Introduction
In mammalian ovaries, granulosa cells have been demonstrated to play a critical role in deciding the destiny of follicles, follicular growth and maintenance as well as their apoptotic process [
1]. More than 99% of follicles degenerate and granulosa cell apoptosis play an important role in the process of follicular atresia [
2]. Granulosa cells apoptosis appears to be an integral part of follicle development, and reflects the mitogenic growth of the follicle [
3]. In addition, an effector caspase (such as
caspase-3) could be activated by an initiator to enforce apoptotic cell death [
2,
4], which could also be activated by
P53. While
P53 activates
BAX and is protected by the regulation of
BCL2 [
4].The relative expression levels of pro-apoptotic and anti-apoptotic factors in granulosa cells determine whether an ovarian follicle will grow or experience atresia in the late preantral stage and affect oocyte ovulation [
5‐
7].
Phospholipases can be found in several different organisms, including bacteria, animals, and viruses [
8]. Phospholipase C (PLC) is a key enzyme in phosphoinositide metabolism that performs cell proliferation/differentiation, the secretion of hormones, fertilization, cell motility and other functions [
9,
10]. PLCβ1, the most extensively investigated PLC isoform, is a critical factor in the regulation of nuclear inositol lipid signaling [
11]. PLCβ plays an important role in the Wnt/Ca
2+ pathway, which promotes the release of intracellular Ca
2+ and affects Ca
2+ sensitive targets, containing protein kinase C (PKC), Ca
2+-calmodulin-dependent protein kinaseII (CAMKII) and Ca
2+-calmodulin-sensitive protein phosphatase calcineurin (Caln) [
12,
13]. Both CAMKII and PKC activate NFκB, and Caln activates cytoplasmic protein nuclear factor associated with T cells (NFAT) via dephosphorylation [
14,
15].
The activations of PLC and PKC can play a role in the physiological cumulus expansion before ovulation in mouse [
16], and involve in mouse embryonic stem-cell proliferation and apoptosis [
17]. But there are little reports about the role of PLC on apoptosis of porcine granulosa cells. Given the pivotal role of granulosa cells apoptosis in follicular development and atresia [
1,
18], we set out to determine whether apoptosis could be regulated by PLC in porcine granulosa cells and how the Ca
2+, several Ca
2+ sensitive proteins and downstream genes could be changed, using the in vitro primary granulosa cells as a model system.
Methods
The animal use protocol was approved by the Institutional Animal Care and Use Committee of the College of Animal Science and Technology, Northwest A&F University, Yang Ling, China.
Preparation of the porcine granulosa cells
The pigs for the experiment were from a local slaughter house. They were a cross of A × (B × C), in which A was the terminal male Duroc, B was the matriarchal father Landrace, and C was the matriarchal mother Yorkshire. All of the pigs were 6–7 months old and weighed approximately 115 kg. Porcine ovaries were collected and washed as described [
19]. Follicular fluid was harvested by aseptic aspiration with a 26 gauge needle [
20] from medium-sized (3–5 mm indiameter) healthy follicles, and porcine granulosa cells were prepared as described [
19].
Culture of the granulosa cells
All reagents and chemicals were obtained from Solarbio Life Sciences (Solarbio, Beijing, China) unless otherwise stated. The porcine granulosa cells were incubated in a basic medium consisting of DMEM/F12 (Gibco, California, USA) with 0.3% bovine serum albumin (BSA) (Roche; Basel, Switzerland), 3% fetal bovine serum(Serapro, Systech Gmbh, Germany), 5 ng/ml sodium selenite, 10 mmol/L NaHCO3, a nonessential amino acid, 50 ng/mL insulin, 0.1 IU/mL FSH, and 1% antibiotics. This medium was used as a control, and the cells were at a density of 1 × 106/mL and incubated in a humidified incubator at 37 °C with 5% CO 2 for 36–44 h before changing to a serum-free culture with 2.5 μg/ml transferrin for 24 h. Then half of the medium (500 μl) was exchanged with fresh solution every 24 h as the experiment required; several doses of U73122 (the PLC inhibitor) in DMF or m-3M3FBS (the PLC activator) in DMSO were added into the culture with final concentration of 0 μM (control), 0.05 μM, 0.5 μM, 5 μM, 50 μM. Expression of genes other than PLCB1, all proteins and intracellular Ca2+ concentration were assessed at 4 h posted treatment, whereas expression of PLCB1 gene and cell apoptosis which was analyzed using flow cytometry were evaluated at 2, 4, 8, 12, 24 and 48 h after incubation.
