The effects of apelin on IGF1/FSH-induced steroidogenesis, proliferation, Bax expression, and total antioxidant capacity in granulosa cells of buffalo ovarian follicles

Apelin (APLN) was believed to be an adipokine secreted from adipose tissue. However, studies demonstrate that it is a pleiotropic peptide and has several effects on the female reproductive system. In this study, We examined the effects of different doses of IGF1 and FSH in the presence of APLN-13 on the production of progesterone in buffalo ovary granulosa cells. Furthermore, different doses of APLN isoforms (APLN-13 and APLN-17) were tested on proliferation, Bax protein expression, and antioxidant capacity in the same cells. Granulosa cells of buffalo ovaries were cultured in the presence of different doses of IGF1 and FSH with or without APLN-13 (10−9 M) to evaluate its effect on the secretion of progesterone tested by ELISA assay. The WST-1 method was used to survey the effect of APLN on granulosa cell proliferation and cytotoxicity. In addition, the antioxidant capacity of the cells in the presence of APLN was assessed using the FRAP method. mRNA and Bax protein levels were measured in granulosa cells treated with APLN using real-time PCR and western blot techniques. APLN-13 (10–9) stimulated the effect of IGF1 on the production of progesterone, and its levels were affected by APLN-13 dose-dependently. However, it did not significantly stimulate the effect of FSH on the secretion of progesterone. APLN-13 (all doses) and APLN-17 (10–8 and 10–9 M) improved the proliferation of granulosa cells. Moreover, preincubation of the cells for an hour by APLN receptor antagonist (ML221, 10 µM) did not significantly affect the proliferation of cells induced by APLN. Neither APLN-13 nor APLN-17 were not cytotoxic for the cells compared to the control treatment. APLN-13 at the doses of 10–6 and 10–8 M substantially up and down-regulated Bax protein expression; however, such effects were not observed when the cells were preincubated with ML221. In addition, APLN-17 did not influence the expression amount of Bax. Furthermore, both APLN-13 and -17 improved the total antioxidant capacity of the ovarian granulosa cells, but such effects were not seen when the cells were preincubated with ML221. According to these results, APLN enhanced the steroidogenesis induced by IGF1 but did not affect the steroidogenesis induced by FSH. APLN also enhanced the cell proliferation and antioxidant capacity of buffalo ovaries follicular granulosa cells; however, its effect on Bax expression was different.


