Abstract
Uterine receptivity to the embryo is crucial for successful implantation. The establishment of uterine receptivity requires a large amount of energy, and abnormal energy regulation causes implantation failure. Glucose metabolism in the endometrium is tissue specific. Glucose is largely stored in the form of glycogen, which is the main energy source for the endometrium. AMP-activated protein kinase (AMPK), an important energy-sensing molecule, is a key player in the regulation of glucose metabolism and its regulation is also tissue specific. However, the mechanism of energy regulation in the endometrium for the establishment of uterine receptivity remains to be elucidated. In this study, we aimed to investigate the energy regulation mechanism of mouse uterine receptivity and its significance in embryo implantation. The results showed that the AMPK, p-AMPK, glycogen synthase 1, and glycogen phosphorylase M levels and the glycogen content in mouse endometrial epithelium varied in a periodic manner under regulation by the ovarian hormone. Specifically, progesterone significantly activated AMPK, promoted glycogenolysis, and upregulated glycogen phosphorylase M expression. AMPK regulated glycogen phosphorylase M expression and promoted glycogenolysis. AMPK was also found to be activated by changes in the energy or glycogen of the endometrial epithelial cells. The inhibition of AMPK activity or glycogenolysis altered the uterine receptivity markers during the window of implantation and ultimately interfered with implantation. In summary, consistency and synchronization of AMPK and glycogen metabolism constitute the core regulatory mechanism in mouse endometrial epithelial cells involved in the establishment of uterine receptivity.
Introduction
Embryo implantation is the process by which a mammalian pregnancy is established through interactions between the blastocyst and the receptive endometrium; it is mediated by the ovarian hormones estrogen and progesterone throughout a precisely controlled period called the ‘window of implantation’ (day 4 of pregnancy in mouse, days 20–24 of pregnancy in human) (Wang & Dey 2006, Zhang et al. 2013, Rosario & Stewart 2016, Ochoa-Bernal & Fazleabas 2020). The World Health Organization has reported that 8–10% of couples worldwide are facing infertility problems, and the incidence of female infertility has been rapidly increasing (Cakmak & Taylor 2011). Accumulating evidence reveals that impaired uterine receptivity is a major reason for unexplained infertility. Therefore, to develop treatments for unexplained infertility, it is necessary to first elucidate the mechanism by which uterine receptivity is established and maintained.
During the window of implantation, specific morphological and functional changes occur in the endometrium, such as in the levels of leukemia inhibitory factor (LIF), mucins (e.g. Muc1), and lactoferrin (Ltf) and the formation of pinopodes (Tu et al. 2014, Rarani et al. 2018, Zupinet al. 2018), all of which require a large amount of energy. However, the vascular remodeling of the endometrium has not yet occurred (Karizbodagh et al. 2017), it is difficult to meet the demand for such a large amount of energy in the short window of implantation from the glucose supply in circulating maternal blood. Studies have shown that the endometrium in minks, rats, and rabbits has large reserves of glycogen, the content of which changes periodically with the phases of the reproductive cycle (Gregoire & Hafs 1971, Dean et al. 2014). This suggests that glycogen metabolism in endometrial epithelial cells (EECs) plays an important role in the implantation process.
Glucose metabolism in the uterus is characterized by a lack of gluconeogenesis and storage of excess glucose in the form of glycogen (Yánez et al. 2003, Dean et al. 2014). Ovarian hormones can directly or indirectly affect the glycogen metabolism in EECs. In mink uterine tissue, estrogen promotes glycogen synthesis, whereas progesterone promotes glycogenolysis (Bowman & Rose 2017). The glycogen content of the endometrium also changes periodically with the periodic changes in endometrial morphology and function. For example, in rodents, the endometrial glycogen level is highest during pro-estrus but low during estrus (Dean et al. 2014). In rats, sheep, rabbits, and minks, the glycogen reserves in the endometrial epithelium are mobilized before and heavily used during embryo implantation (Murdoch 1970, Gregoire & Hafs 1971, Demers et al. 1972, O’Shea & Murdoch 1978, Dean et al. 2014). In the human endometrium, glycogen also accumulates in the epithelium during the late-proliferation to early-secretory phases, whereas glycogen is mobilized during the late-secretory phase to supply energy for embryo implantation (Verma 1983, Spornitz 1992). Compared with women of healthy childbearing age, the endometrial glycogen content in women with infertility issues or miscarriage is significantly reduced (Maeyama et al. 1977). These reports indicate that glycogen storage and utilization, involving the processes of glycogen synthesis and glycogenolysis, constitute a core mechanism in EECs implicated in ensuring the energy supply required for successful embryo implantation. However, how the glycogen metabolism in EECs adapts to the energy requirements of functional activities related to embryo implantation remains to be elucidated.
AMP-activated protein kinase (AMPK) is a serine/threonine-protein kinase that is expressed in a wide variety of cells and organisms, including yeasts, plants, and mammals (Hardie & Carling 1997). The role of AMPK as an energy-sensing molecule has been confirmed in a variety of cells, such as skeletal muscle cells and adipocytes (Ahmadian et al. 2011, Hardie 2011, Mihaylova & Shaw 2011). The regulation of AMPK in cells is tissue specific. The two-way feedback regulation between AMPK and glycogen metabolism is an important regulatory mechanism involved in cellular glucose metabolism. Changes in cell energy requirements can lead to changes in AMPK phosphorylation and its functional activity, which not only affect the activities of enzymes involved in glucose metabolism, thus causing rapid regulatory changes in glucose metabolism, but also affect the expression of these enzymes, resulting in a long-term regulatory effect on glucose metabolism (Ha et al. 2015). However, the regulatory effect of AMPK on tissue-specific glucose metabolism in EECs remains to be elucidated.
In this study, we first detected the levels of total and phosphorylated (p)-AMPK, the content of glycogen, and the expression of the glycogen metabolism-related enzymes glycogen synthase 1 (GYS1) and glycogen phosphorylase M (PYGM) in the mouse endometrial epithelium to study their periodic fluctuations and their regulation by ovarian hormones. We next treated EECs with an AMPK activator or inhibitor to explore the role of AMPK in the regulation of glycogen metabolism in mouse EECs. We then investigated the effects of changes in intracellular glycogen metabolism or the energy state on AMPK activity to demonstrate the role of AMPK as an energy-sensing molecule in mouse EECs. Finally, we treated the endometrial epithelium with an AMPK inhibitor or a glycogen phosphorylase inhibitor to evaluate the role of AMPK and glycogen metabolism in mouse EECs within the context of uterine receptivity and embryo implantation. The findings from this study clarify not only the specificity of the regulatory mechanisms of local glucose metabolism adapted to the energy-consuming functional activities of mouse EECs during the window of implantation but also the specific regulatory mechanism of AMPK.
Materials and methods
Ethics statement
Female ICR mice were used in this study. The study protocol was approved by the Ethics and Scientific Research Committee of Sichuan University. All possible efforts were made to minimize the suffering of the experimental animals.
Experimental design
The experimental design of this study is shown in Fig. 1.
