Abstract
Vaspin, also known as visceral adipose tissue-derived serine protease inhibitor, is a member of the serine protease inhibitor family. Its expression is associated with obesity, insulin resistance and type 2 diabetes, and elevated concentration is observed in polycystic ovary syndrome. However, vaspin has never been studied in the ovary. Here, we identified vaspin in two prolific breeds of pigs: fat Meishan (MS) and lean Large White (LW). We then investigated the molecular mechanism involved in the regulation of its expression in response to gonadotropins, insulin, insulin-like growth factor type 1 (IGF-1) and steroids (progesterone, testosterone and oestradiol) in ovarian follicles cells. Using real-time PCR and Western blot, we found higher vaspin mRNA and protein expression in the ovarian follicles and adipose tissue at 10–12 days of the oestrous cycle in MS compared to LW. Moreover, vaspin expression, as well as its concentration in plasma and follicular fluid, decreased in ovarian follicles of LW during days of the oestrous cycle, while the opposite results were noted in MS. Immunohistochemistry showed vaspin in granulosa, theca, cumulus cells and oocytes as well as in adipocytes. Vaspin level in the ovary increased by gonadotropin, insulin, IGF-1 and steroids stimulation through kinases JAK/Stat, ERK1/2, PI3K and AMPK, as well as factor NF-κB. These findings all show vaspin expression and regulation in the pig ovary, indicating vaspin as a new regulator in female reproduction. Future studies will be necessary for understanding the role of vaspin on ovarian physiology providing new insights into the pathology of ovaries.
Introduction
Vaspin is known as a visceral adipose tissue-derived serine protease inhibitor and is a member of the serine protease inhibitor family which has insulin-sensitising effects (Hida et al. 2005). Rat, mouse and human vaspins are made up of 392, 394 and 395 amino acids, respectively. They exhibit approximately 40% homology with α1-antitrypsin and are related to the serine protease inhibitor family. The gene that encodes for vaspin is termed OL-64 and is present at the long arm of chromosome 14 (14q32.1) where the cDNA consists 1245 bases and encodes for 415 amino acids (Hida et al. 2000, 2005). Vaspin is located in visceral white adipose tissue (WAT) of Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model characterised by abdominal obesity with type 2 diabetes (Hida et al. 2005). Expression of vaspin is also found in gastric mucosa, liver, pancreas, cerebrospinal fluid, skin and in the experimental models of obese db/db and lean C57BL/6 mice (Klöting et al. 2011). Vaspin mRNA expression is regulated by nutritional status, metformin, gender and pituitary factors in rat WAT (González et al. 2009). Moreover, pioglitazone significantly induced vaspin levels in adipose 3T3-L1 cells (Handisurya et al. 2010), and rosiglitazone in brown adipose tissue (Weiner et al. 2017).
Vaspin has also been linked to metabolic syndrome (Heiker 2014) and ovarian pathology such as polycystic ovary syndrome (PCOS), because serum levels of vaspin are enhanced in PCOS patients (Tan et al. 2008, Koiou et al. 2011a , Cakal et al. 2011). It is important to note that PCOS is often associated with insulin resistance, obesity or type 2 diabetes (Rojas et al. 2014), i.e. parameters with direct effects on vaspin levels and the variability in the data may arise from homogenous study cohorts and apply diagnostic criteria (Koiou et al. 2011b ). Plasma vaspin levels are higher in women than in men (Youn et al. 2008, González et al. 2009), suggesting a possible involvement of sex steroids in regulating vaspin production. However, the vaspin expression, regulation and physiological role in the female reproduction have still not been determined.
It is a well-known fact that adipokines regulate female reproduction and more precisely, the ovarian follicles function (Tersigni et al. 2011). Additionally, increasing evidence shows that the dysregulated ovarian expression of adipokines plays an important role in the pathology of PCOS. For example, apelin and its receptor APJ are expressed in ovarian follicle cells, including theca (Tc), granulosa (Gc) and oocyte of different species such as porcine, bovine, rhesus monkey and human (Shirasuna et al. 2008, Schilffarth et al. 2009, Xu et al. 2012, Rak et al. 2017a , Roche et al. 2017). Moreover, ovarian expression of adipokines is dependent on hormonal status. Progesterone (P4) and follicle-stimulating hormone (FSH) stimulated the expression of APJ in the cultured Gc, while luteinising hormone (LH) induced the expression of apelin/APJ in cultured Tc (Shimizu et al. 2009). Resistin and adiponectin are also expressed in the ovarian cells, and several studies have shown their effect on follicular development, steroidogenesis, proliferation/apoptosis and oocyte maturation (Pierre et al. 2009, Richards et al. 2012, Rak et al. 2015a , 2017b ).