Introduction
As a new adipokine produced by adipose tissue, APLN is derived from prepro-APLN (77 amino acids). Prepro-APLN is cleaved into a 55-amino-acid fragment and later into different APLN isoforms such as APLN-36, APLN-17, APLN-13, and the pyroglutamyl APLN -13 (Pyr-APLN-13; (Pitkin et al. 2010)). (Pyr) APLN-13 and -17 are more active isoforms in more quantity in the bloodstream (Tatemoto et al. 1998). The receptor of APLN is APJ (an orphan G protein-coupled receptor) with a similar structure to the angiotensin II type 1 receptor (AT1; (O'Dowd et al. 1993)). APJ can bind to different isoforms 1 3 of APLN with various affinities and activate several signaling pathways that lead to the appearance of different functions in the body (Shokrollahi et al. 2021). In addition to adipose tissue, APLN and APJ are extensively distributed in the various tissues of the body, such as the heart, brain, lung, blood vessels, spleen, intestine, breast, and reproductive tract (De Falco et al. 2002;Estienne et al. 2019;Mercati et al. 2018;Palmioli et al. 2021;Shokrollahi et al. 2021). Moreover, APLN and APJ control angiogenesis, cardiovascular function, fluid homeostasis, cell proliferation, food intake, and regulation of energy metabolism (Heinonen et al. 2005;Kasai et al. 2004;Liu et al. 2020;Shokrollahi et al. 2021). The reproductive roles of APLN have been reported in recent years, in addition to its localization in the gonads and the arcuate, supraoptic, and paraventricular hypothalamic nuclei (Sandal et al. 2015). Through attempts over the last 15 years, APLN has been verified in the granulosa (GCs), theca luteal cells, and oocytes of bovine, human, and porcine Roche et al. 2016Roche et al. , 2017. The considerable effects of APLN have been reported on follicle selection in the bovine species (Shimizu et al. 2009), steroidogenesis in different species such as rats, humans, cattle, sheep, and pigs (Mercati et al. 2019;Rak et al. 2017;Roche et al. 2016Roche et al. , 2017Shirasuna et al. 2008). It is stated that APLN, in the presence of different factors such as IGF1 and FSH, has meaningful effects on the secretion of progesterone (P4) and estradiol (E2) in cattle (Roche et al. 2017) and porcine ) as well as buffalos (Shokrollahi et al. 2022) through various signaling pathways. There are few reports regarding the effect of APLN on the reproduction of buffalos. Recently, Gupta et al. (2023) found that APLN and APJ are expressed in the follicular and luteal cells of buffaloes. They discovered that APLN positively affects steroidogenesis and the survival of follicular cells. In the previous work, we indicated that APLN and APJ are expressed in GCs of different follicular stages. In addition, the results showed that IGF1 and FSH increased the expression of APLN and APJ, and APLN improved the secretion of E2 and P4 in GCs (Shokrollahi et al. 2022). However, we did not examine the effect of APLN on secretion of P4 induced by different doses of IGF1 or FSH.
Despite a lot of work, the information regarding the effect of APLN on IGF1/FSH-induced steroidogenesis and cell proliferation, apoptosis, and scavenging activity of ovarian follicular GCs in buffaloes is scanty. Therefore, the present study hypothesized that APLN could have such an effect on the GCs of buffalo. To verify this hypothesis, the impact of APLN-13 on IGF1/FSH-induced steroidogenesis was evaluated. In addition, the effects of APLN-13/-17 proliferation, cytotoxicity, Bax expression, and scavenging activity of GCs were studied.

Reagents
Unless otherwise stated, all chemicals and media used in the current study were obtained from Sigma-Aldrich (MO, USA).

Follicle collection and granulosa cells culture
Buffalo ovaries were collected from a local slaughterhouse and transported on ice within 2 h after slaughter to the laboratory in phosphate-buffered saline (PBS) supplemented with 0.05 mg/mL streptomycin and 0.06 mg/mL penicillin. In the laboratory, the ovaries were washed adequately with physiological saline solution.
To assess the effect of the APLN on IGF1/FSH-induced steroidogenesis, cell proliferation, apoptosis, and cell redox status, the GCs culture model was established. Therefore, all the healthy (well vascularized and having transparent follicular wall and fluid) and visible follicles (at the sizes of 6-9 mm) were aspirated by a 17-gauge needle attached to a 10-mL syringe. The aspirates were transferred to a 60-mm dish under sterile conditions with PBS, and all cumulusoocyte complexes were removed. The remaining cells and liquids were centrifuged in 15-mL conical tubes at 700 g for 5 min. Then, GCs were resuspended in Dulbecco's Modified Eagle Media (DMEM) media containing 10% fetal bovine serum (FBS) and antibiotics and antimycotic solution (penicillin 100 U/mL, streptomycin 100 mg/mL, amphotericin B 0.25 mg/mL). Cell viability was evaluated using trypan blue exclusion dye, exceeding 80%. The cells were then seeded in a 48/96-well plate in a humidified CO2 (5%) incubator at 37.5 °C , with approximately 1.5 × 10 5 viable cells per well. The cells were allowed to attach and grow (75%-80% confluence) for 48 h. Then cells were treated with fresh media (FBS free) containing different doses of porcine FSH (0, 10 -6 , 10 -7 , 10 -8 , 10 -9 , and 10 -10 M) and/or human recombinant IGF1 (0, 10 -6 , 10 -7 , 10 -8 , 10 -9 , and 10 -10 M) singly or in the presence of 10 -9 M APLN-13 and were maintained for 48 h. Control cells were grown in similar conditions as other cells except for adding the mentioned peptides. After 48 h, the spent media were collected and stored for P4 assay. As part of investigating whether Bax mRNA and protein expression was affected by APLN-13/-17 after culturing for 48 h with 10% FBS, GCs were treated with different doses of these isoforms (0, 10 -6 , 10 -8 , 10 -9 M) after 48 h in a sera free medium then mRNA and protein were extracted from cells.