The experimental design. This study is divided into three parts. Part 1: to investigate the periodic fluctuations in AMPK, phosphorylated (p)-AMPK, and glycogen metabolism-related molecules in the mouse endometrial epithelium and their regulation by ovarian hormones. The uterine tissue or EECs of mice were used to detect the expression of AMPK, p-AMPK, GYS1 and PYGM, and glycogen content. Part 2: to elucidate the regulatory effect of AMPK on glycogen metabolism and verified whether cellular glycogen or the energy state affects AMPK. The endometrial epithelium or primary EECs were treated with AICAR or Compound C, the expression of GYS1 and PYGM and glycogen and ATP content were detected. Or primary EECs were treated with glycogen phosphorylase inhibitor or starvation, the expression of AMPK and p-AMPK were detected. Part 3: to explore the role of AMPK and glycogen metabolism in uterine receptivity and implantation, the endometrial epithelium of mice was treated with Compound C or glycogen phosphorylase inhibitor, the marker of uterine receptivity and embryo implantation were detected. AICAR, AMPK activator; Compound C, AMPK inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The experimental design. This study is divided into three parts. Part 1: to investigate the periodic fluctuations in AMPK, phosphorylated (p)-AMPK, and glycogen metabolism-related molecules in the mouse endometrial epithelium and their regulation by ovarian hormones. The uterine tissue or EECs of mice were used to detect the expression of AMPK, p-AMPK, GYS1 and PYGM, and glycogen content. Part 2: to elucidate the regulatory effect of AMPK on glycogen metabolism and verified whether cellular glycogen or the energy state affects AMPK. The endometrial epithelium or primary EECs were treated with AICAR or Compound C, the expression of GYS1 and PYGM and glycogen and ATP content were detected. Or primary EECs were treated with glycogen phosphorylase inhibitor or starvation, the expression of AMPK and p-AMPK were detected. Part 3: to explore the role of AMPK and glycogen metabolism in uterine receptivity and implantation, the endometrial epithelium of mice was treated with Compound C or glycogen phosphorylase inhibitor, the marker of uterine receptivity and embryo implantation were detected. AICAR, AMPK activator; Compound C, AMPK inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The experimental design. This study is divided into three parts. Part 1: to investigate the periodic fluctuations in AMPK, phosphorylated (p)-AMPK, and glycogen metabolism-related molecules in the mouse endometrial epithelium and their regulation by ovarian hormones. The uterine tissue or EECs of mice were used to detect the expression of AMPK, p-AMPK, GYS1 and PYGM, and glycogen content. Part 2: to elucidate the regulatory effect of AMPK on glycogen metabolism and verified whether cellular glycogen or the energy state affects AMPK. The endometrial epithelium or primary EECs were treated with AICAR or Compound C, the expression of GYS1 and PYGM and glycogen and ATP content were detected. Or primary EECs were treated with glycogen phosphorylase inhibitor or starvation, the expression of AMPK and p-AMPK were detected. Part 3: to explore the role of AMPK and glycogen metabolism in uterine receptivity and implantation, the endometrial epithelium of mice was treated with Compound C or glycogen phosphorylase inhibitor, the marker of uterine receptivity and embryo implantation were detected. AICAR, AMPK activator; Compound C, AMPK inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Animal treatment and in vivoexperiments
Ninety-six ICR mice were purchased from Chengdu Dashuo Biological Technology (Chengdu, China) and housed under standard environmental conditions (temperature of 20°C, 12 h of light per day) and provided with food and water. This part includes five experiments, as shown in Fig. 2.
In vivo experimental design. The in vivo experiment was divided into five parts. ① and ②: Ten ovariectomized mice and 20 pregnant mice on days 1-4 were used to detect the expression of AMPK, p-AMPK, GYS1, PYGM and glycogen content in endometrial epithelium; ③: Twenty pregnant mice on day 2 were used to detect the expression of GYS1, PYGM and glycogen content in EECs; ④Thirty-six pregnant mice on day 2 were used to detect the marker of uterine receptivity and embryo implantation; ⑤ Ten pseudopregnant mice on day 2 were used to detect the embryo implantation.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
In vivo experimental design. The in vivo experiment was divided into five parts. ① and ②: Ten ovariectomized mice and 20 pregnant mice on days 1-4 were used to detect the expression of AMPK, p-AMPK, GYS1, PYGM and glycogen content in endometrial epithelium; ③: Twenty pregnant mice on day 2 were used to detect the expression of GYS1, PYGM and glycogen content in EECs; ④Thirty-six pregnant mice on day 2 were used to detect the marker of uterine receptivity and embryo implantation; ⑤ Ten pseudopregnant mice on day 2 were used to detect the embryo implantation.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
In vivo experimental design. The in vivo experiment was divided into five parts. ① and ②: Ten ovariectomized mice and 20 pregnant mice on days 1-4 were used to detect the expression of AMPK, p-AMPK, GYS1, PYGM and glycogen content in endometrial epithelium; ③: Twenty pregnant mice on day 2 were used to detect the expression of GYS1, PYGM and glycogen content in EECs; ④Thirty-six pregnant mice on day 2 were used to detect the marker of uterine receptivity and embryo implantation; ⑤ Ten pseudopregnant mice on day 2 were used to detect the embryo implantation.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
In the first experiment, the mice were ovariectomized at 6 weeks of age. Two weeks later, 10 mice were primed by s.c. injection of 100 ng of the ovarian hormone estradiol (E2; Sigma-Aldrich) diluted in 100 μL of sesame oil on 2 consecutive days (Wang et al. 1994, Nakayama et al. 2020) and was then randomly divided into two groups to study the regulatory effect of ovarian hormone on AMPK activity and glycogen metabolism-related molecules. One group was given 2 mg of progesterone (P4; Sigma-Aldrich) in 100 μL of sesame oil on day 3 (the E+P group), while the other was given an equal volume of sesame oil as a control (the E group). After 24 h, uterine tissues of the mice were collected to detect the glycogen content, AMPK activity, and the expression of glycogen metabolism-related molecules.
In the second experiment, 8 weeks of female mice were mated with normal male mice of the same strain background at a 2:1 ratio to induce pregnancy. Day 1 of pregnancy was determined by the appearance of a vaginal plug. Uterine tissues were collected from 20 female mice on days 1–4 of pregnancy and used to study periodic variations in the levels of total and p-AMPK, glycogen, and glycogen metabolism-related molecules in the mouse endometrial epithelium. Glycogen in the endometrial epithelium was detected by periodic acid-Schiff (PAS) staining, while GYS1, PYGM, AMPK, and p-AMPK were detected by immunohistochemistry (IHC).
In the third experiment, at 10:00 h on day 2 of pregnancy, 20 mice were used in two experiments to explore the role of AMPK in the regulation of glycogen metabolism in EECs. Briefly, 10 of these mice were injected with 20 μL of the AMPK activator AICAR (10 mmol/L, A9978; Sigma-Aldrich) into one uterine horn and an equal volume of solvent as a control into the contralateral horn. The remaining 10 mice were injected with 5 μL of the AMPK inhibitor Compound C (5 mmol/L, P5449; Sigma-Aldrich) following the same procedure. On day 4 of pregnancy, the expression of GYS1 and PYGM and the level of glycogen in the endometrium were detected using IHC and PAS staining, respectively.
In the fourth experiment, to evaluate the role of AMPK-glycogen interaction in EECs within the context of embryo implantation, 16 mice were included in an experiment to explore the role of AMPK in EECs during embryo implantation. Briefly, at 10:00 h on day 2 of pregnancy, each mouse was injected with 5 μL of the AMPK inhibitor Compound C (5 mmol/L, P5449; Sigma-Aldrich) into one uterine horn and with an equal volume of solvent as a control into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected from all mice, and pinopode formation was evaluated by scanning electron microscopy (s.e.m.), and total RNA was extracted. Quantitative reverse transcription-PCR (RT-qPCR) was performed to detect the expression of Ltf and Muc1 mRNAs in the EECs, and IHC was performed to detect the expression of LIF proteins in the endometrial glandular epithelium. Finally, the embryo implantation sites in the mice were observed on day 5 of pregnancy. Next, 20 mice were included in an experiment to explore the role of glycogen metabolism in EECs during embryo implantation. Briefly, each mouse was injected with 5 μL of a glycogen phosphorylase inhibitor (GPI, 10 mmol/L, 17578-1; Cayman Chemical, Ann Arbor, MI, USA) into one uterine horn and with an equal volume of solvent as a control into the contralateral horn. At 20:00 h on day 4, the experimental indicators and methods are as above.