Thus, the aim of this study was to identify vaspin expression in the porcine ovarian follicles and WAT at 10–12 days of the oestrous cycle. Next, vaspin concentration in plasma and follicular fluid (FF) as well as vaspin expression in the ovarian follicles during the oestrous cycle were analysed. For animal models, we used two prolific breeds of pigs: fat Meishan (MS) and lean Large White (LW), differing in fat content (MS > LW). The Chinese MS breed is one of the most prolific pig breeds known, farrowing between three and five more live piglets per litter than European commercial breeds, such as the LW. However, the MS is not commercially viable in Europe due to its poor growth rate and high carcass fat content (Bidanel et al. 1990, Haley et al. 1992, Serra et al. 1992). In the next part of our study, we investigated the regulation of vaspin expression by gonadotropin, insulin, insulin-like growth factor 1 (IGF-1) and steroid hormones such as P4, testosterone (T) and oestradiol (E2) in vitro coculture of granulosa and theca cells. In mammals, gonadotropin, insulin, IGF-1 and steroids are well known to play a key role on oestrous cycle regulation, ovarian cells activity i.e. folliculogenesis, steroidogenesis, cell proliferation and ovulation (Silva et al. 2009). We investigated whether vaspin expression and secretion are dependent on the activation of several kinases such extracellular signal-regulated kinase (ERK1/2), phosphatidylinositol 3′-kinase (PI3K)/Akt, Janus kinase/signal (Stat/JAK2), adenosine 5′-monophosphate-activated protein kinase α (AMPKα) and nuclear factor-κB (NF-κB). We chose these kinases because their phosphorylation is important in the regulation of adipokines expression. For example, 17β-E2 stimulates (Chen et al. 2006) but IGF-1 inhibits (Chen et al. 2005) resistin gene expression in 3T3-L1 adipocytes via ERK1/2 pathway.
Materials and methods
Reagents
Foetal bovine serum (FBS, heat inactivated) and electrophoresis marker were purchased from ThermoFisher Scientific. Phosphate buffered saline (PBS) was purchased from BioWest (Riverside, MO, USA). Medium M199, antibiotic-antimycotic solution, Tris, trypsin, FSH (cat no. F4021), LH (cat no. L5259), IGF-1 (cat no. I3769), insulin (cat no. I3505), P4, T, E2 (cat no. P0130, 86500 and E2257, respectively), AG 490 (cat no. T3434), Compound C (cat no. P5499), JSH-23 (cat no. J4455) and Laemmli buffer (cat. no. 38733) were all obtained from Sigma-Aldrich. PD98059 (cat no. 1213) was obtained from Tocris and LY294002 (cat no. 9901) from Cell Signaling Technology. Bradford protein assay kit, 4–20% gels (cat no. 456-1093) and membranes (cat no. 1704156) were obtained from Bio-Rad.
Sample collection
Porcine ovarian follicles, WAT around the kidney and plasma were collected from MS and LW sexually mature pigs at a local abattoir under veterinarian control, less than 20 min after slaughter. LW gilts with an average weight of 91.76 ± 8.2 kg and the same number of MS gilts with an average body weight of 30.62 ± 5.8 kg were used in this study. Tissues were transported to the laboratory in PBS with antibiotic-antimycotic solution within 30 min of collection. Ovarian follicles, FF and blood samples were collected from 36 LW and 36 MS pigs on days 4–6 (n = 12), 10–12 (n = 12) and 16–18 (n = 12) of oestrous cycle by morphological examination of the ovaries (Akins & Morissette 1968), while WAT, used as a positive control, was collected at days 10–12 of oestrous cycle. To determine vaspin mRNA expression, ovarian follicles, after excision from the ovary, and WAT were immediately frozen in liquid nitrogen and stored at −70°C. To quantify vaspin protein expression, tissues were homogenised twice in ice-cold lysis buffer. Lysates were cleared by centrifugation at 15,000 g at 4°C for 30 min, and the protein contents were determined by a protein assay using bovine serum albumin as a standard (Bradford method). Ovarian follicles and WAT at days 10–12 of oestrous cycle were fixed in 4% paraformaldehyde, dehydrated in an increasing gradient of ethanol, and then embedded in paraplast for vaspin analysis by immunolocalisation. Both FF and plasma samples were prepared by low-speed centrifugation (2,000 g at 4°C for 10 min) and stored at −20°C to determine the vaspin concentration.