Total RNA extraction, cDNA synthesis
Total RNA was extracted from follicular GCs using TRIZOL reagent by the manufacturer's instruction. Briefly, 200 ml of TRIZOL was added to cells and incubated for 3 min and transferred to a RNase free tube, and 80 ml of chloroform was added, mixed slowly for 15 s, incubated for 3 min, and centrifuged for 25 min at 4˚C at 12,000 g. The aqueous phase was transferred to a new RNase-free tube, and isopropanol (2.5 times supernatant) was added, mixed, incubated for 30 min at room temperature, and centrifuged for 20 min at room temperature 4˚C at 12,000 g. The supernatant was discarded, the pellet was washed with 200 µl ethanol (70%), centrifuged in 7500 g for 8 min at 4˚C, ethanol discarded, pellet air-dried for 5 min and dissolved in preheated DEPC (20 µl) at 60 ˚C. Then a fixed amount of RNA (100 ng) was directly reverse-transcribed into a 20 μL first-strand cDNA using a PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time, TAKARA BIO INC, Japan) following the manufacturer's instructions.

Quantitative real-time PCR analysis
Rt-qPCR was done in a total volume of 20 μL, containing equally distributed cDNA (100 ng), 10 mM each of the forward and reverse primers, and 10 μL of 2 × SYBR Green Master Mix (SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, TAKARA, Japan). All reactions for all genes of interest were performed in triplicate and were run on the light Cycler 480 system (Roche Diagnostics) under the following conditions: 95 ℃ for 30 s, followed by 40 cycles at 95℃ for 5 s and 60℃ for 30 s. β-actin and RPS15 were used as the internal control (reference genes) to normalize the relative gene expression levels (Gupta et al. 2023;Shokrollahi et al. 2022). The gene expression levels were analyzed with the Fig. 1 A comparison of buffalo protein sequences with the sequences of the different proteins used in this study. Buffalo FSH beta subunit (Acc. No: NP_001277871.1) was 92.25% similar to porcine equivalent (Acc. No: NP_999040.1) (A). IGF1 from Buffalo (Acc. No: XP_006058875.1) and human IGF1 (Acc. No: CAG46659.1) had 96.08% similarity (B). There was 77.92% similarity between the APLN of buffalo (Acc. No: XP_025132686.1) and that of human (Acc. No: AAH21104.2 (C). The asterisk (*), colon (:), dot (.) and gaps () indicate identical amino acid residues, conserved, and semiconserved and non-conserved substitutions in the sequences used in the alignment, respectively A FSH beta subunit. B IGF1 C APLN 1 3 2 −ΔΔCT method described previously by Livak and Schmittgen (2001) concerning the housekeeping genes. The details of the selected genes and the primer pairs used in the study are provided in Table 1.

Western blot analysis
Total proteins were obtained from cultured GCs of different experiments by lysing in RIPA buffer containing PMSF (R0010; Solarbio, China) at 4 °C for 30 min followed by collection and centrifugation at 12,000 g for 5 min at 4 °C. The pellet was eliminated, and lysates were diluted with 6X protein loading buffer (DL101-02; TransGen, China) and heated to 100 °C for 5 min. After cooling on ice, the samples were stored at -80 °C until the western blotting. Western blotting was started by loading the samples on a 12% gradient polyacrylamide gel (P0012AC; Beyotime, China) and then transferred to a PVDF membrane (ISEQ00011; Millipore, China), followed by blocking in 8% (wt/vol) Difco Skim Milk in Tris-buffered saline containing 0.1% (vol/ vol) Tween-20 (TBST) for 2 h. Overnight incubation with the primary antibody (Bax and anti-beta-Actin) was performed. Then, after four washes, 10 min each, with TBST, membranes were incubated for 1 h at 37℃ with goat antimouse IgG for beta-Actin and with goat anti-rabbit IgG for Bax. The membranes were washed four more times in TBST for 10 min each, then developed using ECL Plus (P0018, Beyotime, China) followed by detection with a multi-function imager (Syngene, Cambridge, UK). The intensities of individual bands were normalized to the expression of beta-Actin.