In the fifth experiment, mouse embryo transfer was performed to determine whether the blastocyst quality would affect the implantation outcome. Female mice of 8 weeks were mated with vasectomized male mice of the same strain background at a 2:1 ratio to induce pseudopregnancy. Day 1 of pseudopregnancy was determined by the appearance of a vaginal plug. On day 2 of pseudopregnancy, 5 of 10 mice were injected with 5 μL of GPI into one uterine horn and with an equal volume of solvent as a control into the contralateral horn. The remaining five mice were injected with 5 μL of the AMPK inhibitor into one uterine horn and with an equal volume of solvent as a control into the contralateral horn. At 20:00 h on day 4, blastocysts from the normal donor mice were transferred into the bilateral uterine horns of these treated pseudopregnant mice. The embryo implantation sites were observed on day 6.
Mouse EECs purification and culture and in vitro experiments
Mouse EECs were isolated enzymatically from uterine tissues according to a previously described method (Pan et al. 2017). The purity of EECs was confirmed by detecting cytokeratin (CK)-19 protein by immunofluorescence. Cultures with a cell purity of ≥90% were identified as EECs. The EECs were used in experiments once the confluence level reached ≥ 80%.
To study the regulatory effect of ovarian hormones on AMPK activity and glycogen metabolism-related molecules, the EECs were pre-treated with E2 and randomly divided into two groups: one group was given 10−6 mol/L of P4 (the E+P group), while the other was given an equal volume of solvent as a control (the E group). After 24 h, the EECs were collected to detect AMPK activity and the expression of glycogen metabolism-related molecules.
We then aimed to evaluate the role of AMPK as an energy-sensing molecule in EECs, including its role in sensing glycogen metabolism and the energy state. The primary cultured EECs were randomly divided into two groups: one group was treated with 50 μmol/L GPI for 24 h, and the other with an equal volume of solvent as control. Western blotting was used to detect the levels of total AMPK and p-AMPK. To evaluate the effect of AMPK on energy requirements, the primary cultured EECs were randomly divided into two groups: one group (starvation group) was incubated in glucose-free DMEM (A1443001; Gibco, China) supplemented with charcoal-stripped 10% fetal bovine serum for 1 h, and the other in the same medium but with 5 mmol/L glucose as a control. Western blotting was used to detect the levels of total AMPK and p-AMPK.
To study the role of AMPK in the regulation of glycogen metabolism in EECs, EECs were pre-treated with E2 and randomly divided into the AICAR and Compound C groups. Cells in the AICAR group were treated with 1 mmol/L AICAR or an equal volume of solvent (control). Cells in the Compound C group were treated with 10 μmol/L Compound C or an equal volume of solvent (control). After 24 h, EECs were collected from both groups. The levels of GYS1 and PYGM were detected by Western blotting, and the ATP content was detected using an ATP Assay Kit (A095-1-1, Jiancheng, Nanjing, China).
RNA extraction and RT-qPCR
Total RNA was extracted from the EECs using TRIzol reagent according to the manufacturer’s instructions. Subsequently, RT-PCR was performed using HiScript II Reverse Transcriptase (R211-01, Vazyme, Nanjing, China), the reaction system is as follows: to make a mixture: RNase-free ddH2O 20 µL, 2× RT Mix 10 µL, HiScript II Enzyme Mix 2 µL, Oligo (dT)23VN (50 µM) 1 µL, Random hexamers (50 ng/µL) 1 µL, Total RNA 1 pg to 1 µg. The reaction conditions: 25°C 5 min, 50°C 15 min, and 85°C 2 min.
qPCR was performed using SYBR Green probes (R123-01, Vazyme) according to the manufacturers’ protocols. The following primers were used for RT-qPCR (TsingKe, Chengdu, China):
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β-actin-forward: 5′- CATCCGTAAAGACCTCTATGCCAAC-3′
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β-actin -reverse: 5′- ATGGAGCCACCGATCCACA-3′
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Ltf-forward: 5′-TGAGGCCCTTGGACTCTGT-3′
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Ltf- reverse: 5′- ACCCACTTTTCTCATCTCGTTC-3′
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Muc1-forward: 5′- GGCATTCGGGCTCCTTTCTT-3′
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Muc1- reverse: 5′- TGGAGTGGTAGTCGATGCTAAG-3′
The 2−ΔΔCt method was used to calculate the relative gene expression according to the threshold cycle. The data for each transcript were normalized to the β-actin mRNA level to compensate for unequal amounts of mRNA in the samples.
Immunohistochemistry (IHC)
IHC was performed using the immunostaining kit SP-9001 (ZSGBBIO, Beijing, China) as previously described (Nie et al. 2019). The uterine tissues were fixed in 4% paraformaldehyde for 48 h. After dewaxing, hydration, and blocking of endogenous peroxidase activity with H2O2 for 20 min and background activity with goat serum for 30 min, the paraffin sections were incubated overnight at 4°C with polyclonal rabbit antibodies. The following antibodies were used: GYS1 (1:250 dilution; ab40810, Abcam), PYGM (1:300; ab203343, Abcam), AMPK (1:300; ab131512, Abcam), p-AMPK (1:300; ab23875, Abcam), and LIF (1:300; 26757-1-AP, Proteintech, Wuhan, China). For the negative control, the primary antibody was replaced with a non-specific IgG. And incubated with a biotinylated secondary antibody for 30 min and with an enzyme for 30 min. A color reaction was performed using diaminobenzidine, and the sections were counterstained with hematoxylin. The staining results were observed through an optical microscope, and Image-Pro Plus 6.0 software was used to assess the area and density of the dyed region, and the integrated optical density (IOD) value of the IHC section. The mean densitometry of the digital image (magnification, x400) was designated as representative target molecules staining intensity. The signal density of the tissue areas from three to five randomly selected fields was counted in a blinded manner and subjected to statistical analysis (Chen et al. 2017).
IHC of the cells was performed as follows. EECs were fixed in 4% paraformaldehyde for 30 min, treated with Triton X-100 for 20 min, and then blocked endogenous peroxidase activity with H2O2 for 20 min, following the same procedure as in IHC for tissue. The following antibodies were used: CK-19 (1:300; 10712-1-AP, Proteintech), vimentin (1:300; bs-8533R, Bioss). The primary antibody with a non-specific IgG was considered as a control.
Immunofluorescence staining
Immunofluorescence staining was performed according to a previously described method (Pan et al. 2017). The EECs were fixed in 4% paraformaldehyde for 10 min, and then blocked for 30 min in 3% bovine serum in PBS. The cells were incubated at 4°C overnight with polyclonal rabbit anti-CK-19 (1:50, 10712-1-AP) diluted in PBS. The cells were then incubated with the secondary antibody for 1 h at room temperature. The secondary antibody used for CK-19 was goat anti-rabbit IgG/FITC (1:300, Bioss). The cell nuclei were stained with DAPI (Beyotime). The staining results were observed via fluorescence microscopy.
Western blotting
EECs were lysed with RIPA protein lysis buffer (P0013B, Beyotime, Shanghai, China). The lysates were homogenized and then centrifuged at 15,700 g for 10 min at 4°C to collect the supernatant. Protein concentrations were detected by BCA protein assay (P0012S, Beyotime). Subsequently, Western blotting was performed according to a standard protocol. Total proteins extracted from EECs were resolved on SDS-PAGE gels (30 μg protein per lane). After electrophoresis, proteins were transferred to PVDF membranes (IPVH00010, Millipore), and then the membranes were blocked with 5% skim milk powder (1172GR100, Biofroxx, Einhausen, Germany) in TBST for 2 h at room temperature. The following primary antibodies were used: rabbit polyclonal antibodies against AMPK (1:600 dilution; ab131512, Abcam), p-AMPK (1:600; ab23875, Abcam), PYGM (1:600; ab203343, Abcam), GYS1 (1:600; ab40810, Abcam), and β-actin (1:3000; AF7018, Affinity, Changzhou, China). For all blots, a horseradish peroxidase-labeled goat anti-rabbit IgG (1:2000; bs-0295G-HRP; Bioss, Beijing, China) was used as the secondary antibody. A chemiluminescence reagent (4AW012; 4A Biotech, Beijing, China) was used to visualize the blots. Image Lab 4.0 software was used to analyze the blots.
