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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Metformin Modulates Steroidogenic and Metabolic Functions in Cultured Ovine Granulosa Cells

P
Prachi Dhaka1,2
G
Govind Sahay Gottam2
S
Shyam Sundar Soni1
N
Neetu Kumari1,3
S
Shivendra Sharma1
S
Satyaveer Singh Dangi1
A
Arun Kumar1
A
Ajit Singh Mahla1,*
0000-0001-9234-4991
1Division of Animal Physiology and Biochemistry, ICAR-Central Sheep and Wool Research Institute, Avikanagar-304 501, Rajasthan, India.
2Department of Veterinary Physiology and Biochemistry, Post-Graduate Institute of Veterinary Education and Research, Jaipur-302 031, Rajasthan, India.
3Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India. 

ABSTRACT

Background: Metformin is an antidiabetic drug used for the treatment of infertility in polycystic ovarian syndrome and has been shown to affect the functions of granulosa cells (GC) in humans and rodents. Recently, metformin has been shown to enhance folliculogenesis, ovulation rate and prolificacy in sheep. The objective of this study was to determine the effect of metformin on GC isolated from sheep ovaries on cell number, proliferation, steroidogenesis and some hormones and genes related to follicular development.

Methods: GC were cultured in 3D culture using hanging drops for 48 hours and then treated with various concentrations of metformin (0.001 mM, 0.01 mM, 0.1 mM), dorsomorphin (0.1 mM), or a combination of both (0.1 mM) for 48 hours. Cell number and proliferation were measured using the trypan blue exclusion dye method and MTT assay. Progesterone, estradiol and insulin concentration in culture media were quantified by RIA. The expression of GLUT4, PPARα and SREBP1c was determined by qPCR.

Result: Metformin enhances the number and proliferation of GC while reducing the concentration of steroid hormones, such as androstenedione, progesterone and estrogen, in a dose-dependent manner. It also decreases the gene expression levels of SREBP1c and upregulates the mRNA expression of PPARα and GLUT4 and dorsomorphin treatment reverses these effects. These results suggest that metformin may modulate ovarian steroidogenesis, metabolism and folliculogenesis, influencing sheep reproductive-performance. However, further studies are required to investigate the detailed mechanism through which metformin exerts its effect.

INTRODUCTION

Improving reproductive efficiency is a major focus in farm animals due to its direct contribution to productivity and profitability. Sheep can contribute to food security by maximizing their reproductive traits, such as ovulation rate, fertility and prolificacy. Prolificacy depends on the number of matured oocytes released from ovulatory follicles during each reproductive cycle and is influenced by the interaction of multiple factors, including genetics, nutritional, hormonal and environmental factors (Scaramuzzi et al., 2006). Various approaches have been explored to improve the reproductive performance of sheep, including nutritional (Somchit-Assavacheep  et al., 2013; Viñoles  et al., 2005), immunological (Campbell and Scaramuzzi, 1995; Scaramuzzi et al., 1980) and pharmacological strategies (Kumawat et al., 2024).
       
Among pharmacological agents recently explored to improve reproductive performance in sheep is metformin (Kumawat  et al., 2023, 2024). Metformin is an insulin-sensitizing drug commonly used in the treatment of type 2 diabetes, cycle disturbance, hyperandrogenism and anovulation in polycystic ovarian syndrome (Palomba et al., 2009; Shpakov, 2021; Velazquez et al., 1997). Recently, metformin treatment for 12 weeks significantly enhanced reproductive performance in ewes, increasing the number of preovulatory follicles, ovulation rate and prolificacy rate with a 2.9-fold rise in multiple births compared to controls. Additionally, metformin treatment also reduces glucose, insulin, total cholesterol, LDL-cholesterol and estradiol concentrations than in the control group (Kumawat et al., 2024). Similarly, in another study on ewes, the combined approach of nutritional flushing with metformin treatment for 6 weeks improved the preovulatory follicular turnover and ovulation rate, leading to increased fetal numbers during early gestation in low body condition score ewes, with a marked reduction in the estradiol and glucose concentration (unpublished data). Both studies indicate that metformin treatment improves both metabolic and ovarian follicular dynamics. Metformin modulates peroxisome proliferator-activated receptors (PPARs), activates the AMPK signalling cascade and inhibits insulin-stimulated cell proliferation (Bai et al., 2019). It improves insulin sensitivity, facilitating glucose uptake, inhibiting the production of androstenedione, estrogen and progesterone by suppressing enzymes such as StAR, 3β-HSD, CYP11A1 and aromatase in human ovarian cells (Palomba et al., 2009). Metformin directly or indirectly inhibits growth, function and cell proliferation in rat (Tosca et al., 2006) and human (Rice et al., 2009), leading to reduced granulosa cell (GC) number, which in turn results in the formation of small follicles that undergo early maturation. However, the precise mechanism through which metformin exerts its effect in ewes remains unclear. Hence, by leveraging an in vitro GC culture model, the present study aimed to investigate the effect of metformin on cell proliferation, steroidogenesis and the expression of metabolic genes and to determine the involvement of AMPK signalling by using dorsomorphin, a pharmacological inhibitor. The present study will enable to determine how metformin influences the follicular development and reproductive performance by linking these cellular effects.