The proliferation and cytotoxicity effects of treatments on GCs
The effects of APLN-13/-17 on GCs proliferation and cytotoxicity were evaluated by WST-1 ( ( 4 -[ 3 -( 4 -i o d o p h e nyl ) -2 -( 4 -n i t r o p h e nyl ) -2 H -5tetrazolium]-1,3-benzene disulfonate) method. WST-1 is an assay that utilizes mitochondrial dehydrogenases to cleave the tetrazolium salt WST-1 to formazan. As the number of viable cells increases, the activity of mitochondrial dehydrogenases increases; thus, formazan dye is produced in greater quantities. Briefly, 4 × 10 4 cells per 200 µL of culture media were seeded in 96-well plates treated with APLN-13/-17 (0, 10 -9 , 10 -8 , 10 -6 M) with or without preincubation of cells with APJ antagonist (ML221 10 µM) for 1 h and incubated for 48 h. According to the product manual, 10 µL of WST-1 was added to the cells during the last 4 h of incubation. Then, the absorbance was detected by a plate reader machine at a wavelength of 440 nm.
The rate of cytotoxicity of APLN-13/-17 was calculated according to the following formulae: where CTA is the control group, and ATC is APLN-treated cells.

Steroids ELISA assay
P4 concentration was estimated in serum-free media from buffalo GCs after 48 h of culture using ELISA kits supplied by Jiangsu Meibiao Biotechnology Co (MB-4772A), China. Cells were plated in 48-well plates (10 5 viable cells/well), and six replicates were tested for each experimental condition (IGF1 and FSH in the presence or absence of APLN-13 or -17). The results were expressed as the concentration of the steroid (pmol/L). The intra-and inter-assay coefficients of variation for progesterone were less than 10%. Results are given as mean ± SEM. Data were obtained from 3 independent cultures that were done separately.

Statistical analysis
All the data have been shown as mean ± SEM. One or twoway ANOVA followed by Duncan's multiple range test was used to test the differences among groups by GLM procedure of SAS software (Version 9.4). A significant difference was considered if P < 0.05. The data were checked for normality and homogeneity of variance between the groups.

Results
The effect of varying IGF1 and FSH doses, with or without APLN-13, on the secretion of P4 from GCs of buffalo ovaries Figure 2 shows the effect of different doses of IGF1 and FSH, with and without APLN-13, on the secretion of P4 in buffalo ovarian GCs. To understand the effects of APLN with two main hormones influencing the biological function of GCs, we treated the GCs with different doses of IGF1 or FSH with or without APLN-13 in a sera-free DMEM culture media for 48 h following the incubation of the cells in the same media with 10% FBS. The results showed that different doses of IGF1 significantly increased the secretion of P4 from buffalo GCs (P < 0.01). APLN significantly increased IGF1-induced secretion of P4. IGF1 affected P4 production from GCs in a dose-dependent manner, increasing with higher doses. Furthermore, treatment of GCs with FSH increased the secretion of P4, even though there was no difference between various doses of FSH. In addition, we observed no significant differences between FSH alone and combined with APLN-13 on P4 concentration in the treated buffalo GCs.