Glycogen Periodic Acid-Schiff (PAS) staining
PAS staining was performed using a Glycogen Periodic Acid-Schiff Staining Kit (G1281, Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, fixed and paraffin-embedded uterine tissues were treated with periodic acid and the Schiff reagent after deparaffinization and rehydration. The tissues were then observed using an optical microscope, where the intracellular glycogen deposits appeared as red-stained areas. Image-Pro Plus 6.0 was used to quantify the staining images.
Determination of the intracellular ATP level
Detection of pinopodes by scanning electron microscope (SEM)
Uterine tissue samples were cut into 1-mm3 sections and fixed in 2.5% glutaraldehyde overnight. The samples were then washed twice with phosphate buffer and once with a 4% sucrose solution, followed by gradient alcohol dehydration and critical point drying. Each sample was observed under a scanning electron microscope.
Embryo transfer
Pseudopregnant mice were produced by mating females with vasectomized males. Day 1 of pseudopregnancy was defined as the day of the appearance of a vaginal plug. On day 2 of pseudopregnancy, each mouse was injected with 5 μL of GPI (10 mmol/L) or an AMPK inhibitor (5 mmol/L) dissolved in sterile PBS into one uterine horn and with an equal volume of sterile PBS as a control into the contralateral horn. At 20:00 h on day 4 of pseudopregnancy (window of implantation), 128 blastocysts were harvested from donor mice and transferred into the uterine horns of eight recipient mice. The blastocyst implantation rates on day 6 were compared between the treated and untreated uterine horns.
Statistical analysis
GraphPad Prism software was used to assess differences between the experimental groups. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. Comparisons involving three or more groups were performed using a one-way ANOVA, followed by Tukey’s multiple comparisons test. All of the data are presented as means ± s.d.. A P value < 0.05 was considered to indicate a statistically significant difference.
Results
AMPK activity, glycogen content, and glycogen metabolism-related molecule expression in the mouse endometrial epithelium varied in a periodic manner on days 1-4 of pregnancy
The IHC results revealed that the expressions of total AMPK and p-AMPK in pregnant mice showed similar trends of a gradual increase on days 1–2 but a decrease on days 3–4 (P < 0.01, Fig. 3A and B). The p-AMPK/AMPK ratio, which reflects AMPK activity, in the pregnant mice was increased on days 1–2 but decreased on days 3–4 and was lowest on day 4 (P < 0.01, Fig. 3B). The expression of GYS1 was highest on day 1 and declined thereafter to the lowest level on day 4 (P < 0.05, Fig. 3A and B). The expression of PYGM was lowest on day 1, increased on day 2, and remained almost unchanged on days 3–4 of pregnancy (P < 0.01, Fig. 3A and B). Negative control was shown in Fig. 3C.
The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium on days 1–4 of pregnant mice. (A) The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium on days 1–4 was detected by IHC, n =5 mice per group, the arrows show the endometrial epithelium. scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of AMPK, p-AMPK, GYS1, and PYGM protein in the endometrial epithelium on days 1–4 were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving three or more groups were analyzed using a one-way ANOVA, followed by Tukey’s multiple comparisons test; (C) A negative control for IHC. Different letters above the columns, such as a and b, indicate P < 0.05, and if two columns have the same letter, it indicates no statistical significance.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium on days 1–4 of pregnant mice. (A) The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium on days 1–4 was detected by IHC, n =5 mice per group, the arrows show the endometrial epithelium. scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of AMPK, p-AMPK, GYS1, and PYGM protein in the endometrial epithelium on days 1–4 were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving three or more groups were analyzed using a one-way ANOVA, followed by Tukey’s multiple comparisons test; (C) A negative control for IHC. Different letters above the columns, such as a and b, indicate P < 0.05, and if two columns have the same letter, it indicates no statistical significance.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium on days 1–4 of pregnant mice. (A) The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium on days 1–4 was detected by IHC, n =5 mice per group, the arrows show the endometrial epithelium. scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of AMPK, p-AMPK, GYS1, and PYGM protein in the endometrial epithelium on days 1–4 were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving three or more groups were analyzed using a one-way ANOVA, followed by Tukey’s multiple comparisons test; (C) A negative control for IHC. Different letters above the columns, such as a and b, indicate P < 0.05, and if two columns have the same letter, it indicates no statistical significance.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The glycogen content in the endometrial epithelium was detected by PAS staining. The glycogen content was highest on day 1 of pregnancy and declined thereafter to the lowest level on day 4 (P < 0.05, Fig. 4A and B). Negative control was showed in Fig. 4C.
Glycogen content in the endometrial epithelium on days 1–4 of pregnant mice. (A) Glycogen content in the endometrial epithelium on days 1–4 was detected by Glycogen PAS staining, n =5 mice per group, the arrows show the endometrial epithelium. scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of glycogen in the endometrial epithelium on days 1–4 were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving three or more groups were analyzed using a one-way ANOVA, followed by Tukey’s multiple comparisons test; (C) A negative control for PAS. Le, luminal epithelium. Different letters indicate P < 0.05.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Glycogen content in the endometrial epithelium on days 1–4 of pregnant mice. (A) Glycogen content in the endometrial epithelium on days 1–4 was detected by Glycogen PAS staining, n =5 mice per group, the arrows show the endometrial epithelium. scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of glycogen in the endometrial epithelium on days 1–4 were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving three or more groups were analyzed using a one-way ANOVA, followed by Tukey’s multiple comparisons test; (C) A negative control for PAS. Le, luminal epithelium. Different letters indicate P < 0.05.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Glycogen content in the endometrial epithelium on days 1–4 of pregnant mice. (A) Glycogen content in the endometrial epithelium on days 1–4 was detected by Glycogen PAS staining, n =5 mice per group, the arrows show the endometrial epithelium. scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of glycogen in the endometrial epithelium on days 1–4 were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving three or more groups were analyzed using a one-way ANOVA, followed by Tukey’s multiple comparisons test; (C) A negative control for PAS. Le, luminal epithelium. Different letters indicate P < 0.05.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Taken together, these results demonstrate that AMPK activity, glycogen metabolism-related molecule levels, and the glycogen content in the endometrial epithelium exhibit periodic fluctuations. This suggests that there is a regulatory relationship between ovarian hormones, AMPK, and glycogen, all of which are altered in response to the morphological and functional changes in the endometrium for implantation.
Ovarian hormones regulate AMPK activity and the expression of glycogen metabolism-related molecules in EECs
The in vivo experimental results showed that compared with the baseline expression levels of GYS1, PYGM, AMPK, p-AMPK, and p-AMPK/AMPK in the endometrial epithelium in the E group, the expression of GYS1 was decreased but that of PYGM, AMPK, p-AMPK, and p-AMPK/AMPK was increased in the E+P group (P < 0.01, Fig. 5A and B). Similarly, compared with the baseline level of glycogen in the endometrial epithelium in the E group, the glycogen content was decreased in the E+P group (P < 0.001, Fig. 6A and B). Negative control was shown in Fig. 6C.