MATERIALS AND METHODS

Collection of ovaries
 
Sheep ovaries (n=60) were collected from the commercial abattoir/ slaughterhouse. The ovaries were placed in chilled Dulbecco’s phosphate-buffered saline (PBS) (HiMedia) containing an antibiotic and antimycotic solution (100 IU/ml of penicillin G sodium, 0.1 mg/ml of streptomycin sulphate, 0.25 μg/ml of amphotericin B, HiMedia) and transported to the laboratory within 2 hours after slaughter (Kumar et al., 2014). Ovaries were washed at least 4-5 times in PBS and processed immediately.
 
Granulosa cell isolation and culture
 
Healthy follicles were assessed by clear amber follicular fluid with no debris. The follicular fluid was aspirated from the medium (3.5-5.0 mm) ovarian follicles into chilled media using a 1 ml sterile insulin syringe into a 1.5 ml tube under sterile conditions. After follicular aspiration, the sample was allowed to stand undisturbed to allow thecal cells to settle and GC-rich supernatant was collected and centrifuged at 1200xg for 10 minutes at 4°C (Kumar et al., 2014). The cell pellet was suspended in 200 µl of culture media, DMEM/F12(Gibco) supplemented with 3.3 mg/ml bovine serum albumin (Sigma Aldrich), 1% ITS supplement (insulin, transferrin and sodium selenite, HiMedia), 10-7M Δ4-Androstene-3,17-dione (TCI), 10% fetal bovine serum (FBS) (HiMedia), 0.05 µg/ml  FSH+0.01 µg/ml LH (Stimufol, Reprobiol SPRL), 100 IU/ml of penicillin G sodium, 0.1 mg/ml of streptomycin sulphate, 0.25 μg/ml of amphotericin B (HiMedia). Cell number and viability were estimated/assessed using the trypan blue dye exclusion method in an automatic cell counter, the Countess™ (Invitrogen) (Soni et al., 2018).
       
The GC were cultured in hanging drops in 24-well culture plates (Nunc™, Thermo Fisher Scientific, Inc.) with a density of 5000 cells in an 8 µl drop supplemented with culture media and cultures were maintained at 37°C for 5% CO2 and 95% RH. After the first 48 hours, cells were either left untreated as a control (CON) or treated with different doses of metformin (MET; 0.001 mM, 0.01 mM, 0.1 mM) (Sigma Aldrich), 0.1 mM dorsomorphin (DM) (Sigma Aldrich), or a combination of both (MET+DM; 0.1 mM) for 48 hours. At the end of the treatment period, hanging drops were collected and processed for RNA isolation, hormonal assay, cell viability and proliferation assay.
 
Cell viability and proliferation assay
 
Cell viability was accessed by taking 8 µl cultured drop mixed with trypan blue dye in a ratio of 1:1. Cell proliferation in 3D GC culture was assayed using MTT Cell Proliferation Kit-1 (Roche, Sigma) as per manufacturer protocol. Initially, 8 µl of hanging drop was plated in 96-well plates and then 10 µl of MTT was added. The final volume of one well was maintained at 100 µl with media making final concentration 0.5 mg/ml of MTT. Then 96-well plates containing spheroids were incubated for 4 hours at 37°C. After incubation, purple color formazan crystals were observed dissolved in 100 µl/well of DMSO and the plate was incubated at 37°C overnight. The absorbance was measured at 565nm using Infinite M Nano+ microplate reader (TECAN).
 
