The in vitro mechanism of Vaspinregulates the proliferation and steroidogenesis of rat lutein cells

Abstract

Objective

Stable luteal cell function is an important prerequisite for reproductive ability and embryonic development. However, luteal insufficiency seriously harms couples who have the desire to have a pregnancy, and the most important thing is that there is no complete solution. In addition, Vaspin has been shown to have regulatory effects on luteal cells, but the complex mechanisms involved have not been fully elucidated. Therefore, this study aimed to explore the effect of Vaspin on rat luteal cells and its mechanism.

Methods

Granulosa lutein cells separated from the ovary of female rats were incubated for 24h with gradient concentrations of Vaspin, and granulosa lutein cells incubated with 0.5% bovine serum albumin were used as controls. The proliferation, apoptosis, angiogenesis, progesterone (P4) and estradiol (E2) were detected by CCK-8, Anneixn-FITC/PI staining, angiogenesis experiment and ELISA. Western blot was applied to observe the expression levels of proteins related to cell proliferation, apoptosis, angiogenesis and MEK/MAPK signaling pathway.

Results

Compared with the Control group, Vaspin could significantly up-regulate the proliferation of granulosa lutein cells and reduce the apoptosis. Moreover, Vaspin promoted the angiogenesis of granulosa lutein cells and the production of P4 and E2 in a concentration-dependent manner. Furthermore, Vaspin up-regulated the CyclinD1, CyclinB1, Bcl2, VEGFA and FGF-2 expression in granulosa lutein cells, and down-regulated the level of Bax. Also, Vaspin increased the p-MEK1 and p-p38 levels.

Conclusion

Vaspin can up-regulate the proliferation and steroidogenesis of rat luteal cells and reduce apoptosis, which may be related to the influence of MEK/MAPK activity.

Keywords:

• Luteal cells
• spontaneous abortion
• proliferation
• steroidogenesis
• vaspin
• mek/mapk

Introduction

Spontaneous abortion refers to the fetal death as a result of the pregnancy loss before 13 weeks of gestation. Spontaneous abortion, also known as early pregnancy abortion or miscarriage, is the most common pregnancy complication in obstetrics [1]. It is estimated that 13.5%- 16.6% of pregnant women experience spontaneous abortion, accounting for the vast majority of all abortions. The risk of spontaneous abortion increases with the age of the mother [2]. Spontaneous abortion may be induced by the interaction of age, heredity, immunity, hormones and environmental factors [3]. In addition, high blood pressure, type 2 diabetes and obesity are also potential factors for spontaneous abortion. In particular, obesity will reduce gonadotropin levels and fertility of women, affect fertilization and reproduction, and increase abortion rates [4]. According to how frequently they occur, spontaneous abortions are typically divided into two categories: sporadic and recurrent [5]. Different countries and regions have different definitions of recurrent spontaneous abortion. One of the consistent facts is that it affects 1-2% of couples, accompanied with as high as 0.6%-2.3% of prevalence rate [6]. Moreover, it is worth noting that poorly understanding to the pathophysiology of spontaneous abortion at present leads to the unsatisfactory therapeutic effect [7]. For a long time, spontaneous abortion has not only seriously affected the physical and mental health of couples with pregnancy expectations, but also placed a great psychological burden to mothers with a willing to pregnancy.

According to previous studies, corpus luteum can produce progesterone (P4) to initiate the secretory phase of endometrium. Corpus luteum not only participates in implantation [8], but also supports early pregnancy [9]. The failure of implantation caused by the dysfunction of luteal cells and insufficient P4 secretion is very common in most of infertile patients [10]. As early as 30 years ago, investigations reported that luteal insufficiency was the main cause of spontaneous abortion [11]. Luteal insufficiency refers to the insufficiency of luteal endocrine function (unable to secrete a normal amount of P4) after ovulation, which is not conducive to the transformation of endometrium from the proliferative phase to the secretory phase [12]. Studies have pointed out that luteal support for patients with recurrent spontaneous abortion is beneficial to the normal pregnancy [13]. It follows that the stable function of luteal cells is particularly important for normal pregnancy. Clinically, the treatments for luteal insufficiency mainly focus on the supplement of P4 and chorionic gonadotropin (CG) [14]. However, possibly owing to unclear mechanism of luteal function regulation, current treatment strategies cannot completely solve luteal insufficiency. Therefore, further exploration of the mechanism of luteal function regulation is required.