Immunofluorescence
Follicle-stimulating hormone receptor (FSHR), which is mainly located in the cytoplasm and appears red under the fluorescent microscope after immunostaining, is the marker protein of porcine granulosa cells. In addition, DAPI dye can penetrate cell membranes and produce fluorescence by binding to nucleic acids in the nucleus, making the cells appear blue under the microscope.
After the cells grew on the glass slide for 36–44 h, they were fixed in 4% paraformaldehyde for 30 min and washed three times with PBS. The porcine granulosa cells were permeabilized with 0.2% Triton X-100 and then blocked using 10% normal serum in 1% BSA in TBS (10 mM tris-HCl, 150 mM NaCl, and 0.1% Tween 20; pH 7.5) for 1 h at room temperature. The cells were incubated with anti-FSHR antibodies (Bioss, Beijing, China; Cat No.: bs-0895R, diluted 1:350) in 1% BSA in TBS overnight at 4 °C. The slides with cells were washed twice for 5 min each time, followed by incubation with Cy3 conjugated Goat Anti-rabbit IgG (H + L) (Servicebio, Wuhan, China) (Cat No.: GB21303, 1:300) for 60 min. The nuclei were identified by 4,6-diamidino-2-phenylindole (DAPI) staining. Nonimmune rabbit IgG was used as the negative control. The slides were imaged using a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan). The data were analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).
RNA extraction and RT-qPCR
The granulosa cells were treated for 2 h, 4 h, 8 h, 12 h, 24 h and 48 h which was used for detecting PLCB1 mRNA expression or for 4 h which was used for detecting other genes, and then total RNA was extracted from the cells using TRIzol reagent (Takara, Kyoto, Japan) as described [
21]. RNA concentration was determined using a spectrophotometer (NanoDrop 2000c, Thermo Scientific, Waltham, Massachusetts, USA) by absorbance at 260 nm [
22]. Then, 1 μg of total RNA was converted to cDNA using 5 × All-In-One RT MasterMix (Abm, Vancouver, Canada) according to the manufacturer’s protocol [
22]. Primers were synthesized by Sangon Biotech (Shanghai, China), and the primer sequences used for genes studied are listed in Table
1. The RT-qPCR reactions were performed in triplicate using the EvaGreen 2 × qPCR MasterMix-no Dye (Abm, Vancouver, Canada) with a Bio-Rad CFX96 system (BioRad, California, USA) according to the manufacturer’s protocol. The mRNA relative expression of each gene was calculated comparing to the housekeeping gene β-actin. The final relative expression fold differences, with gene expression NC as a control, were calculated with the 2
− ∆∆Ct method for each gene [
19].
Table 1
Primer Sequences used in this study
β-Actin | F-5′- ATCAAGATCATCGCGCCTCC-3’ | XM_003124280.4 | 169 |
R-5′- AATGCAACTAACAGTCCGCCT-3’ |
PLCB1 | F-5′-TGAGAAGGAGGGCAGCTTTGGA-3’ | XM_013985062.1 | 108 |
R-5′-CAGCGAGGTCTTGCTCAATGGT-3’ |
Cdc42 | F-5′- ATGTGGAGTGTTCTGCACTCA-3’ | XM_005656039.2 | 132 |
R-5′-GGCTCTGGAGAGACGTTCAT −3’ |
NFATC1 | F-5′-GAAAACCGACGGAGACCTGT-3’ | NM_214161.1 | 172 |
R-5′-TATGACTGGAGCGTTGGCAG-3’ |
NFATC2 | F-5′-TCCTACCCCACGGTCATTCA-3’ | NM_001113452.1 | 179 |
R-5′-TGTATCCAGCTAAGGTGTGTGTC −3’ |
NFKB | F-5′-GAGGTGCATCTGACGTATTC − 3’ | NM_001048232.1 | 138 |
R-5′- CACATCTCCTGTCACTGCAT-3’ |
NLK | F-5′-GCGGCTTACAATGGCGGTACAT-3’ | XM_013981185.1 | 122 |
R-5′-TGAAGATGGTGCTGAGGGTGGT-3’ |
CTNNB1 | F-5′-TCGCCTTCACTACGGACTACCA-3’ | NM_214367.1;XM_013981492.1; XM_005669377.2;XM_013981494.1 | 180 |
R-5′-TGATGAGCACGAACCAGCAACT-3’ |
BAK1 | F-5′-GACAGAAGTGGGGCAAGATCA-3’ | XM_001928147.3 | 190 |
R-5′-CGCTCCAATCAGCTCCCTCT-3’ |
BAX | F-5′-GGCCTCCTCTCCTACTTTGG −3’ | XM_013998624.2; XM_003127290.5 | 103 |
R-5′-CTCAGCCCATCTTCTTCCAG −3’ |
BCL2 | F-5′-ATCGCCCTGTGGATGACTGAGT-3’ | XM_003121700.4 | 140 |