Analysis of hormones by radioimmunoassay
 
The concentrations of ovarian steroid hormones, particularly progesterone, estradiol and insulin, in the culture medium of GC were measured after 48 hours of treatment to culture by competitive radioimmunoassay (RIA, 125I-labeled) procedure using RIA kits (Immunotech). The analytical sensitivity of progesterone was 0.04 ng/ml and coefficients of intra and inter-assay variation were ≤9.48 and ≤16.85%, respectively, whereas for estradiol was 10.41 pg/ml, ≤10.0 and ≤16.4%, respectively. Insulin concentrations were determined using an immunoradiometric  assay (Immunotech). The kit had an analytical sensitivity of 0.49 µIU/ml and functional sensitivity of 1.35 µIU/ml, while the intra-assay coefficient of variation was ≤3.99%. Two replicates were performed for each sample.
 
RNA isolation and real-time PCR
 
Total RNA was extracted using RNAiso plus (Takara) using an optimized protocol to yield high-quality RNA from the cultured GC. In brief, 300 µl RNAiso plus was added to the GC pellet, vortexed briefly, then incubated for 10 min at room temperature. Addition of 400 µl of chloroform to the samples, vortexed for 15-20 seconds and incubated for 15 minutes, followed by centrifugation. The aqueous phase was collected and an equal amount of 100% ethanol was added to it with 2 µl of linear acrylamide (Invitrogen) and kept overnight for incubation at -20°C. The samples were centrifuged and the RNA pellet was washed twice with 75% ethanol, centrifuged, air-dried and dissolved in 20 µl nuclease-free water. RNA was quantified by measuring absorbance using an Infinite M Nano+ microplate reader (TECAN). Samples were stored at -80°C until processed.
       
Total RNA samples were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Real-time PCR was run using SYBR Green Master mix (Thermo Fisher Scientific). Each reaction was done in duplicate. The mRNA levels of GLUT4, PPARα and SREBP1c were analysed by the ΔCt (threshold cycle) method, where ΔCt was calculated by subtracting the Ct value of the internal control gene (RPL19) (Schulze et al., 2017) from the Ct value of target genes. Relative gene expression levels were then determined using the 2(-ΔΔCT) method (Livak and Schmittgen, 2001). The primer sequence is summarized in Table 1.

Table 1: Sequence of theprimers used in real-time PCR.



Statistical analysis
 
All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data were expressed as median ± interquartile range (IQR) due to non-normal distribution. The Kruskal-Wallis test, a non-parametric alternative to one-way ANOVA, was used to compare differences among groups, followed by Dunn’s post hoc test to compare differences between the groups. A p-value less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Cell proliferation and viability
 
GC of sheep were incubated with different concentrations of MET (0.001, 0.01, or 0.1 mM), DM and a combination of both MET+DM for 48 hrs and a dose-dependent increase in cell numbers was observed with metformin treatments (Fig 1A). Statistical analysis revealed there was a significant difference between groups (p = 0.03). The cell number tended to be greater at 0.1mM MET (p = 0.09) than that of DM. Similarly, the proliferation % of cells showed a significant difference among the groups (p<0.01). Post hoc analysis between the groups showed a higher proliferation at 0.1 mM MET than that of DM (p = 0.02) and MET+DM (p = 0.09) (Fig 1B).

Fig 1: Effect of different concentrations of metformin and dorsomorphin on cell number (A) and cell proliferation in ovine granulosa cells.