The effects of various doses of APLN-13 and APLN-17 on the buffalo GCs proliferation and cytotoxicity
The effects of different doses of APLN-13/-17 on the GCs proliferation and cytotoxicity are presented in Fig. 3. APLN-13 caused a significant increase in cell proliferation compared to control cells; however, there were no substantial differences among various doses of APLN-13 in cell proliferation (Fig. 3A). APLN-17 also increased the proliferation of GCs at 10 -8 and 10 -9 M doses but not at 10 -6 (Fig. 3B). APLN-13 and APLN-17 were not cytotoxic for the GCs (with and/or without ML221; Fig. 3C, 3D). Results also showed that preincubation of the cells with APJ antagonist (ML221 10 µM) before APLN-13/-17 given to buffalo GCs at 0, 10 -10 , 10 -9 , 10 -8 , 10 -7 , 10 -6 doses alone or combined with APLN-13 (10 -9 ). Different capital and lower-case letters on the bars reveal the significant effects of different concentrations of IGF1 or FSH and in combination with APLN-13, respectively. Data represent the mean ± SEM of 6 replicates/treatments repeated in 3 different experiments treatment did not affect the GC proliferation (Except APLN-17 at 10 -6 M).

The effects of various doses of APLN-13 and APLN-17 on the expression abundance of Bax in the GCs of buffalo ovaries
The effects of various doses of APLN-13/-17 on the expression amount of Bax are shown in Fig. 4. APLN-13, when administered at 10 -8 and 10 -6 M, significantly decreased and increased the mRNA expression of Bax without preincubating the cells with ML221 (Fig. 4A). The same effects were not detected when the cells were preincubated with ML221 (10 µM; Fig. 4B), and APLN-13 at 10 -6 M significantly decreased the mRNA and protein expression levels of Bax. On the other hand, different doses of APLN-17 did not considerably affect the abundances of Bax mRNA/protein with or/and without preincubation of the cells with ML221 for an hour (Fig. 4C, 4D).

The effects of various doses of APLN-13 and APLN-17 on the total antioxidant capacity of GCs
The effects of different doses of APLN-13/-17 on the antioxidant capacity of GCs are shown in Fig. 5. APLN-13 at the doses of 10 -9 , 10 -8 M significantly increased the reduction power of GCs compared to the control cells; however, it did not have a significant effect at the dose of 10 -6 M (Fig. 5A).
Contrastingly, all doses of APLN-17 had a statistically significant effect on the antioxidant power of GCs (Fig. 5B). The cells treated with APLN-13 and APLN-17 at doses of 10 -8 had the highest impact on the antioxidant power of GCs. When cells were preincubated with ML221 (10 µM) for 1 h before being treated with APLN-13/-17, the antioxidant capacity of the cells was not significantly affected.