Ovarian cells in vitro culture
Ovarian Gc and Tc were subsequently prepared from the follicles at days 10–12 of oestrous cycle of LW pigs. Gc and Tc from the same follicles were prepared by technique described by Stoklosowa et al. (1978). The coculture model of both cell types in ovarian follicles is better than a monoculture of one cell type because all interaction (structural and functional) between Gc and Tc cells are preserved in vitro. This in vitro model was used in previous studies examining the role of IGF-1, ghrelin and adipokines in the porcine ovary (Kolodziejczyk et al. 2003, Rak & Gregoraszczuk 2008, 2015a , Rak-Mardyla & Gregoraszczuk 2010). Herein, viability (Trypan blue exclusion test) was found to be 92% for Gc and 84% for Tc. For coculture experiments, Gc and Tc were inoculated at concentrations of 6 × 104 and 1.5 × 104 cells/well, respectively, in 96-well tissue culture plates. Therefore, the ratio of both types of cells was comparable to that observed in vivo (Gc:Tc = 4:1). All cultures were maintained at 37°C in a humidified atmosphere of 5% CO2/95% O2.
For the determination of the regulation of vaspin expression, ovarian cells were incubated for 48 h in M199 supplemented with 5% FBS as a control medium or with FSH or LH at 50–150 ng/mL, insulin or IGF-1 at 10–100 ng/mL and steroids P4, T or E2 at 10−8–10−6 M. Doses of hormones were chosen based on literature and our previous study (Rak et al. 2015b ). After incubation, medium was stored at −20°C for vaspin concentration, while the cells were boiled in Laemmli buffer for 4 min and then stored at −20°C for vaspin protein expression analysis. We performed four independent culture. For one culture we used ovarian cells collected from seven pigs (ten follicles from one pig, per each hormone: control and three doses, in single repetition), so the total number of pigs was 28.
To investigate whether vaspin expression is dependent on kinases activation, cells were plated in 96-well culture plates in M199 medium with 10% FBS for 24 h. Next, the media were changed to 5% FBS and the cells were pre-treated for 1 h with the JAK inhibitor AG490 at 50 µM, the ERK1/2 inhibitor PD098059 at 50 µM, the PI3K inhibitor LY294002 at 10 µM, the AMPKα inhibitor compound C at 10 μM and the NF-κB inhibitor JSH-23 at 50 μM. Then, gonadotropins at 100 ng/mL, insulin or IGF-1 at 50 ng/mL and steroids at 10−7 M were added for 48 h. The concentrations of the inhibitors were chosen based on previous data (Rak et al. 2015b ) and an unpublished study. After incubation, medium was stored at −20°C for vaspin concentration, while the cells were boiled in Laemmli buffer for 4 min and then stored at −20°C for vaspin protein expression analysis. We performed five independent culture. For one culture we used ovarian cells collected from 14 pigs (20 follicles from two pigs, per each hormone: control and three doses, in single repetition), so the total number of pigs was 70.
Real-time PCR
Total RNA was extracted from ovarian follicles and WAT using TRIzol reagent (Reverchon et al. 2014). Reverse transcription procedure was subsequently conducted. Briefly, 1 µg of total RNA was reverse transcribed for 1 h at 37°C in a final reaction volume of 20 μL, containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 200 μM of each deoxynucleotide triphosphate (Amersham, Piscataway, NJ, USA), 50 pmol of oligo(dT) 15, 5 U of ribonuclease inhibitor and 15 U of MMLV reverse transcriptase. Afterwards, porcine cDNA was diluted 1:5.
Real-Time PCR was performed in a 20 μL final volume containing 10 μL iQ SYBR Green supermix (Bio-Rad), 0.25 μL of each primer (10 μM), 4.5 μL of water and 5.0 μL of template. The cDNA templates were amplified and detected using the MYIQ Cycler real-time PCR system (Bio-Rad) following the protocol previously described by Rak et al. (2017a). The abundance of housekeeping gene PPIA (cyclophilin A) was examined and normalised according to Vandesompele et al. (2002). The descriptions of the different primers are as follows: Vaspin (SERPINA12) (forward 5′-GCTGTGAGTCGTGACCAAGT-3′ and reverse 5′-CACAGAGATGCTCCAAGGG-3′) and PPIA (forward 5′-GCATACAGGTCCTGGCATCT-3′ and reverse 5′-TGTCCACATGCAGCAATGGT-3′). The specificity of the amplified fragment sequence was assessed by Beckman Coulter Genomics. The efficiency was between 1.8 and 2.0.