Vaspin, a visceral adipose tissue-derived serine protease inhibitor, belongs to the serine protease inhibitor (serpin) family [15]. Previous studies have shown that Vaspin is expressed in the cytotrophoblast and syncytiotrophoblast of the human placenta during the first trimester while it is localized in the syncytiotrophoblast during the third trimester, and that Vaspin has the highest expression levels at the end of pregnancy [16]. In addition, a team pointed out that the expression level of Vaspin increased significantly in sows during estrus, and its highest expression was observed in the middle and late luteal phases. Therefore, they believed that Vaspin was a luteotropic factor [17]. Furthermore, they investigated the effect of Vaspin on P4, HSD3B and prostaglandin levels, as well as the involvement of vaspin receptor and kinases pathways (MAP3/1 and PKA) on Vaspin action [17]. The above studies indicated that Vaspin is a potential factor for regulating the function of luteal cells. In this study, luteal cells were isolated from rats to observe the effect and mechanism of Vaspin on the function of luteal cells in vitro. This study will provide new clues for exploring the mechanism of luteal insufficiency-induced abortion.

Materials and methods

Isolation and culture of luteal cells

Firstly, mature normocyclic virgin SD rats (3 to 4 months old) were selected for this study. This study was approved by the Ethics Committee of Guangdong Medical Experimental Animal Center (C202307-1). Next, luteal cells of rats were separated according to the previous described method [18]. Briefly, rats were injected with 50 IU of pregnant mare serum gonadotropin (PMSG), and 48 h later, 50 IU of human CG (hCG) was given. After 7 days, the rats were euthanized by CO₂ anesthesia, and granulosa lutein cells were separated from corpora lutea. To be specific, the ovaries were taken out after opening the abdomen of the rats and then placed in sterile phosphate buffered saline (PBS). After upon removing excess adipose and cystic tissues, the corpora lutea were transferred to a DMEM/F12 medium and cut into pieces as much as possible with sterile ophthalmic scissors. Subsequently, 0.1% collagenase and 15 IU/mL DNase I were supplemented to the tissue samples, followed by an incubation in a shaking water bath at 37 °C for 60 min. Later, a filter with a diameter of 38 μm was employed to filter the debris in the digested suspension. The filtrate was collected and centrifuged at 500 g for 10 min, and the obtained cell precipitate was washed twice with the DMEM/F12 medium. The obtained cell precipitate was followed by the determination of 97.7% purity of the isolated granulosa lutein cells using flow cytometry (Supplementary Figure 1). The granulosa lutein cells were then cultured in DMEM/F12 medium containing 100 IU/mL penicillin and 100 μg/mL streptomycin at 37 °C and 5% CO_2, allowing the cells to attach to the plate. The cells were then washed with DMEM/F12 medium, after which the cells were cultured in DMEM/F12 medium supplementing with 0.5% bovine serum albumin, ascorbic acid (20 μg/mL), transferrin (5 μg/mL) and sodium selenite (5 ng/mL, ICN, Poland) [19].

For observing the effect of Vaspin (Abbexa, Cambridge, UK), the rat granulosa lutein cells were incubated using different concentrations of Vaspin (80 ng/ml, 160 ng/ml and 320 ng/ml) for 24 h [20]. Besides, the rat granulosa lutein cells cultured in a DMEM/F12 medium supplemented with 0.5% bovine serum albumin for 24 h were set as a control group.