The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium of ovariectomized mice. (A) The expression of p-AMPK, AMPK, GYS1, and PYGM in the endometrial epithelium was detected by IHC, n = 5 mice per group, the arrows show the endometrial epithelium. Scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of p-AMPK, AMPK, GYS1, and PYGM protein in the endometrial epithelium counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving two groups were analyzed using a paired-samples t-test. **P < 0.01, ***P < 0.001. E, estrogen group; E+P, estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium of ovariectomized mice. (A) The expression of p-AMPK, AMPK, GYS1, and PYGM in the endometrial epithelium was detected by IHC, n = 5 mice per group, the arrows show the endometrial epithelium. Scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of p-AMPK, AMPK, GYS1, and PYGM protein in the endometrial epithelium counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving two groups were analyzed using a paired-samples t-test. **P < 0.01, ***P < 0.001. E, estrogen group; E+P, estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The expression of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium of ovariectomized mice. (A) The expression of p-AMPK, AMPK, GYS1, and PYGM in the endometrial epithelium was detected by IHC, n = 5 mice per group, the arrows show the endometrial epithelium. Scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of p-AMPK, AMPK, GYS1, and PYGM protein in the endometrial epithelium counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving two groups were analyzed using a paired-samples t-test. **P < 0.01, ***P < 0.001. E, estrogen group; E+P, estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The content of glycogen in the endometrial epithelium of ovariectomized mice. (A) The content of glycogen in the endometrial epithelium were detected by PAS, n = 5 mice per group, the arrows show the endometrial epithelium. Scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of glycogen in the endometrial epithelium counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving two groups were analyzed using a paired-samples t-test. ***P < 0.001. (C) The negative control for PAS. E, estrogen group; E+P: estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The content of glycogen in the endometrial epithelium of ovariectomized mice. (A) The content of glycogen in the endometrial epithelium were detected by PAS, n = 5 mice per group, the arrows show the endometrial epithelium. Scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of glycogen in the endometrial epithelium counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving two groups were analyzed using a paired-samples t-test. ***P < 0.001. (C) The negative control for PAS. E, estrogen group; E+P: estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The content of glycogen in the endometrial epithelium of ovariectomized mice. (A) The content of glycogen in the endometrial epithelium were detected by PAS, n = 5 mice per group, the arrows show the endometrial epithelium. Scale bars: 20 μm; (B) Statistical analysis of (A). Relative density analysis of glycogen in the endometrial epithelium counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. Differences in comparisons involving two groups were analyzed using a paired-samples t-test. ***P < 0.001. (C) The negative control for PAS. E, estrogen group; E+P: estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The purity of primary EECs in vitro was confirmed by staining for CK-19 (Fig. 7A). Besides, CK-19 and vimentin were used for IHC of epithelial cells, respectively, to further confirm the purity of epithelial cells (Fig. 7B). In vitro experiments were used to further verify the regulation of ovarian hormones on glycogen metabolism-related molecules in EECs. The in vitro results were consistent with the in vivo results (P < 0.05, Fig. 7C and D).
The expression of AMPK, p-AMPK, GYS1, and PYGM in EECs. (A) Identification of mouse EECs in vitro. The expression of CK19 in EECs was detected by IF. Scale bars: 100 μm. (B) The expression of CK19 and vimentin in EECs was detected by IHC. (C) The expression of GYS1 and PYGM in EECs was detected by Western blot and statistical analysis of GYS1 and PYGM protein in EECs. The experiment was repeated three times, n = 3. Relative density analysis of GYS1 and PYGM protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; (D) The expression of p-AMPK and AMPK in EECs was detected by Western blot, and statistical analysis of p-AMPK and AMPK protein in EECs. The experiment was repeated three times, n = 3. Relative density analysis of p-AMPK and AMPK protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean±SD. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; *P < 0.05. E, estrogen group; E+P, estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The expression of AMPK, p-AMPK, GYS1, and PYGM in EECs. (A) Identification of mouse EECs in vitro. The expression of CK19 in EECs was detected by IF. Scale bars: 100 μm. (B) The expression of CK19 and vimentin in EECs was detected by IHC. (C) The expression of GYS1 and PYGM in EECs was detected by Western blot and statistical analysis of GYS1 and PYGM protein in EECs. The experiment was repeated three times, n = 3. Relative density analysis of GYS1 and PYGM protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; (D) The expression of p-AMPK and AMPK in EECs was detected by Western blot, and statistical analysis of p-AMPK and AMPK protein in EECs. The experiment was repeated three times, n = 3. Relative density analysis of p-AMPK and AMPK protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean±SD. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; *P < 0.05. E, estrogen group; E+P, estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
The expression of AMPK, p-AMPK, GYS1, and PYGM in EECs. (A) Identification of mouse EECs in vitro. The expression of CK19 in EECs was detected by IF. Scale bars: 100 μm. (B) The expression of CK19 and vimentin in EECs was detected by IHC. (C) The expression of GYS1 and PYGM in EECs was detected by Western blot and statistical analysis of GYS1 and PYGM protein in EECs. The experiment was repeated three times, n = 3. Relative density analysis of GYS1 and PYGM protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; (D) The expression of p-AMPK and AMPK in EECs was detected by Western blot, and statistical analysis of p-AMPK and AMPK protein in EECs. The experiment was repeated three times, n = 3. Relative density analysis of p-AMPK and AMPK protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean±SD. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; *P < 0.05. E, estrogen group; E+P, estrogen and progesterone group.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Overall, these results demonstrated that progesterone significantly activates AMPK, promotes glycogenolysis, and upregulates PYGM expression (or downregulates GYS1 expression). This suggests that ovarian hormones can regulate AMPK activity and glycogen metabolism in EECs.
AMPK regulates the glycogen content and ATP levels of EECs
Although the above results suggest that ovarian hormones can regulate glycogen metabolism, it is not clear whether this regulatory effect of ovarian hormones occurs directly on glycogen metabolism, through AMPK, or in both ways. We used AMPK activator (AICAR) or an AMPK inhibitor (Compound C) to directly activate or inhibit AMPK activity in the endometrial epithelium of pregnant mice and evaluated the effect on the glycogen content. The results demonstrated that in comparison with the control, AICAR treatment reduced the glycogen content in EECs (P < 0.05, Fig. 8A), whereas Compound C increased the glycogen content (P < 0.05, Fig. 8B). Further, compared with the control, AICAR increased the ATP levels in EECs (P < 0.05, Fig. 8C), whereas Compound C decreased the ATP levels (P < 0.05, Fig. 8D). These results suggest that the regulation of glycogen by ovarian hormones, especially progesterone, is mediated through AMPK.
AMPK regulates glycogen and ATP content of EECs. (A) AICAR group: glycogen content and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 20 μL injection of AICAR (10 mM) into one uterine horn and the same volume solvent into the contralateral horn as a control. On day 4 of pregnancy, the level of glycogen in the endometrial epithelium were detected by using PAS staining. n = 4 mice per group. Relative density analyses of glycogen in the endometrial epithelium was counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. Le, lumen epithelium; (B) Compound C group: glycogen content and statistical analysis; n = 4 mice per group. (C) AICAR group: ATP level. The experiment was repeated three times, n = 3. (D) Compound C: ATP level. The experiment was repeated three times, n = 3. *P < 0.05 vs control, ***P < 0.001 vs control. AICAR, AMPK activators; compound C, AMPK inhibitors.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
AMPK regulates glycogen and ATP content of EECs. (A) AICAR group: glycogen content and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 20 μL injection of AICAR (10 mM) into one uterine horn and the same volume solvent into the contralateral horn as a control. On day 4 of pregnancy, the level of glycogen in the endometrial epithelium were detected by using PAS staining. n = 4 mice per group. Relative density analyses of glycogen in the endometrial epithelium was counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. Le, lumen epithelium; (B) Compound C group: glycogen content and statistical analysis; n = 4 mice per group. (C) AICAR group: ATP level. The experiment was repeated three times, n = 3. (D) Compound C: ATP level. The experiment was repeated three times, n = 3. *P < 0.05 vs control, ***P < 0.001 vs control. AICAR, AMPK activators; compound C, AMPK inhibitors.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
AMPK regulates glycogen and ATP content of EECs. (A) AICAR group: glycogen content and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 20 μL injection of AICAR (10 mM) into one uterine horn and the same volume solvent into the contralateral horn as a control. On day 4 of pregnancy, the level of glycogen in the endometrial epithelium were detected by using PAS staining. n = 4 mice per group. Relative density analyses of glycogen in the endometrial epithelium was counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. Le, lumen epithelium; (B) Compound C group: glycogen content and statistical analysis; n = 4 mice per group. (C) AICAR group: ATP level. The experiment was repeated three times, n = 3. (D) Compound C: ATP level. The experiment was repeated three times, n = 3. *P < 0.05 vs control, ***P < 0.001 vs control. AICAR, AMPK activators; compound C, AMPK inhibitors.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
AMPK regulates the expression of PYGM in EECs
EECs were treated with AICAR or Compound C in vivo and in vitro to activate or inhibit AMPK activity, respectively, and investigate the role of AMPK in the regulation of glycogen metabolism. The in vivo experimental results showed that AICAR treatment upregulated the expression of PYGM in the endometrial epithelium (P < 0.01, Fig. 9A and B) but had no significant effect on the expression of GYS1 (P > 0.05, Fig. 9A and B). The results of the in vitro experiments were consistent with these in vivo results (P < 0.05, Fig. 9C). Compound C treatment decreased the expression of PYGM in the endometrial epithelium (P < 0.01, Fig. 9D and E) but had no significant effect on the expression of GYS1 (P > 0.05, Fig. 9D and E). The results of the in vitro experiments were consistent with these in vivo results (P < 0.05, Fig. 9F). These results suggest that before implantation, AMPK regulates PYGM expression and promotes glycogenolysis in the EECs of pregnant mice.