Western blot
Tissue preparation, lysis, Western blotting and quantification were performed as previously described (Rak et al. 2015a , b ). For each sample, 30 μg of protein were reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad), and then transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20, and then incubated overnight at 4°C with anti-vaspin antibody (cat no. PA5-30989, ThermoFisher Scientific) diluted at 1:1,000. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated antibody (cat. no. 7074, Cell Signaling Technology) diluted at 1:1000. An anti-β-actin antibody (cat no. A5316, Sigma-Aldrich) was used as loading control. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (cat. no. K-12043 D20, Advansta Inc., Menlo Park, USA) and visualised using the ChemidocTM XRS + System (Bio-Rad). All visible bands were quantified using a densitometer and ImageJ software (US National Institutes of Health).
Immunohistochemistry
To determine vaspin localisation in the ovary or WAT, immunohistochemistry was applied (Reverchon et al. 2014). Sections (5 μm thick) were mounted onto APES-coated slides, deparaffinised in xylene, and then gradually rehydrated through a series of ethanol dilutions. The sections were immersed in 0.01 M citrate buffer and heated in a microwave oven for antigen retrieval. Endogenous peroxidase activity and nonspecific binding were blocked. Next, the sections were incubated overnight at 4°C with anti-vaspin antibody (cat no. PA5-30989, ThermoFisher Scientific) then washed in TBST. After, they were incubated with biotinylated goat anti-rabbit IgG (1:400; Vector Lab) followed by avidin-biotin-peroxidase complex (1:1:100; Strept ABC complex/HRP, DAKO/AS). The sections were dehydrated, mounted in DPX (Fluka, Chemie GmbH, Buchs) and then photographed using the Nikon Eclipse E200 microscope attached to the Coolpix 5400 digital camera (Nikon) with corresponding software.
ELISA assay
A commercially available pig vaspin ELISA (cat. no. MBS267502; MyBioSource, San Diego, USA) was used to quantify vaspin concentrations in plasma, FF and culture medium. The sensitivity of the vaspin assay was 0.05 ng/mL, and the inter- and intra-experimental coefficients of variation were <12 and <8%, respectively. Samples were run in triplicate within the same assay.
Statistical analysis
Statistical data are presented as means ± s.e.m. of four/five independent experiments performed in threefold. Distribution of normality was checked by Shapiro–Wilk test. Statistical analysis was carried out using two-way ANOVA, followed by Tukey’s test (GraphPad Software) to compare vaspin levels between breeds of pigs (LW and MS) and days of the estrous cycle. A one-way ANOVA was used for multiple comparisons involving more than two treatment groups; to compare hormones/inhibitors to control or hormones + inhibitors compare to inhibitors. Statistical significance is indicated by different letters (P < 0.05) or by *P < 0.05, **P < 0.01 and ***P < 0.001.
Results
Vaspin expression and cellular immunolocalisation in the ovarian follicles and adipose tissue as well as concentration in plasma and FF
Comparing mRNA expression of vaspin between ovaries and WAT collected at 10–12 days of the oestrous cycle of both pig breeds, we observed significantly higher expression in MS ovarian follicles (ratio Vaspin/PPIA mRNA expression: 1.301 ± 0.013) and WAT (ratio Vaspin/PPIA mRNA expression: 3.846 ± 0.165) compared to LW ovarian follicles (0.0189 ± 0.0001) and WAT (0.427 ± 0.03) (P < 0.05); these results were confirmed on protein expression (Fig. 1A, P < 0.05). Immunohistochemistry with porcine follicle sections collected from LW showed that vaspin protein is present in Gc but also in cumulus cells, oocytes and Tc (Fig. 1B). Similar results were observed in MS ovarian cells (data not shown). We observed intense immunostaining for vaspin in WAT (Fig. 1B) collected from LW. As a negative control, we replaced the primary antibody with PBS or normal serum. No immunoreaction was observed for the negative controls. As shown in Fig. 1C, we found that mRNA expression of vaspin decreased in ovarian follicles of LW pigs during days of the oestrous cycle, while the opposite results were observed in MS pigs, where vaspin mRNA expression was significantly higher at 16–18 days of the oestrous cycle (P < 0.05). Immunoblotting results confirmed the data of vaspin mRNA (Fig. 1D, P < 0.05).