R-5′-GCCTTCAGAGACAGCCAGGAGA-3’ |
CASP3 | F-5′-ATACACGTACTCATGGCGAAC-3’ | XM_005671704.2 | 102 |
R-5′-TCCAGAGTCCACTGATTTGCT-3’ |
CASP8 | F-5′-CCAGGATTTGCCTCCGGTTA −3’ | NM_001031779.2 | 144 |
R-5′-TGTGGGATGTAGTCCAGGCT −3’ |
TP53/P53 | F-5′-TCGGCTCTGACTGTACCACCAT −3’ | NM_213824.3 | 155 |
R-5′-GGCACAAACACGCACCTCAAAG-3’ |
Western blotting
Western Blotting was measured as described previously [
19] after treatment for 4 h. The protein concentrations were determined by BCA protein assay (Beyotime, Hangzhou, China), and the proteins used were 35 μg (for PLCB1) or 15 μg (for others). The primary antibodies were PLCB1 (Ommi mAbs, USA; OM116324,1:1000 dilution), PKC beta1 + 2 (Bioss, Beijing, China; bs-0267R, 1:2000 dilution), CAMKIIα (WanleiBio, Shen Yang, China; WL03453, 1:1200 dilution), calcineurin A (WanleiBio, Shen Yang, China; WL03449,1:600 dilution) and β-actin (Santa Cruz Biotechnology, CA, USA; SC-1615, 1:500 dilution).The secondary antibodies were Goat anti-Rabbit lgG(H + L)-HRP (Sungene Biotech, Tian Jin, China; LK2001, 1:4000 dilution) for PLCB1, PKC beta1 + 2, CAMKIIα and calcineurin A or Goat anti-Mouse lgG(H + L)-HRP (Sungene Biotech, Tian Jin, China; LK2003, 1:4000 dilution) for β-actin. The membranes were visualized using ECL (CW Bio, Beijing, China). The data were analyzed with Quantity One (BioRad, California, USA).
Flow cytometry analysis of the cell apoptosis
The cells were seeded into 6-well culture plates at a density of 1 × 10 6/mL cells per well. The cells were treated with 0.5 μM U73122 or m3M3FBS for 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, respectively, and then the cells were washed three times with cold PBS by centrifugation at 800 g for 5 min. Cell apoptosis was assessed with an annexin V-FITC/propidium iodide apoptosis detection kit (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions, with the cell pellet resuspended in 100 μL of 1 × binding buffer and adjusted to the density to 1 × 10 6/mL. The stain was detected within 1 h by a flow cytometry instrument (Beckman Coulter, CA, USA).
Determination of intracellular Ca2+ concentration
The cells at a density of 8 × 10
5/mL cells per well were treated with 0.5 μM U73122 or m3M3FBS for 4 h. The concentration of intracellular Ca
2+ was monitored by a fluorescent Ca
2+ indicator Fluo 3-AM (Sigma, St. Louis, MO, USA), and was measured as described previously [
23]. The intracellular Ca
2+ changes were measured with the ratio of fluorescence intensities excited at 488 nm and 530 nm using flow cytometry (CyFlow Cube, PARTEC, Germany).
Statistical analysis
All experiments were carried out at least three times.GraphPad Prism 6.0 was used to graph the results. The data were analyzed using SPSS 19.0 software (SPSS science, Chicago, IL, USA), multiple comparisons among the groups were evaluated by Duncan’s test. One-way ANOVA that conforms to normal distribution and homogeneity of variance was used to determine differences among multiple groups which was marked by letters, and an independent sample t-test that conforms to normal distribution was used to determine the difference between two groups which was marked by asterisk. The data are presented as the means ± SEM. A value of p < 0.05 was considered statistically significant. For each treatment, the means without common letters are significantly different (p < 0.05), and means with * indicate a difference at p < 0.05, means with ** indicate a difference at p < 0.01, and means with *** indicate a difference at p < 0.001. The data of cell apoptosis and intracellular Ca2+ concentration were analyzed with FlowJo V10 (Leonard Herzenberg’s laboratory, Stanford University, USA).