       
The increased MET-induced cell number and proliferation results in our study align with the findings of Sonntag et al., (2005), who reported that metformin preincubation of primary GC significantly increases the basal cell viability as compared to the control and hypothesized that metformin modulates the insulin receptor, which leads to activation of AKT preferentially as compared with the MAPK pathway. Also, metformin promoted cell viability and proliferation in mouse female germline stem cells by enhancing histone 3 lysine 27 modification at the Traf2 to regulate MAPK signalling, contributing to increased cell proliferation (Chen et al., 2023). Both MAPK/AKT signalling pathways are closely related to cell proliferation. The reduced cell number and proliferation following dorsomorphin treatment in our study are consistent with Garulli et al., (2014), where dorsomorphin inhibits BMP signalling, resulting in reduced cell proliferation. However, in contrast to our results, metformin treatment has also been reported to reduce cell proliferation and protein synthesis in bovine GC (Tosca et al.,  2006, 2007, 2010).
 
Steroidogenesis
 
The effect of treatment on the concentration of progesterone showed a significant difference among groups (p<0.01). Post hoc analysis indicated that its concentration was significantly lower with 0.01 mM MET (p = 0.04) and 0.1 mM MET (p = 0.02) treatments than that of DM (Fig 2A). A significant effect of treatments was observed in estrogen levels (p = 0.02) between different groups. The MET treatment caused a dose-dependent decrease in the estradiol concentration in the culture media, the concentrations being lower (p = 0.09) with 0.1 mM MET than that of CON. Notably, the estradiol concentration was significantly lower (p = 0.03) with DM than that of CON (Fig 2B). The treatments had no significant effect on insulin levels in GC culture medium (Fig 2C).

​

Fig 2: Effect of different concentrations of metformin and dorsomorphin on progesterone (A), estrogen (B) and insulin (C) levels in ovine granulosa cells.


       
Metformin has been shown to reduce estradiol synthesis by inhibiting aromatase expression through MAPK signalling pathways in human GC (Rice et al., 2009; Fuhrmeister et al., 2014). Similar to our study, metformin treatments showed a dose-dependent inhibitory effect on estrogen and progesterone secretion in rat GC, possibly through inhibiting some steroidogenic factors (Tosca et al.,  2006, 2007) and a decrease in the abundance of pERK and pAkt in addition to downregulation of FSHR, StAR, CYP11A1, HSD3B steroidogenic genes (Weaver and Ramachandran, 2023). Our findings indicated stable insulin levels across treatment groups, implying a lack of insulin uptake or metabolism by GC under the given culture conditions. Although insulin was not supplemented exogenously to the culture, the presence of insulin in our culture may have been through serum-derived components, such as FBS and ITS supplement. These findings suggest that metformin modulates insulin signalling pathways in GC independent of the changes in extracellular insulin.
 
Gene expression
 
A significant difference in GLUT4 mRNA expression was observed between the treatment groups (p<0.01). The expression was highest in the 0.1 mM MET group, which was significantly higher (p = 0.01) than that in DM, while numerically (p = 0.07) elevated compared to the MET+DM group (Fig 3A). The gene expression of PPARα also significantly differed among the groups (P<0.01). Multiple comparison tests showed a significant upregulation of PPARα mRNA expression in 0.1mM MET (p = 0.01) and 0.01 mM MET (p = 0.05) groups as compared to the DM group (Fig 3B). MET treatments resulted in a dose-dependent downregulation in SREBP1c mRNA expression, which was reduced in 0.01 mM MET (p = 0.09) and 0.1 mM MET (p = 0.02) groups as compared to the DM group. Additionally, a numerical reduction was observed in the 0.1 mM MET group compared to the MET+DM group (p = 0.09) (Fig 3C).

​

Fig 3: Effect of different concentrations of metformin and dorsomorphin on GLUT4 (A), PPARá (B) and SREBP1c (C) gene expression in sheep granulosa cells.


       
GLUT 4, a member of the glucose transporter family, is expressed in the GC of sheep (Williams et al., 2001). In insulin-resistant conditions, expression and translocation of GLUT4 are often impaired; resulting in decreased cellular glucose uptake and insulin-sensitizing agents can enhance GLUT4 expression (Carvajal et al., 2013; Ferreira et al., 2014; Rice et al., 2011; Zhai et al., 2012). In GC, metformin increases insulin-stimulated GLUT4 translocation to the plasma membrane through PI3K-mediated Akt activation (Rice et al., 2011). The downregulation in GLUT4 expression in the D treatment group in our study is in line with findings of Kou et al., (2021) wherein GLUT4 expression in 3T3-L1 cells following dorsomorphin treatment was suppressed through the involvement of the AMPK signalling pathway. Dorsomorphin treatment reverses the effect of AMPK activator SN-4 on GLUT4 translocation (Toma et al., 2024) suggesting that the reverse effect on GLUT4 in our study following dorsomorphin treatment may be through the AMPK pathway.
       