Vaspin levels in the ovarian follicles and visceral adipose tissue (WAT) of two pig breeds: Large White (LW) and Meishan (MS). (A) Vaspin protein expression in the ovarian follicles and WAT collected from ovarian follicles at 10–12 days of the oestrous cycle. (B) Immunolocalisation of vaspin in oocyte (Oo), granulosa (Gc), theca (Tc) and cumulus (Cc) cells of ovarian follicles and WAT collected at 10–12 days of the oestrous cycle of LW. Scale bar: 100 μM for ovarian follicles and 40 μM for WAT. (C) Vaspin mRNA and (D) protein expression in the ovarian follicles and (E) concentration in FF and plasma during the oestrous cycle (days 4–6, 10–12 and 16–18) in LW and MS. Figures and Images shown are representative of the four experiments on 12 LW and 12 MS different animals per stages. Statistical analysis was carried out using two-way ANOVA, followed by Tukey’s test (GraphPad Software). The data are plotted as the mean ± s.e.m. Different letters indicate significant differences at P < 0.05.
Citation: Reproduction 158, 2; 10.1530/REP-19-0034
We also measured the concentration of vaspin in plasma and FF during the oestrous cycle. As shown in Fig. 1E, levels of vaspin in LW pigs decreased, while in MS, they increased significantly both in plasma and FF during the oestrous cycle. Furthermore, we observed that vaspin levels were higher both in plasma and FF in MS than LW pigs (Fig. 1E, P < 0.05). Interestingly, concentrations of vaspin were significantly higher in FF than plasma of both pig breeds, except at 10–12 days of the oestrous cycle in LW.
Vaspin expression and secretion is regulated by gonadotropin, insulin, IGF-1 and steroid hormones in the ovarian follicles cells
We observed dose-dependent effect of gonadotropin on vaspin expression in the ovarian follicles cells. FSH significantly increased vaspin protein expression at 100 and 150 ng/mL. Furthermore, LH had stimulatory effect at 50 and 100 ng/mL, while inhibitory at 150 ng/mL (Fig. 2A, P < 0.05, P < 0.01). Vaspin secretion into the culture medium was significantly higher after FSH and LH stimulation, at both 100 ng/mL doses (Fig. 2A, P < 0.05).
Effect of (A) follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at doses 50–150 ng/mL, (B) insulin and insulin-like growth factor type 1 (IGF-1) at doses 10–100 ng/mL and (C) steroids progesterone (P4), testosterone (T) and oestradiol (E2) at doses 10−8–10−6 M on vaspin protein expression and concentration in the culture medium of ovarian cells from Large White pigs. Statistical analysis was carried out using one-way ANOVA, followed by Tukey’s test (GraphPad Software) of four independent cultures. Statistical significances are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.
Citation: Reproduction 158, 2; 10.1530/REP-19-0034
After 48 h of ovarian cell culture, insulin at all investigated doses had stimulatory effect on vaspin protein expression and secretion to the culture medium (Fig. 2B, P < 0.05, P < 0.01). IGF-1 significantly stimulated vaspin protein expression at 50 ng/mL and secretion at 10 and 50 ng/mL (Fig. 2B, P < 0.05, P < 0.01). We documented also that IGF-1 at 100 ng/mL decreased vaspin protein expression (Fig. 2B, p < 0.05).
As shown in Fig. 2C, protein expression of vaspin was strongly dependent on steroid hormones doses. We observed that P4 at 10−7 and 10−8 M, T at 10−6 and 10−7 M and E2 at all investigated doses increased vaspin protein expression (Fig. 2C, P < 0.01, P < 0.001). However, concentration of vaspin in culture medium was significantly higher after P4 at 10−7 M, T at 10−6 M and E2 at 10−7 M (Fig. 2C, P < 0.05, P < 0.001).