AMPK regulates glycogen metabolism in EECs. (A,B) AICAR group: the expression of GYS1 and PYGM in the endometrial epithelium in vivo and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 20 μL injection of AICAR (10 mM) into one uterine horn and the same volume solvent into the contralateral horn as a control. On day 4 of pregnancy, the expression of GYS1 and PYGM in the endometrial epithelium was detected using IHC. Relative density analysis of GYS1 and PYGM protein in the endometrial epithelium were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. scale bars: 20 μm; (C) AICAR group: the expression of GYS1 and PYGM in EECs in vitro and their statistical analysis. EECs were treated with 1 mM AICAR or an equal volume of solvent (control). After 24 h, GYS1 and PYGM protein in EECs were detected by Western blot. The experiment was repeated three times, n = 3; (D,E) Compound C group: the expression of GYS1 and PYGM in endometrial epithelium in vivo and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of compound C (5 mM) according to the same procedure. On day 4 of pregnancy, the expression of GYS1 and PYGM in endometrial epithelium were detected by using IHC. scale bars: 20 μm; (F) compound C group: the expression of GYS1 and PYGM in EECs in vitro and their statistical analysis. EECs were treated with 10 μM Compound C or an equal volume of solvent (control). After 24 h, GYS1 and PYGM protein in EECs were detected by Western blot. The experiment was repeated three times, n = 3. *P < 0.05 vs control, ***P < 0.001 vs control. AICAR, AMPK activators; Compound C, AMPK inhibitors.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
AMPK regulates glycogen metabolism in EECs. (A,B) AICAR group: the expression of GYS1 and PYGM in the endometrial epithelium in vivo and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 20 μL injection of AICAR (10 mM) into one uterine horn and the same volume solvent into the contralateral horn as a control. On day 4 of pregnancy, the expression of GYS1 and PYGM in the endometrial epithelium was detected using IHC. Relative density analysis of GYS1 and PYGM protein in the endometrial epithelium were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. scale bars: 20 μm; (C) AICAR group: the expression of GYS1 and PYGM in EECs in vitro and their statistical analysis. EECs were treated with 1 mM AICAR or an equal volume of solvent (control). After 24 h, GYS1 and PYGM protein in EECs were detected by Western blot. The experiment was repeated three times, n = 3; (D,E) Compound C group: the expression of GYS1 and PYGM in endometrial epithelium in vivo and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of compound C (5 mM) according to the same procedure. On day 4 of pregnancy, the expression of GYS1 and PYGM in endometrial epithelium were detected by using IHC. scale bars: 20 μm; (F) compound C group: the expression of GYS1 and PYGM in EECs in vitro and their statistical analysis. EECs were treated with 10 μM Compound C or an equal volume of solvent (control). After 24 h, GYS1 and PYGM protein in EECs were detected by Western blot. The experiment was repeated three times, n = 3. *P < 0.05 vs control, ***P < 0.001 vs control. AICAR, AMPK activators; Compound C, AMPK inhibitors.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
AMPK regulates glycogen metabolism in EECs. (A,B) AICAR group: the expression of GYS1 and PYGM in the endometrial epithelium in vivo and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 20 μL injection of AICAR (10 mM) into one uterine horn and the same volume solvent into the contralateral horn as a control. On day 4 of pregnancy, the expression of GYS1 and PYGM in the endometrial epithelium was detected using IHC. Relative density analysis of GYS1 and PYGM protein in the endometrial epithelium were counted with Image-Pro Plus 6.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test. scale bars: 20 μm; (C) AICAR group: the expression of GYS1 and PYGM in EECs in vitro and their statistical analysis. EECs were treated with 1 mM AICAR or an equal volume of solvent (control). After 24 h, GYS1 and PYGM protein in EECs were detected by Western blot. The experiment was repeated three times, n = 3; (D,E) Compound C group: the expression of GYS1 and PYGM in endometrial epithelium in vivo and statistical analysis. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of compound C (5 mM) according to the same procedure. On day 4 of pregnancy, the expression of GYS1 and PYGM in endometrial epithelium were detected by using IHC. scale bars: 20 μm; (F) compound C group: the expression of GYS1 and PYGM in EECs in vitro and their statistical analysis. EECs were treated with 10 μM Compound C or an equal volume of solvent (control). After 24 h, GYS1 and PYGM protein in EECs were detected by Western blot. The experiment was repeated three times, n = 3. *P < 0.05 vs control, ***P < 0.001 vs control. AICAR, AMPK activators; Compound C, AMPK inhibitors.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of glycogenolysis or change in the energy state activates AMPK in EECs
We further found that cellular glycogen or the energy state also affects AMPK. The results showed that the total AMPK level was significantly decreased and the p-AMPK/AMPK ratio was significantly increased in the GPI-treated group relative to the control group (P < 0.05, Fig. 10A and B). However, these groups did not differ in terms of the p-AMPK level (P > 0.05, Fig. 10B). These results suggest that the glycogen metabolism of EECs affects AMPK.