The purity of granulosa luteal cells was analyzed by flow cytometry

Simply put, the purified cells were pretreated with Fix & Perm Cell osmosis buffer (eBioscience, San Diego, CA). Anti-fshr monoclonal antibody (absin, Shanghai, China) was added and incubated at 4 °C for 30 min in the dark. Subsequently, a second FITC-labeled antibody (absin, Shanghai, China) was added and incubated at 4 °C for 30 min in the dark. Finally, the cells were suspended in 300 μL PBS and detected by FACSCantoTM II flow cytometry (BD Biosciences) [21].

CCK-8 assay

The detection of the luteal cell viability was performed using CCK-8 assay. To be specific, cell samples were seeded in a 96-well plate at a density of 2000 cells per 100 μL and cultured under a condition of 37 °C and 5% CO₂ for 24 h. Next, the culture medium was replaced by a new one, and Vaspin with different concentrations (80, 160, 320 ng/ml) was added. Subsequently, the 96-well plate was cultured again at 37 °C and 5% CO₂. 24 h later, an hour of incubation with 10 μL of CCK-8 solution (TJ35001, Biolite bioteeh, China) was carried out under the conditions of light avoidance, 37 °C and 5% CO₂. The absorbance at 450 nm was measured by a microplate reader (RNE90002, REAGEN, USA), and the cell viability of each group was calculated based on the absorbance.

Anneixn-FITC/PI staining

Firstly, 1 × 10⁵ granulosa lutein cells were seeded in a 12-well plate. Next, the cells were cultured with different concentrations of Vaspin (80, 160, 320 ng/ml) for 24 h, followed by a Annexin V-FITC/PI staining (BL110A, Biosharp, China). The suspended cells were collected in a 1.5 ml centrifuge tube, centrifuged at 4 °C and 1000 g for 5 min, and resuspended twice with PBS. Subsequently, 3 × 10⁵ granulosa lutein cells were resuspended with 500 μL of Anneixn-FITC buffer, and the resuspension solution was incubated with 5 μL of Annexin V-FITC and propidium iodide (PI) for 5 min after mixing them evenly. The binding of the granulosa lutein cells to Annexin V-FITC/PI was then analyzed using high-throughput flow cytometry (iQue® 3, Sartorius, Germany); the binding of the cells to PI was observed at an excitation wavelength of 488 nm and an emission wavelength of 520 nm; and the binding of the cells to Annexin V-FITC was observed at an excitation wavelength of 535 nm and an emission wavelength of 617 nm.

Angiogenesis experiment

To observe the angiogenesis ability of granulosa lutein cells, a 24-well plate embedded with matrigel was prepared. Specifically, 150 μL matrigel was added to the 24-well plate, and cell samples were incubated at 37 °C for 30 min. Next, 2 × 10⁴ granulosa lutein cells were inoculated in the 24-well plate, and Vaspin with different concentrations (80, 160, 320 ng/ml) was added for 24 h of incubation. Finally, the angiogenesis of cells in each group was observed under an inverted optical microscope (DMi8 M, Leica, Germany).

ELISA

The cell supernatant was collected and the concentrations of P4 and E2 in the medium were determined by enzyme immunoassay [20]. The experiment was conducted according to the instructions of the ELISA detection kit for P4 (KS18474, Keshun, China) and E2 (E-OSEL-R0001, Elabscience, China). The detection range of the rat P4 ELISA kit was 3.13-200 g/L, and the highest concentration of the standard curve was 200 g/L. The resulting solution was then diluted to different gradients, and the standard dilution solution (0 g/L) was directly used as a blank control. Subsequently, the absorbance at 450 nm was measured on a microplate reader (Thermo Fisher Scientific, Waltham, USA), and the levels of P4 and estradiol (E2) in cells were quantified. Each sample was made in triplicate.