Involvement of JAK/Stat, ERK1/2, PI3K and AMPKα kinases as well as NF-κB factor in action gonadotropin, insulin, IGF-1 and steroids on vaspin protein expression and concentration in the ovarian follicles cells
As shown in Fig. 3, both vaspin expression and secretion were strongly reduced in cells treated with FSH and PD098059, Compound C or JSH (Fig. 3, P < 0.05, P < 0.01, P < 0.001). We observed that LH with AG490 significantly increased, while LH with JSH decreased vaspin expression and secretion (Fig. 3, P < 0.05).
Effect of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at dose 100 ng/mL on protein expression (A) and concentration in medium (B) of vaspin in cultured ovarian cells, at 10–12 days of the oestrous cycle of Large White pigs after using pharmacological inhibitors of Janus kinase (JAK/STAT; AG490, 50 µM), extracellular signal-regulated kinase (MAPK/ERK1/2; PD098059, 50 µM), phosphatidylinositol 3′-kinase (PI3K; LY294002, 10 µM), adenosine 5′-monophosphate-activated protein kinase (AMPK; Compound C, 10 µM) and nuclear factor-κB (NF-κB; JSH-23, 50 µM). Statistical analysis was carried out using one-way or two-way ANOVA, followed by Tukey’s test (GraphPad Software) of five independent cultures. Statistical significances are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.
Citation: Reproduction 158, 2; 10.1530/REP-19-0034
The expression and secretion of vaspin under insulin with all investigated inhibitors were at the same levels. We observed that IGF-1 with LY29402 significantly decreased vaspin levels, while IGF-1 with JSH increased both vaspin expression and secretion (Fig. 4, P < 0.05, P < 0.01, P < 0.001).
Effect of insulin and insulin-like growth factor type 1 (IGF-1) at dose 50 ng/mL on protein expression (A) and concentration in medium (B) of vaspin in cultured ovarian cells, at 10–12 days of the oestrous cycle of Large White pigs after using pharmacological inhibitors of Janus kinase (JAK/STAT; AG490, 50 µM), extracellular signal-regulated kinase (MAPK/ERK1/2; PD098059, 50 µM), phosphatidylinositol 3′-kinase (PI3K; LY294002, 10 µM), adenosine 5′-monophosphate-activated protein kinase (AMPK; Compound C, 10 µM) and nuclear factor-κB (NF-κB; JSH-23, 50 µM). Statistical analysis was carried out using one-way or two-way ANOVA, followed by Tukey’s test (GraphPad Software) of five independent cultures. Statistical significances are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.
Citation: Reproduction 158, 2; 10.1530/REP-19-0034
We showed that vaspin expression and concentration in the culture medium were higher in E2 with AG490 and P4 with Compound C treatments (Fig. 5, P < 0.05, P < 0.01, P < 0.001). In other cases, levels of vaspin were blocked by inhibitors, suggesting activation of these kinases in the regulation of vaspin in the ovary (Fig. 5). Additionally, we observed that all the inhibitors we used had no effect on vaspin expression and secretion.
Effect of progesterone (P4), testosterone (T) and oestradiol (E2) at dose 10−7 M on protein expression (A) and concentration in medium (B) of vaspin in cultured ovarian cells, at 10–12 days of the oestrous cycle of Large White pigs after using pharmacological inhibitors of Janus kinase (JAK/STAT; AG490, 50 µM), extracellular signal-regulated kinase (MAPK/ERK1/2; PD098059, 50 µM), phosphatidylinositol 3′-kinase (PI3K; LY294002, 10 µM), adenosine 5′-monophosphate-activated protein kinase (AMPK; Compound C, 10 µM) and nuclear factor-κB (NF-κB; JSH-23, 50 µM). Statistical analysis was carried out using one-way or two-way ANOVA, followed by Tukey’s test (GraphPad Software) of five independent cultures. Statistical significances are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.
Citation: Reproduction 158, 2; 10.1530/REP-19-0034
Discussion
Vaspin is a recently discovered visceral adipose tissue-derived factor that exhibits insulin-sensitising effects, since its administration to obese rodents ameliorated certain obesity-associated disorders (Hida et al. 2005). However, it is unknown whether vaspin has an effect on reproduction. In this study, we report for the first time that vaspin is expressed in the ovary, more specifically in porcine ovarian follicles. Immunohistochemistry analysis documented vaspin protein in ovarian granulosa, theca, cells and cumulus, oocytes as well as in WAT cells. Results of our data showed that vaspin expression is dependent on the breed of pigs (fat mobilisation), days of the oestrous cycle and its regulation by gonadotropin, insulin, IGF-1 and steroids hormones, interestingly through the activation of different kinases such as JAK/Stat, ERK1/2, PI3K and AMPK, as well as the NF-κB factor.
