGACTTCAACAGCGACACTCAC-3’ GAPDH

203

XM_004014340.3

119

NM_001190390.1

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R:5’-CCCTGTTGCTGTAGCCGAATTC-3’

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YAP1: yes associated protein 1; BAX: Bcl-2-associated X protein; BCL2: B-cell lymphoma 2; CASP3: caspase3; CASP9: caspase9; PCNA:

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proliferating cell nuclear antigen; GAPDH: glyceraldehyde-3-phosphate dehydrogenase

Table 2

Antibodies name

Cat NO.

YAP1 Rabbit Polyclonal antibody

13584-1-AP

Source Proteintech Group, Rosemont

Dilutions

1:500

(IL,USA)

BAX Rabbit Polyclonal antibody

50599-2- Ig

Proteintech Group, Rosemont

1:2000

BCL2 Rabbit Polyclonal antibody

12789-1-AP

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(IL,USA) Proteintech Group, Rosemont

1:2000

(IL,USA)

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Abcam

Anti-PCNA antibody

ab15497

1:600

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GAPDH Mouse Monoclonal antibody

Sango Biotech

D151452

lP

Anti-YAP1 (Phospho-Ser127) Polyclonal antibody

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(Cambridge, UK)

60004-1-Ig

HRP-conjugated Affinipure Goat Anti- Rabbit

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SA00001-2

1:800 (Shanghai, China) Proteintech Group, Rosemont (IL,USA) Proteintech Group, Rosemont (IL,USA)

HRP-conjugated Affinipure Goat Anti-Mouse

Proteintech Group, Rosemont

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IgG(H+L)

SA00001-1

IgG(H+L)

Details of antibodies used for western blot

1:8000

(IL,USA)

1:4000

1:4000

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lP

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