Western blot

After different treatments, granulosa lutein cells were added with lysis buffer and stood on ice for a 30-min lysis. Following that, cell samples were centrifuged at 10000 g and 12 °C for 10 min, and the supernatant was extracted. Then, the concentration of protein samples was determined using Pierce™ BCA protein detection kit (23227, Thermo Scientific, USA). Subsequently, an equal amount of protein samples (30 μg) were evenly mixed with protein loading buffer (LT103, Epizyme, China) and heated in a boiling water bath for 5 min. Next, the protein was separated with 12% sodium dodecyl sulfate- polyacrylamide gel electropheresis (SDS-PAGE), and then transferred onto the polyvinylidene fluoride membrane through the Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (221BR, BIO-RAD, USA). The membrane was sealed in 5% skimmed milk for 24 h at ambient temperature. Later, the membrane was incubated with primary antibodies, including CyclinD1 (Rabbit, A19038, ABclonal, 1:1000), CyclinB1 (Rabbit, A19037, ABclonal, 1:1000), Bax (B-cell lymphoma 2 associated X, Rabbit, A12009, ABclonal, 1:1000), Bcl2 (B-cell lymphoma 2, A0208, ABclonal, 1:1000), p-MEK1 (Rabbit, AP1021, ABclonal, 1:1000), MEK1 (Rabbit, A19565, ABclonal, 1:1000), p-p38 (Rabbit, AP0526, ABclonal, 1:1000), p38 (Rabbit, A14401, ABclonal, 1:1000), VEGFA (vascular endothelial growth factor A, Rabbit, A12303, ABclonal, 1:1000) and FGF-2 (fibroblast growth factor-2, Rabbit, A0235, ABclonal, 1:1000). Goat Anti-Rabbit IgG- Horseradish Peroxidase (AS014, ABclonal, 1:5000) was added for the next incubation. The target bands were detected using ultra chemiluminescence reagents (Willget Biotech, Shanghai, China). Taking GAPDH (Rabbit, AC001, ABclonal, 1:1000) as an internal control, ImageJ software was employed for grayscale scanning and standardization of the protein bands.

Statistics and analysis

All data were expressed as means ± standard deviation (SD). All samples were made in triplicate, and each experiment was repeated three times. By using the Shapiro Wilk test to determine whether the data conforms to a normal distribution, the results indicate that each group of data meets the characteristics of a normal distribution. Therefore, one-way ANOVA was used for inter group difference analysis. If the results of one-way ANOVA were significant, the Tukey method was further used for inter group comparison. p < 0.05 indicates statistically significant. All statistical analysis was carried out using Statistical Product and Service Solutions (SPSS) Statistics 23.0.

Results

Vaspin promotes the viability of luteal cells

To clarify whether Vaspin affects luteal cells, the CCK-8 assay was used to observe the effect of different concentrations of Vaspin on the viability of granulosa lutein cells. The observation outcomes revealed that, compared with the control group, Vaspin treatment increased the viability of granulosa lutein cells in a concentration-dependent manner (Figure 1A, p < 0.01). Besides, the western blot results showed that the expression levels of CyclinD1 and CyclinB1 increased significantly with the increase of Vaspin concentration (Figure 1B-C, p < 0.01). Therefore, Vaspin could enhance the viability and proliferation of luteal cells.

Figure 1. Vaspin promotes the viability of luteal cells. (A) CCK-8 to observe the effect of different concentrations of Vaspin on the viability of granulosa lutein cells (n = 3); (B-C) Western blot to assess the effects of different concentrations of Vaspin on the expression levels of CyclinD1 and CyclinB1 in granulosa lutein cells (n = 3). **p < 0.01, vs. Control.

Vaspin inhibits the apoptosis of luteal cells

The impact of Vaspin on the apoptosis of granulosa lutein cells was observed by Anneixn-FITC/PI staining. As shown in Figure 2A, relative to the control group, the apoptosis level of granulosa lutein cells decreased significantly with the increasing Vaspin concentration (Figure 2A, p < 0.01). Similarly, the levels of apoptosis-related proteins (Bax and Bcl2) were observed by western blot. The results showed that, in contrast to the control group, the protein expression of Bax in granulosa lutein cells gradually decreased while that of Bcl2 gradually increased, with the increase of Vaspin concentration (Figure 2B, p < 0.01). The above results indicated that Vaspin could inhibit the apoptosis of luteal cells.