By comparing levels of vaspin between both pig breeds, fat MS and lean LW, we observed significantly higher expression of vaspin in the ovarian follicles and WAT collected from MS compared to LW, which may reflect the different strategies employed by the two breeds to achieve prolificacy and fat mobilisation. Body weight, length, weight and width of uterine horns, as well as surface area and weight of endometrium and ovaries are greater in LW gilts (Bazer et al. 1988), while MS is characterised by an abundance of fat. Thus, our results indicate that vaspin levels are strongly dependent on fat quantity. Data of González et al. (2009) documented that vaspin levels in WAT in female rats are mediated by serum levels of leptin, which is the first fat cell-derived hormone. Multivariate regression analysis revealed increased body fat mass as the strongest predictor for vaspin gene expression (Klöting et al. 2006), confirming that expression of this adipokine is induced by increased abundance of fat, impaired glycaemic control and metabolic syndrome and as such, may represent a compensatory molecule in obesity and related disorders such as inflammation and insulin resistance (Wada 2008). A number of studies have investigated that vaspin levels decreased significantly after weight loss and fat reduction, for example after bariatric surgery or lifestyle intervention (Handisurya et al. 2010, Golpaie et al. 2011, Vink et al. 2017).
To our knowledge, the presented data showed for the first time expression of vaspin in the ovarian tissue. Strong immunolocalisation of vaspin protein was observed in granulosa, cumulus and theca cells, as well as in oocytes. We have shown that in similar concentration of vaspin in plasma and FF, both mRNA and protein expression of vaspin in the ovarian follicles are decreased in LW pigs during days of the oestrous cycle compared to MS pigs, in which vaspin levels were significantly higher at 16–18 days of the oestrous cycle, reflecting possibly different strategies in reproduction. MS breed reaches maturity very fast, where the number of oestrous cycles and ovulation rate are much higher than LW (data not yet published). Additionally, sows carry traits that make them one of the most reproductive breeds in the world. Previous studies have compared follicular development between MS and LW (Dufour & Mariana 1993). In gilts with low recruitment of antral follicles, as observed in MS, ovulatory follicles can be selected from a wide range of size classes containing few follicles. Whereas, in gilts with high recruitment of antral follicles, as observed in LW, ovulatory follicles appear to be selected from a narrow range of mostly large size classes. In addition to a decrease in the number of follicular size classes, increased level of atresia seems to limit ovulations in such breeds. It is still not known whether or not FSH is involved in the selection of ovulatory follicles. However, in the absence of equine chorionic gonadotropin (eCG), few LH-sensitive follicles are recruited and selected in the LW compared to MS. On the other hand, recruitment and selection have become identical in the two breeds after eCG injection. The process of ovulation in pigs implies different strategies for recruitment, selection and atresia according to the breed (Dufour & Mariana 1993). Additionally, Miller et al. (1998) documented higher FF E2 concentrations observed in preovulatory follicles, as well as number of follicles in the ovaries collected from MS. Moreover, Driancourt and Terqui (1996) also reported that follicles from hyperprolific animals secreted more E2 than those from control animals. Taken together, higher levels on E2 and different ovulatory rate can be factors that induce vaspin ovarian levels during oestrous cycle in MS.