Inhibition of glycogenolysis or changes in energy demand activates AMPK in EECs. (A) The expression of AMPK and p-AMPK in EECs were detected by Western blot. EECs were treated either with 50 μM GPI or an equal volume of solvent (control), the EECs were collected after 24 h, and Western blotting was used to detect the protein levels of total AMPK and p-AMPK; (B) Statistical analysis of p-AMPK and AMPK protein in EECs in (A). The experiment was repeated three times, n = 3. Relative density analysis of AMPK and p-AMPK protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; (C) The expression of AMPK and p-AMPK in EECs was detected by Western blot. EECs were incubated in glucose-free DMEM for 1 h, and Western blotting was used to detect the protein levels of total AMPK and p-AMPK; (D) Statistical analysis of p-AMPK and AMPK protein in EECs in (C). The experiment was repeated three times, n = 3. *P < .05 vs control, **P < .01 vs control. GPI, glycogen phosphorylase inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of glycogenolysis or changes in energy demand activates AMPK in EECs. (A) The expression of AMPK and p-AMPK in EECs were detected by Western blot. EECs were treated either with 50 μM GPI or an equal volume of solvent (control), the EECs were collected after 24 h, and Western blotting was used to detect the protein levels of total AMPK and p-AMPK; (B) Statistical analysis of p-AMPK and AMPK protein in EECs in (A). The experiment was repeated three times, n = 3. Relative density analysis of AMPK and p-AMPK protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; (C) The expression of AMPK and p-AMPK in EECs was detected by Western blot. EECs were incubated in glucose-free DMEM for 1 h, and Western blotting was used to detect the protein levels of total AMPK and p-AMPK; (D) Statistical analysis of p-AMPK and AMPK protein in EECs in (C). The experiment was repeated three times, n = 3. *P < .05 vs control, **P < .01 vs control. GPI, glycogen phosphorylase inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of glycogenolysis or changes in energy demand activates AMPK in EECs. (A) The expression of AMPK and p-AMPK in EECs were detected by Western blot. EECs were treated either with 50 μM GPI or an equal volume of solvent (control), the EECs were collected after 24 h, and Western blotting was used to detect the protein levels of total AMPK and p-AMPK; (B) Statistical analysis of p-AMPK and AMPK protein in EECs in (A). The experiment was repeated three times, n = 3. Relative density analysis of AMPK and p-AMPK protein in EECs was counted with Image Lab 4.0 software. Error bars represent the mean ± s.d. The statistical significance of differences between pairs of groups was analyzed using a paired-samples t-test; (C) The expression of AMPK and p-AMPK in EECs was detected by Western blot. EECs were incubated in glucose-free DMEM for 1 h, and Western blotting was used to detect the protein levels of total AMPK and p-AMPK; (D) Statistical analysis of p-AMPK and AMPK protein in EECs in (C). The experiment was repeated three times, n = 3. *P < .05 vs control, **P < .01 vs control. GPI, glycogen phosphorylase inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
To evaluate the effect of AMPK on the energy state of EECs, the primary EECs were incubated in glucose-free DMEM. The results showed that the p-AMPK level and the p-AMPK/AMPK ratio were significantly increased in the starvation group relative to the control group (P < 0.05, Fig. 10C and D). However, these groups did not differ in terms of the total AMPK level (P > 0.05, Fig. 10D). These results suggest that the energy state of EECs affects AMPK.
Taken together, these results suggest that ovarian hormones, AMPK, and glycogen (or the energy state) form multi-level feedback regulation. Specifically, glycogenolysis and AMPK form a negative feedback loop that promotes glycogenolysis to improve the efficiency of energy supply during the window of implantation.
Inhibition of AMPK activity in EECs affects the establishment of uterine receptivity and inhibits embryo implantation
Compound C or an equal volume of solvent was injected into the opposite uterine horns of each mouse on day 2 of pregnancy. Endometrial epithelial pinopodes were observed by s.e.m. on day 4 of pregnancy (window of implantation). Notably, the number of pinopodes was significantly lower in the Compound C-treated horns than in the control horns (Fig. 11A). Furthermore, a qRT-PCR analysis revealed that the expression of Ltf and Muc1 mRNA was significantly increased in the compound C-treated horns relative to the control horns (P < 0.05, Fig. 11B). IHC revealed that the expression of LIF in the endometrial glandular epithelium was significantly lower in the Compound C-treated horns than in the control horns (P < 0.05, Fig. 11C). On day 5, the number of implantation sites was significantly lower in the Compound C-treated horns than in the control horns (P < 0.05, Fig. 11D). The mouse embryo transfer experiment further confirmed the significant reduction in the embryo implantation rate in the Compound C-treated horns (P < 0.05, Fig. 11E). These results suggest that timely activation of AMPK is necessary for the establishment of uterine receptivity and successful implantation of embryos.
Inhibition of AMPK activity in EECs affects the establishment of uterine receptivity and further affects embryo implantation. (A) The morphology of pinopodes was observed by s.e.m.. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of Compound C (5 mM) into one uterine horn and an equal volume of solvent into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected, and pinopode formation was evaluated; (B) The expression of Ltf and Muc1 mRNAs was detected by qRT-PCR; (C) The expression of LIF and statistical analysis. GE: glandular epithelium. scale bars: 20 μm. (D) Left: embryo implantation was observed on day 5. Right: statistical analysis of embryo implantation; n = 4 mice per group. (E) Left: embryo implantation on day 6 of embryo transfer. Right: statistical analysis of embryo implantation rate. n = 4 mice per group. *P < 0.05 vs control. **P < 0.01 vs control.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of AMPK activity in EECs affects the establishment of uterine receptivity and further affects embryo implantation. (A) The morphology of pinopodes was observed by s.e.m.. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of Compound C (5 mM) into one uterine horn and an equal volume of solvent into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected, and pinopode formation was evaluated; (B) The expression of Ltf and Muc1 mRNAs was detected by qRT-PCR; (C) The expression of LIF and statistical analysis. GE: glandular epithelium. scale bars: 20 μm. (D) Left: embryo implantation was observed on day 5. Right: statistical analysis of embryo implantation; n = 4 mice per group. (E) Left: embryo implantation on day 6 of embryo transfer. Right: statistical analysis of embryo implantation rate. n = 4 mice per group. *P < 0.05 vs control. **P < 0.01 vs control.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of AMPK activity in EECs affects the establishment of uterine receptivity and further affects embryo implantation. (A) The morphology of pinopodes was observed by s.e.m.. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of Compound C (5 mM) into one uterine horn and an equal volume of solvent into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected, and pinopode formation was evaluated; (B) The expression of Ltf and Muc1 mRNAs was detected by qRT-PCR; (C) The expression of LIF and statistical analysis. GE: glandular epithelium. scale bars: 20 μm. (D) Left: embryo implantation was observed on day 5. Right: statistical analysis of embryo implantation; n = 4 mice per group. (E) Left: embryo implantation on day 6 of embryo transfer. Right: statistical analysis of embryo implantation rate. n = 4 mice per group. *P < 0.05 vs control. **P < 0.01 vs control.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of glycogenolysis in EECs affects the establishment of uterine receptivity and inhibits implantation of embryos
The results showed that the number of pinopodes was significantly lower in the GPI-treated horns than in the control horns (Fig. 12A). A qRT-PCR analysis revealed that the expression of Ltf and Muc1 mRNA was significantly increased in the GPI-treated horns relative to the control horns (P < 0.05, Fig. 12B). IHC revealed that the expression of LIF in the endometrial glandular epithelium was significantly lower in the GPI-treated horns than in the control horns (P < 0.05, Fig. 12C). On day 5, the number of implantation sites was significantly lower in the GPI-treated horns than in the control horns (P < 0.05, Fig. 12D). The mouse embryo transfer experiment further confirmed the significant reduction in the embryo implantation rate in the GPI-treated horns (P < 0.05, Fig. 12E). These results suggest that significant and timely upregulation of glycogenolysis is necessary for the establishment of uterine receptivity and successful implantation of embryos.