Figure 2. Effect of Vaspin on the apoptosis of luteal cells. (A) Anneixn-FITC/PI staining to observe the effects of different concentrations of Vaspin on the apoptosis of granulosa lutein cells (n = 3); (B-C) Western blot to detect the effects of different concentrations of Vaspin on the expression levels of Bax and Bcl2 in granulosa lutein cells (n = 3). **p < 0.01, vs. Control. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 associated X.

Vaspin stimulates the angiogenesis of luteal cells

In addition, we revealed the impact of Vaspin on the angiogenesis of luteal cells. According to the angiogenesis experiment, there was no doubt that, relative to the control group, the angiogenesis of granulosa lutein cells gradually increased with the increase of Vaspin concentration (Figure 3A, p < 0.01). Furthermore, the expression levels of angiogenesis-related proteins (VEGFA and FGF-2) were observed by western blot. Briefly, compared with the control group, Vaspin significantly up-regulated the expression levels of VEGFA and FGF-2 in granulosa lutein cells in a concentration-dependent manner (Figure 3B, p < 0.01). Hence, Vaspin could stimulate the angiogenesis of luteal cells.

Figure 3. Vaspin stimulates the angiogenesis of luteal cells. (A) Angiogenesis experiment to observe the effect of different concentrations of Vaspin on the angiogenesis ability of granulosa lutein cells (n = 3); (B-C) Western blot to estimate the effects of different concentrations of Vaspin on the expression levels of VEGFA and FGF-2 in granulosa lutein cells (n = 3). **p < 0.01, vs. Control. VEGFA, vascular endothelial growth factor A; FGF-2, fibroblast growth factor-2. Red arrows indicate formed blood vessels.

Vaspin increases the steroidogenesis in luteal cells

Interestingly, we also observed that compared with the control group, granulosa lutein cells incubated with Vaspin exhibited much higher levels of P4 (42.09 ± 2.45, 37.82 ± 2.32, and 33.05 ± 2.12 vs. 25.50 ± 2.91 g/L) and E2 (88.43 ± 2.94, 79.92 ± 3.98, and 66.55 ± 5.23 vs. 58.29 ± 7.32 ng/L), and these levels increased with the rising Vaspin concentration (Figure 4A-B, p < 0.01). Collectively, Vaspin could increase the steroidogenesis in luteal cells.

Figure 4. Vaspin up-regulates the steroidogenesis in luteal cells. A-B, ELISA to observe the impact of different concentrations of Vaspin on the production of P4 and E2 in granulosa lutein cells (n = 3). **p < 0.01, vs. Control. P4, progesterone; E2, estradiol.

Vaspin activates the activity of MEK/MAPK signaling pathway

Western blot was applied to reveal the mechanism by which Vaspin affects granulosa lutein cells. Compared with the control group, the ratios of p-MEK1/MEK1 and p-p38/p38 were raised with the increase of Vaspin concentration (Figure 5A-B, p < 0.01). In a nutshell, Vaspin could activate the MEK/MAPK signaling pathway activity.

Figure 5. Vaspin activates the activity of MEK/MAPK signaling pathway. (A-B) Western blot to observe the effects of different concentrations of Vaspin on the expression of MEK/MAPK signaling pathway-related proteins (p-MEK1, MEK1, p-p38, p38) (n = 3). **p < 0.01, vs. Control.

Discussion

The dangers of spontaneous abortion are often underestimated, and there is no timely treatment or prevention measures for it [22]. If a woman has a miscarriage but does not receive treatment timely, her mental health may be severely damaged. Generally, miscarried women and their partners inquire about the causes and solutions of miscarriage to soothe the pain of losing their fetus [23]. Previous studies have stated that the stable function of the corpus luteum is essential for conception and pregnancy as well as maintaining a regular ovarian cycle [24]. Luteal insufficiency is one of the causes of abortion. Therefore, revealing the mechanism of luteal cell function regulation may be a new direction to decipher the mechanism of abortion.