Discussion
In recent years, adipokines have been the most focused peptides in the reproduction system. The effect of the APLN/ APJ system on various aspects of reproduction has been studied in different species, but the findings are contradictory. These contradictory results could be attributed to the species, the environmental effects of the reproduction status of different species, and lab conditions. There are few reported results regarding the effects of APLN on the reproduction of buffaloes. In our previous study, we detected the expression of APLN and its receptor in the GCs of buffalo. Additionally, we showed that APLN isoforms increase the secretion of P4 and related enzymes. Recently another report demonstrated that APLN and APJ are expressed in ovarian follicles and corpora lutea, and APLN had a stimulatory effect on P4 secretion and improved GC survival in buffalo (Gupta et al. 2023). As part of the current study, we surveyed for the first time the effects of different FSH and Capital and lower-case letters on the bars indicate significant differences (P < 0.01) between APLN doses and ML221 groups, respectively, calculated by ANOVA and Duncan test IGF1 doses on P4 production in buffalo ovaries with and without APLN-13. We have chosen the APLN-13 isoform for this purpose because, according to our previous study, APLN-13 had a distinguished more impact on steroidogenesis compared to APLN-17, and regarding the literature, APLN-13 has more biological impact than other isoforms. Also, we evaluated the effects of APLN-13 and APLN-17 on cell proliferation, Bax protein expression, cytotoxicity, and antioxidant capacity in the same cells. Our findings showed that APLN-13 triggered the effect of IGF1 on the production of P4. In the presence of APLN-13, P4 was not significantly affected by FSH doses from buffalo GCs. These results partly agree with the literature (Roche et al. 2016(Roche et al. , 2017. Roche et al. (2016) showed that APLN-13/-17 did not affect the E2 or P4 secretion in the presence of FSH in cultured human GCs; also, these researchers found that the secretion of P4 from cultured bovine GCs did not influence by APLN in the presence of FSH (Roche et al. 2017). As one of the main adipokines, APLN has a crucial role in energy metabolism (Bertrand et al. 2015). It affects glucose transfer and mimics the insulin effect in skeletal muscles (Dray et al. 2008;Yue et al. 2010). As insulin and IGF1 have similar structures and receptors, APLN can enhance IGF1 responsiveness in GCs and increase E2 and P4 production (Roche et al. 2016). Furthermore, previous studies showed that APLN enhances the phosphorylation of PI3K/Akt induced by IGF1 (Roche et al. 2016), which causes steroidogenesis regulation in GCs (Hoffmann et al. 2019;Zhou et al. 2013). Roche et al. (2016) suggested that the effects of APLN on IGF1-induced steroidogenesis in GCs are exerted through PI3K/Akt signaling pathway. Our results showed an increasing impact of APLN-13 on IGF1-induced P4 secretion in a dose-dependent manner, but whether these increases in P4 concentrations in the medium are due indirectly to increases in cell numbers or directly owing to stimulation of steroidogenesis will require further study. Moreover, in our previous study, we showed that IGF1 caused the enhancement of the expression of APLN and IGF1 in buffalo GCs (Shokrollahi et al. 2022). These findings strongly support the association between APLN and IGF1 in the buffalo GCs.
The current results showed that APLN-13 and-17 significantly increased the proliferation of buffalo GCs, although APLN-17 at 10 −6 M did not substantially influence GCs proliferation compared to the control group, and the same results were gained when the cells were preincubated with ML221 for 1 h. Additionally, we showed that APLN-13/-17 was not cytotoxic to buffalo GCs. Literature demonstrated that APLN restrains cell apoptosis and retina angiogenesis and improves the proliferation of vascular smooth muscle cells, osteoblasts, and microvascular endothelial cells (Kasai et al. 2010, Olszanecka-Glinianowicz et al. 2013, Xie et al. 2006. Likewise, the proliferative effect of APLN on various carcinoma cell lines has been verified in different studies (Peng et al. 2015;Rene Gonzalez et al. 2009;Yang et al. 2014). In concurrence with our results, APLN promoted the GCs proliferation in the rat (Shuang et al. 2016), bovine (Roche et al. 2017), and porcine . However, the results of one study suggested that APLN does not affect human GCs proliferation, but in line with our findings, they explained that it does not affect the viability of GCs (Roche et al. 2016). Another report declared that APLN prevents the proliferation and migration of pulmonary arterial smooth muscle cells ). We did not investigate the cellular pathways of APLN's effect on proliferation or apoptosis. However, the reported results showed that APLN promoted in vitro cell proliferation through activation of the ERK1/2 signaling pathway in porcine GCs , lung adenocarcinoma (Yang et al. 2016), MCF-7 (breast cancer cells) (Tang et al. 2007), and ovarian cancer cells (Hoffmann et al. 2019). On the other hand, in some other studies, it is illustrated that cell proliferation promotion can occur via the IP3/Akt signaling pathway (Qin et al. 2013;Zhang et al. 2015). ERK1/2 signaling pathway activation is the main pathway for cell proliferation improvement by APLN. ERK1/2, as an essential pathway to cell proliferation, triggers the expression of cyclin proteins (mainly cyclin D1) and improves cell cycle move on (Chen et al. 2015). APLN also improves steroidogenesis and proliferation of GCs through increasing IGF1 sensitivity (Dray et al. 2008;Roche et al. 2016).