Inhibition of glycogenolysis in EECs affected the establishment of uterine receptivity and further affects embryo implantation. (A) The morphology of pinopodes was observed by s.e.m. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of GPI (10 mM) into one uterine horn and an equal volume of solvent into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected, and pinopode formation was evaluated; (B) The expression of Ltfand Muc1 mRNAs was detected by qRT-PCR; (C) The expression of LIF and statistical analysis. GE, glandular epithelium. scale bars: 20 μm. (D) Left: embryo implantation was observed on day 5. Right: statistical analysis of embryo implantation; n = 4 mice per group. (E) Left: embryo implantation on day 6 of embryo transfer. Right: statistical analysis of embryo implantation rate. n = 4 mice per group. *P < 0.05 vs control. **P < 0.01 vs control. ***P < 0.001 vs control. GPI, glycogen phosphorylase inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of glycogenolysis in EECs affected the establishment of uterine receptivity and further affects embryo implantation. (A) The morphology of pinopodes was observed by s.e.m. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of GPI (10 mM) into one uterine horn and an equal volume of solvent into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected, and pinopode formation was evaluated; (B) The expression of Ltfand Muc1 mRNAs was detected by qRT-PCR; (C) The expression of LIF and statistical analysis. GE, glandular epithelium. scale bars: 20 μm. (D) Left: embryo implantation was observed on day 5. Right: statistical analysis of embryo implantation; n = 4 mice per group. (E) Left: embryo implantation on day 6 of embryo transfer. Right: statistical analysis of embryo implantation rate. n = 4 mice per group. *P < 0.05 vs control. **P < 0.01 vs control. ***P < 0.001 vs control. GPI, glycogen phosphorylase inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Inhibition of glycogenolysis in EECs affected the establishment of uterine receptivity and further affects embryo implantation. (A) The morphology of pinopodes was observed by s.e.m. At 10:00 h on day 2 of pregnancy, mice received a 5 μL injection of GPI (10 mM) into one uterine horn and an equal volume of solvent into the contralateral horn. At 20:00 h on day 4, which was considered the window of implantation, the endometrium was collected, and pinopode formation was evaluated; (B) The expression of Ltfand Muc1 mRNAs was detected by qRT-PCR; (C) The expression of LIF and statistical analysis. GE, glandular epithelium. scale bars: 20 μm. (D) Left: embryo implantation was observed on day 5. Right: statistical analysis of embryo implantation; n = 4 mice per group. (E) Left: embryo implantation on day 6 of embryo transfer. Right: statistical analysis of embryo implantation rate. n = 4 mice per group. *P < 0.05 vs control. **P < 0.01 vs control. ***P < 0.001 vs control. GPI, glycogen phosphorylase inhibitor.
Citation: Reproduction 163, 5; 10.1530/REP-21-0382
Discussion
In this study, we obtained several important findings. First, we observed that the AMPK, p-AMPK, GYS1, and PYGM levels and the glycogen content in the mouse endometrial epithelium varied in a periodic manner under regulation by the ovarian hormone. Specifically, progesterone significantly activated AMPK, promoted glycogenolysis, and upregulated PYGM expression in EECs. Second, AMPK regulated PYGM expression and promoted glycogenolysis in EECs. Third, AMPK was also activated by changes in the energy state or glycogen content of EECs. Finally, the inhibition of AMPK activity or glycogenolysis altered pinopodes and the expression of the uterine receptivity markers during the window of implantation and ultimately interfered with embryo implantation. Overall, these findings demonstrated that progesterone upregulates PYGM through AMPK to ensure timely glycogenolysis. Meanwhile, glycogenolysis was found to have a negative feedback effect on AMPK. This bidirectional regulation ensures a large supply of energy to the endometrium in a short period of time during the window of implantation to ensure the establishment of uterine receptivity and successful embryo implantation.
Many functional activities in the endometrial epithelium exhibit periodicity in response to the regulatory effects of E2 and P4. In this study, we first determined the periodic fluctuations in AMPK, p-AMPK, and glycogen metabolism-related molecules in the mouse endometrial epithelium under regulation by ovarian hormones. To our knowledge, this is the first study to evaluate periodic characteristics of AMPK, p-AMPK, GYS1, and PYGM in the endometrial epithelium. We further found that the glycogen content in the mouse endometrial epithelium also varied in a periodic manner, which is consistent with previous findings in rats, rabbits, and minks (Gregoire & Hafs 1971, Dean et al. 2014). In addition, our results showed that progesterone significantly activated AMPK, promoted glycogenolysis, and upregulated PYGM expression. Notably, the regulatory effects of estrogen and progesterone on glycogen metabolism are controversial. In rats, rabbits, and minks, estrogen promotes glycogen synthesis, and progesterone promotes glycogenolysis (Demers et al. 1973a), whereas in cats and primates, progesterone promotes glycogen synthesis (Demers et al. 1973b, Jaffe et al. 1985). We speculate that these differences in estrogen- and progesterone-mediated regulation of glycogen metabolism may be related to species differences. In this study, we focused on evaluating the regulatory effects of ovarian hormones on glycogen and related molecules in the endometrial epithelium and their role in the establishment of uterine receptivity in pregnant mice. As expected, progesterone showed a direct regulatory effect on AMPK, but its regulatory mechanism is still unclear and warrants further investigation.
A growing body of evidence suggests that AMPK and glycogen metabolism are interrelated (Janzen et al. 2018). Specifically, the regulation of AMPK activity is affected by glycogen utilization, and glycogen storage dynamics are in turn regulated by AMPK activity. A large amount of glycogen accumulation in cells can inhibit AMPK activity and thus reduce glycogenolysis. Meanwhile, AMPK activation can increase the activity of glycogen phosphorylase to promote glycogenolysis and inhibit the activity of glycogen synthase to inhibit glycogen synthesis. In rat skeletal muscle tissues, AMPK activation was found to increase the activity of glycogen phosphorylase and promote glycogenolysis while decreasing AMPK-mediated glycogen synthesis (Carling & Hardie 1989, Young et al. 1996). Another study revealed significantly reduced GYS1 activity in rat skeletal muscle after treatment with AICAR (Wojtaszewski et al. 2002). AICAR was also shown to induce the phosphorylation and inactivation of GYS1 at serine-7 in muscle tissues from an AMPKα2-knockout mouse, which further identified AMPK as an inhibitor of GYS1 activity and glycogen synthesis (Jørgensen et al. 2004). Here, we demonstrated that activated AMPK phosphorylated PYGM, leading to altered glycogenolysis and reduced glycogen content in EECs. However, no significant changes were observed in the expression of GYS1, which warrants further investigation.
During the window of implantation, the establishment of uterine receptivity requires a large amount of energy. Glycogen is the main source of energy supplied to the endometrium during peri-implantation, and the characteristic regulatory mechanism of glycogen metabolism in the endometrium plays a vital role in successful embryo implantation. Although some studies have shown that uterine AMPK activity is regulated by ovarian estrogen and progesterone and plays an important role in uterine receptivity (Kim & Moley 2009, Griffiths et al. 2020), the regulatory role of AMPK in uterine energy supply has not been clarified. In this study, we found that changes in the energy state or inhibition of glycogenolysis in EECs could activate AMPK. We further inhibited AMPK activity or glycogenolysis to explore its effects on uterine receptivity and implantation. The results showed that the inhibition of AMPK activity or glycogenolysis in EECs interfered with the establishment of uterine receptivity during the window of implantation and ultimately inhibited embryo implantation. These results emphasize the importance of energy supply regulated by AMPK-glycogen interactions in EECs for embryo implantation. For example, one study showed that knockout or low expression of SGLT1 in the endometrial epithelium affects energy metabolism and embryo implantation by reducing glycogen content (Salker et al. 2017, Zhang et al. 2021). Other studies have demonstrated a close relationship between endometrial glycogenolysis and infertility. A retrospective study of the endometrial protein components of patients who underwent in vitro fertilization demonstrated that the levels of endometrial glycogen phosphorylase B were significantly higher in pregnant patients than in non-pregnant patients (Azkargorta et al. 2018). In light of our findings and those of previous studies, we speculate that the consistency and synchronization of AMPK and glycogen in EECs play a crucial role in embryo implantation by maintaining the energy supply essential to establish uterine receptivity during the window of implantation.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Author contribution statement
Li Nie designed research and mainly performed experiments and analyzed results and wrote the manuscript. Li-xue Zhang, Yong-dan Ma,Yun Long, Huan Liu, Zhao-qi Wang and Zi-yang Ma discussed the results. Lin-chuan Liao and Xin-hua Dai analyzed results. Yi-cheng Wang and Zhi-hui Cui revised the manuscript. Dong-zhi Yuan and Li-min Yue designed research and approved final version of manuscript.
Acknowledgements
The authors gratefully acknowledge the support from the National Natural Science Foundation of China (No. 81771542 and No. 31471105, URL: http://www.nsfc.gov.cn/) and Sichuan Science and Technology Program (No.2020YJ0486, URL: http://202.61.89.120/) and the Fundamental Research Funds for the Central Universities (No. 2021SCU12078).
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