Interestingly, Kurowska et al. observed that Vaspin could regulate the secretion of steroid hormones in porcine luteal cells [25]. Their team also proved that Vaspin could significantly promote angiogenesis and proliferation while inhibiting apoptosis of porcine luteal cells [17], indicating the regulation potential of Vaspin to luteal cell function. Therefore, the effect of Vaspin on the viability of granulosa lutein cells was observed first in this study. The observation results displayed that Vaspin could remarkably increase the viability of granulosa lutein cells and the expression levels of CyclinD1 and CyclinB1. However, Li et al. discovered that Vaspin could significantly inhibit the expression of CyclinD1 in the study of its effect in the glucose-induced proliferation and chemotaxis of vascular smooth muscle, which may be put down to its inhibition of the vascular smooth muscle cell proliferation [26]. It is clear that Vaspin has dual effects on cell proliferation or viability, possibly owing to function differences between cells with different transcriptome levels. Nevertheless, Vaspin was considered to significantly increase the viability of granulosa lutein cells in this study. This suggests that in pregnant women with luteal insufficiency caused by decreased granulosa lutein cells, Vaspin can maintain luteal function and prevent miscarriage in pregnant women.

Apoptosis is an essential assurance of a regular cell cycle [27]. Notably, programmed cell death of luteal cells plays a crucial role in structural luteolysis [28]. In unfertilized animals, the apoptosis of luteal cells encourages the initiation of the next ovulation. However, in pregnant animals, the apoptosis level of luteal cells is closely related to the function of luteal cells; inhibiting apoptosis aids in protecting the function of luteal cells [29, 30]. Xu et al. believed that melatonin alleviated benzo(a)pyrene-induced corpus luteum dysfunction by suppressing the apoptosis of luteal cells [29]. Additionally, Al-Gubory et al. concluded that antioxidant enzymes could reduce reactive oxygen species-induced apoptosis and ensure the involvement of corpus luteum in embryonic development [30]. In our research, Vaspin significantly down-regulated the apoptosis level of granulosa lutein cells, as well as decreased the expression level of Bax while increasing that of Bcl2. Therefore, Bcl2 is an anti-apoptosis factor, while Bax is a pro-apoptosis factor that can activate Caspase-9 and cut Caspase-3 [31, 32]. More interestingly, Zhu et al. proved that Vaspin could lower the apoptosis level of osteoblasts induced by serum-free culture. They also found that Vaspin inhibited the Bax expression and promoted the Bcl2 expression [33]. In the investigation of Kurowska et al. Vaspin significantly promoted the proliferation of porcine granulosa cells and the cell cycle progression into the S phase and G2/M phase and reduced apoptosis. It was also found that siRNA silencing glucose-regulated protein (GRP78) receptor and pharmacological inhibitors of mitogen-activated kinase (MAP3/1/ERK1/2), Janus kinase (STAT3) and protein kinase B (AKT) pharmacological inhibitors could block the proliferation of vascular cells and enhance the activity of caspase-3/7 [17, 34]. In short, Vaspin can regulate the apoptosis level of luteal cells.

VEGF-A and FGF2 have also been revealed to be involved in regulating endothelial cells and promoting the formation of blood vessels [35, 36]. Some investigators have found extensive angiogenesis in both maternal and fetal placental tissues, with VEGF and FGF family molecules and their receptors serving as effective angiogenesis mediators in the placenta. And their research further disclosed that the VEGFA gene rs699947 was associated with an increased risk of recurrence in women receiving assisted reproductive technology (ART) for infertility [37]. In addition, it is worth noting that angiogenesis of luteal cells is a key prerequisite for their secretory function [38]. The rich capillary network is not only the medium for luteal cells to receive external signals such as nutrients and hormones, but also the hub for the output of luteal secretion substances, P4 and E2 [39]. Studies have shown that incomplete angiogenesis cannot guarantee the normal development of luteal cells, and it will affect the concentration of P4 in serum and result in abortion [40]. Hence, the angiogenesis of luteal cells is necessary for embryo development. In addition to limiting the development of atherosclerosis [41], Vaspin has also been reported to promote the angiogenesis of porcine luteal cells [17]. Here, we observed that Vaspin could boost the angiogenesis ability of granulosa lutein cells in a concentration-dependent manner. Also, as Vaspin concentration increased, so did the expression of VEGFA and FGF-2 in granulosa lutein cells. Collectively, Vaspin also participates in the regulation of angiogenesis of luteal cells.