Discussion
PLCs are important enzymes that is thought to play a role in the physiological regulation of organisms [
9,
10], and could participate in the apoptosis of mouse embryo stem-cells [
17].Whether PLC play a part in apoptosis of porcine granulose cells was unclear [
24]. U73122 is an inhibitor of the PLC protein widely used in mammals [
25,
26], and m-3M3FBS is an activator of PLC protein which could participate in several physiology regulation [
27,
28]. In this study, the influence of PLC inhibitor and activator on PLC mRNA and protein expression, apoptosis, intracellular Ca
2+ concentration and several targeted proteins and genes in porcine granulosa cells in vitro was investigated to explore the role of PLC in porcine granulosa cells.
The apoptosis of granulosa cells is mainly regulated by the caspase-dependent signaling pathways, and most of the apoptosis leads to the terminal differentiation at the antral follicle surface in granulosa cells [
3]. Mitochondria are involved in mammalian cell apoptosis [
29]. Previous studies found that the activation of PLC has the effect of anti-apoptosis [
30], and the inhibition of PKC increases apoptosis [
17], which are consistent with our work. The results in this study were in line with the results from previous research that the inhibition of BCL2 family proteins was accompanied by the promotion of BAX and BAK which might be regulated by cell death signals [
31,
32], and the activation of BAX, which might regulate downstream genes including caspase 3, could induce apoptosis [
33]. An increased abundance of
BCL2 was reported to be accompanied by an increase in
NFκB expression rather than
BAX expression [
34], and the expression of
BCL2 was the opposite to
BAX mRNA expression [
35], which was the same in our results. In this study, we can speculate that apoptosis of porcine granulosa cells was induced by the supplementation of U73122 at each time point, and mainly inhibited by the m-3M3FBS treatment after 8 h. Annexin/PI binding could indicate that apoptosis and necrosis increase with atresia progression [
36].
The PLC enzymes are responsible for hydrolysis of phosphatidylinositol-4, 5- bisphosphate (PIP
2), an inner membrane component which could produce the second messenger IP3 and DAG, and then released the intracellular Ca
2+ [
37]. The effect of inhibitor U73122 or m-3M3FBS on PLC mRNA and protein expression was affected by both the culture time and their concentration of treatment. The activation of PLC β could increase intracellular Ca
2+ [
38], which is in line with this study that PLC activator increased the intracellular Ca
2+ concentration and PLC inhibitor decreased the intracellular Ca
2+ concentration. The PKC activator PLC could be activated by prostaglandin f (2alpha) in the ovary [
39]. It was reported that the Wnt/Ca
2+ signaling pathway could reduce cisplatin resistance, and CaMKII might be an underlying therapeutic target in chemoresistant ovarian cancers [
40]. Caln, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of tumor cells in mice, a phenotype which coincides with increased apoptosis in vivo [
41]. In this study, the results showed that PLC mediated the abundance of three Ca
2+-sensitive proteins and affected apoptosis.
We found that
CDC42 mRNA expression was down-regulated by the PLC inhibitor U73122 and up-regulated by the PLC activator m-3M3FBS in porcine granulosa cells, which indicated that CDC42 gene was a target of PLC. The CDC42-PAKs-ERK1/2 MAPK signaling cascade in the prehierarchical follicles of the chicken ovary could mediate the suppression of granulosa cell proliferation, differentiation and follicle selection [
42]. In this study, the expression of
NFkB was inhibited by U73122, while the rate of early apoptosis was increased which was in line with previous studies [
43]. The expression of
NLK mRNA was slightly higher, while β-catenin mRNA expression was decreased with m-3M3FBS supplementation. The result was consistent with previous study [
44]. On the other hand, the rapid activation of membranous glucocorticoid receptor (mbGR)/PLC/PKC further lead to the activation of β-catenin [
45]. It is possible that different cell types have different regulation mechanisms, and the relationship between PLC and β-catenin still needs further study.
Taken together, these result proved that PLC acts in porcine granulosa cells via several target proteins and genes, and participates in the regulation of apoptosis, which may provide new information to understand the role of PLC in porcine granulosa cells. The regulation of PLC signaling might be instructive to protect the function of mammal ovaries and monitor the drug action in clinical application. Future studies might focus on the effects of the specific mechanism of the regulation.
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