Based on our findings that indicated differences in vaspin expression during the oestrous cycle, we focused on determining the factors that can regulate vaspin expression in the ovary. Results of our in vitro study clearly showed that gonadotropin, insulin, IGF-1 and steroids (P4, T and E2) significantly increased vaspin protein expression in the ovarian follicles and secretion to the culture medium. However, both LH and IGF-1 at higher doses 150 and 100 ng/mL, respectively, have inhibitory effect on vaspin protein expression, suggesting dose-dependent effect of these hormones. Similar results were observed in response to other adipokines such as resistin, where IGF-1 decreased in a dose-dependent manner the resistin level in porcine ovarian cells (Rak et al. 2015b ). To our knowledge, our results demonstrated for the first time that both gonadotropins regulate vaspin expression. However, previous studies have reported that gonadotropins are involved in the regulation of expression of other adipokines in ovarian cells, such as leptin (Siawrys & Smolinska 2013), adiponectin and its receptors (Chabrolle et al. 2007, Wickham et al. 2013), apelin and its receptor (APJ) (Shimizu et al. 2009) and resistin (Rak et al. 2015b ). Several studies showed that the hormonal interactions of the hypothalamic–pituitary–ovarian axis are accountable for normal ovarian activity. FSH plays a role in regulating the number of ovarian follicles that mature and in Gc proliferation and apoptosis (Wheaton et al. 1998), whereas LH is the principal luteotrophic signal in pigs, cows and sheep and is necessary for normal development of the corpus luteum and maintenance of its function (Gregoraszczuk 1991, Stouffer 2006). For the first time, these observations suggest that gonadotropins are also involved in the regulation of vaspin expression in ovarian follicles. Next, we observed that insulin and IGF-1 also have stimulatory effect on vaspin expression. Our results are in agreement with the data that demonstrated that vaspin expression in WAT was induced by insulin (Hida et al. 2005) and metformin, an insulin sensitiser (González et al. 2009). Previously, several studies documented connection between vaspin expression and steroids hormone. For example, vaspin serum levels were significantly enhanced in women using oral contraceptives (von Loeffelholz et al. 2010). Additionally, combined therapies using metformin and oestrogen/progestogen enhanced vaspin serum levels in non-obese women with hyperinsulinaemic androgen excess (Ibáñez et al. 2009). Elevated serum levels and adipose tissue expression were also observed in obese women with PCOS, where dehydroepiandrosterone sulphate significantly induced vaspin protein expression and secretion in adipose tissue explants (Tan et al. 2008).
Finally, we investigated the molecular mechanism involved in the regulation of vaspin in the ovarian cells in response to gonadotropins, insulin, IGF-1 and steroids by blocking different kinases activation. We observed that specific inhibitors of kinases JAK/Stat (AG490), ERK1/2 (PD098059), PI3K (LY294002) and AMPK (Compound C) as well as factor NF-κB (JSH-23) significantly changed the levels of vaspin, suggesting that expression of vaspin in the ovarian cells can be mediated via different kinases activation. To support these observations, Tang et al. (2014) found that resistin mRNA expression in 3T3-L1 adipocytes was mediated via ERK MAPK, JNK, PI3K and JAK2 pathways in response to endothelin 1. Similarly, endothelin 1 regulates adiponectin gene expression and secretion in 3T3-L1 via ERK1/2 and PKA activation (Juan et al. 2007). Results of our study are consistent with the previous data showing that resistin expression was regulated by 17β-E2 (Chen et al. 2006) or IGF-1 (Chen et al. 2005) by activation of ERK pathway. To our knowledge, phosphorylation of JAK/Stat, ERK1/2, PI3K or AMPK is necessary to ovarian physiology and its function, including steroid hormones secretion, proliferation, embryogenesis, cell migration and apoptosis (Makarevich et al. 2000, Mertens-Walker et al. 2010, Rak et al. 2017b ). Additionally, it is interesting in our findings that several kinases are also activated to regulate vaspin expression in ovarian cells.
In summary, our data indicated that (a) vaspin expression was significantly higher in the ovarian follicles and adipose tissue from MS compared to LW; (b) mRNA and protein expression of vaspin as well as its concentration in plasma and FF decreased in ovarian follicles of LW pigs during days of the oestrous cycle, while the opposite results were observed in ovarian follicles from MS pigs, where vaspin mRNA expression was significantly higher at 16–18 days of the oestrous cycle; (c) vaspin is present not only in granulosa, theca and cumulus cells but also in oocytes; (d) expression of vaspin was increased by gonadotropin, insulin, IGF-1 and steroids hormone and (e) different kinases such as JAK/Stat, ERK1/2, PI3K and AMPK, as well as factor NF-κB, were activated in vaspin regulation in the cultured ovarian cells. Further studies are necessary to better understand the role of vaspin on reproduction and ovarian physiology, for example steroid secretion, cell proliferation/apoptosis and oocyte maturation, thus providing new insights into the pathology of ovaries like PCOS. For example, recent published studies clearly documented that vaspin could be a potential novel biomarker for the prediction and early diagnosis of pregnancy pathology (Mm et al. 2014, Mierzyński et al. 2019).
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
Grant support: National Science Centre, Poland (Grant HARMONIA no. 2016/22/M/NZ9/00316).
Acknowledgements
The authors thank Christelle Rame and Eric Venturic for their technical support. The present publication contains parts of Patrycja Kurowska’s PhD dissertation.
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