This study also revealed that APL-13 only at the doses of 10 −8 M (without ML221) and 10 −6 M (with ML221) decreased Bax protein expression; nonetheless, the dose of 10 −6 M (in the absence of ML221) elevated the protein expression of Bax compared to control cell; therefore, the results were divergent. Bcl-22 protein family is a necessary governor for apoptosis in various types of cells (Greenfeld et al. 2007). Different members of the BCL2 family are expressed in growing follicles, such as anti-apoptotic and proapoptotic factors (Hussein 2005). It is shown that Bax, as a proapoptotic protein, is engaged in the apoptosis of follicle cells under different conditions and, along with Bcl-2 as an anti-apoptotic factor, plays a critical role in the apoptosis of different cells. The anti-apoptotic effect of APLN has previously been shown in mouse (Tang et al. 2007) and human (Xie et al. 2006) osteoblasts, rat cardiomyocytes (Zhang et al. 2009), and rat neurons (Zeng et al. 2010). Furthermore, Shuang et al. (2016) showed that APLN decreases apoptosis of rat ovarian GCs. However, in contrast to parts of our study, they found that APLN upregulated the expression of Bax. In an older study, Shimizu et al. (2009) found that the mRNA expression of APJ was increased in apoptosis-induced bovine GCs. They concluded that there is a correlation between GCs atresia and the expression of APJ. However, Roche et al. (2017) did not find any effect of APLN-13/-17 on the apoptosis of bovine ovarian GCs. In line with our findings, APLN diminished mRNA and protein expression of proapoptotic proteins (including Bax) in BeWo cells (Mlyczyńska et al. 2021). APLN decreases apoptosis through PI3K/Akt and ERK1/2 signaling pathways, and activation of these pathways causes suppression of BAX/BAD proapoptotic proteins (Rastaldo et al. 2011). Moreover, it is demonstrated that APLN enhances Bcl-2 expression levels, reduces Bax protein's expression amount, and arrests the release of cytochrome C (Rastaldo et al. 2011). We did not assess the expression of Bax in luteinizing GCs. However, Shirasuna et al. (2008) implied that down-regulation of the APLN-APJ system could be associated with cell apoptosis and leutolysis in bovine.
The present study results revealed that all doses of APLN-13/-17 significantly improved the antioxidant power of buffalo ovarian GCs, with 10 -8 dose having the highest effect. Overall, APLN improved the antioxidant potential of GCs. Oxidative stress is a crucial factor causing reactive oxygen species (ROS) aggregation and subsequent cell death (Dawid et al. 2022). In human adipocytes, Than et al. (2014) found APLN reduced ROS production and release, as well as oxidative stress and oxidative-induced cellular degeneration. Likewise, in agreement with the results of this study, APLN enhanced the response of rat adipocytes and adrenal medulla cells to oxidative stress and decreased the levels of oxidative stress indicators (Foroughi et al. 2019;Niknazar et al. 2019). Mlyczyńska et al. (2021) outlined that APLN reduced oxidative stress by enhancing extracellular oxygen utilization. APLN increases GC antioxidant power and is directly correlated with improving cell viability and decreasing apoptosis. Another possible explanation for APLN's positive effect on cell death could be its antioxidant properties.

Conclusion
Our results showed that APLN stimulated the IGF1-induced production of progesterone, but it did not affect FSH-induced progesterone secretion in the buffalo ovarian granulosa cells. Moreover, APLN improved the proliferation and antioxidant power of the same cells. However, the effect of APLN on the Bax protein as a proapoptotic factor varied. These data demonstrate that APLN may have an essential role in steroidogenesis and proliferation of follicular granulosa cells of buffalos; nonetheless, these roles, in addition to Bax expression and antioxidant power of the cells, require more investigation.
Authors' contribution BS designed, conducted the study and data analysis, and also wrote the manuscript. HYZ and XYM helped with some lab work and JHS was the project leader. All authors contributed to the article and approved the submitted version. Data availability All data supporting the conclusions of this study are included in the article.

Declarations
Ethical approval The Animal Ethics Committee at the Guangxi Buffalo Research Institute handled all aspects of the experiments and ensured they complied with animal research ethical regulations.
Competing interests There are no conflicts of interest to declare.