In this paper, Vaspin was also found to significantly increase the production of P4 and E2 in granulosa lutein cells. Both P4 and E2 play an important role in the normal development of embryos [42, 43]. Kurowska et al. described that Vaspin stimulated synthesis of P4 and E2 in porcine granulosa cells [44], and that the increased P4 sythesis accelerated the oocyte maturation [20]. Similar observation was noted in human luteinized granulosa cells by Bongrani et al. [45]. After Vaspin treatment, the secretion of P4 and E2 from luteal cells was increased [25], so Vaspin may be a potential signal molecule for inducing luteal cells to secrete P4 and E2. Silva et al. demonstrated that the increase of MEK/MAPK signaling pathway activity was related to the increase of secretion of P4 and E2 from dog luteal cells [46]. Furthermore, Guo et al. found that microcystin-LR reduced the number and volume of embryos implanted in pregnant mice, which was related to the decrease of angiogenesis in luteal cells as a result of the decreased p-MEK level [47]. The phosphorylation level of MAPK increased significantly during the maturation of luteal cells in rats, as revealed by Maizels et al. who also confirmed that the content of p-MARK was closely connected to the maturation of luteal cell [48]. In our study, Vaspin activated the MEK/MAPK signaling pathway activity in granulosa lutein cells. This study provided the first evidence that Vaspin could raise the phosphorylation level of MEK. Previous experiments have also proved that Vaspin protects mouse mesenchymal stem cells from oxidative stress-induced apoptosis via activating MAPK/p38 [49]. Some studies, on the other hand, believed that MAPK activity is inhibited or not affected by Vaspin [26, 33]. Howsoever, the regulatory effect of Vaspin on the MAPK activity is very complicated.

There are some limitations in this study. First off, we did not further explore the specific regulatory mechanism of Vaspin on the MEK/MAPK signaling pathway. Additionally, we only observed the regulatory effect of Vaspin on luteal function in vitro. We truly found that Vaspin could maintain or even enhance luteal function, reduce the apoptosis of luteal cells, as well as promote placental angiogenesis and the secretion of P4 and E2, so as to protect the fetus and avoid abortion in pregnant women. However, we did not conduct in vivo experiments to observe whether Vaspin affects luteal function or embryonic development by targeting protein receptors in luteal cells. As a result, a thorough experimental design must be created for the upcoming research. Last but not least, depending on animal evolution, Vaspin may be generalizable to the role of gestational luteum in other mammals. To be certain, we still need to carry out pertinent research.

Conclusion

To sum up, Vaspin is an important signal molecule to maintain the function of luteal cells. Briefly, Vaspin can stimulate the proliferation of luteal cells, down-regulate the level of apoptosis, enhance the ability of angiogenesis and promote the secretion of steroid hormones. This may be put down to the activation of the MEK/MAPK signaling pathway by Vaspin. But the mechanism needs to be further clarified in future studies by including MAPK inhibitors as positive controls. Overall, Vaspin deserves further exploration and may be a potential target for the prevent or avoid of abortion or luteal insufficiency.

Author contributions

XF conceptualized and designed the study, drafted the initial manuscript. LL, and XZ collected the data and carried out the initial analyses. JW critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Guangdong Medical Experimental Animal Center (C202307-1). All methods were performed according to the international, national and institutional rules considering animal experiments, clinical studies and biodiversity rights.

Disclosure statement

The authors declare that they have no competing interests.

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.

Additional information

Funding

This study did not receive any funding in any form.