Previous studies reported that the insulin-sensitising drugs upregulate the PPARα mRNA expression (Arruda et al., 2020; Lee et al., 2006; Sozio et al., 2011). Insulin enhances both phosphorylation and transcriptional activity of PPARá and suggests that this might be modulated by insulin-mediated phosphorylation (Shalev et al., 1996). Similar to our study, metformin treatment of H4IIEC3 cells could activate AMPK to inhibit the activity of PPARα and this effect could be reversed by the treatment of compound C (Sozio et al., 2011). Further, Kang et al., (2016) reported an increase in PPARα mRNA levels in metformin-administered mice and this effect was markedly diminished by treatment with compound C in primary hepatocytes.
       
Insulin signalling is closely related to lipid metabolism and lipids are essential for the development and maintenance of oocytes. Metformin is involved in the regulation of lipogenesis by downregulating SREBP1c gene expression (Zhou et al., 2001). Metformin increases AMPK phosphorylation, which in turn enhances the phosphorylation of SREBP1c. This phosphorylation inhibits the nuclear translocation of SREBP1c, thereby reducing lipid synthesis (Li et al., 2011). Similar to our study, Zhang et al., (2018) observed that the effect of metformin on SREBP2 expression is not completely inhibited by compound C.

CONCLUSION

In conclusion, our findings suggest that metformin treatment in sheep GC results in an increase in cell proliferation with reduced steroidogenic activity in a dose-dependent manner and this effect is reversed by dorsomorphin. The metformin treatment downregulated the lipogenic gene SREBP1c expression while upregulating the metabolic genes GLUT4 and PPARα. However, the exact pathways through which metformin regulates gene expression in our study need further investigation.

ACKNOWLEDGEMENT

The authors acknowledge the Director, ICAR-Central Sheep and Wool Research Institute, Avikanagar, Tonk, Rajasthan, India, for providing the necessary permissions, funds and facilities to conduct this research and the administration of Rajasthan University of Veterinary and Animal Sciences, Jobner, Jaipur, Rajasthan, India, for the necessary approvals.
 
Disclaimers
 
All views and findings discussed in this article reflect the authors’ interpretations only and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Funding
 
This research work was supported by the Indian Council of Agricultural Research under the institute of ICAR-Central Sheep and Wool Research Institute, Avikanagar, Tonk, Rajasthan, India (PHY/01/02/20-25).

CONFLICT OF INTEREST

None of the authors had any conflict of interest to declare.

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    Full Research Article

    Metformin Modulates Steroidogenic and Metabolic Functions in Cultured Ovine Granulosa Cells

    P
    Prachi Dhaka1,2
    G
    Govind Sahay Gottam2
    S
    Shyam Sundar Soni1
    N
    Neetu Kumari1,3
    S
    Shivendra Sharma1
    S
    Satyaveer Singh Dangi1
    A
    Arun Kumar1
    A
    Ajit Singh Mahla1,*
    0000-0001-9234-4991
    1Division of Animal Physiology and Biochemistry, ICAR-Central Sheep and Wool Research Institute, Avikanagar-304 501, Rajasthan, India.
    2Department of Veterinary Physiology and Biochemistry, Post-Graduate Institute of Veterinary Education and Research, Jaipur-302 031, Rajasthan, India.
    3Department of Veterinary Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India. 

    ABSTRACT

    Background: Metformin is an antidiabetic drug used for the treatment of infertility in polycystic ovarian syndrome and has been shown to affect the functions of granulosa cells (GC) in humans and rodents. Recently, metformin has been shown to enhance folliculogenesis, ovulation rate and prolificacy in sheep. The objective of this study was to determine the effect of metformin on GC isolated from sheep ovaries on cell number, proliferation, steroidogenesis and some hormones and genes related to follicular development.

    Methods: GC were cultured in 3D culture using hanging drops for 48 hours and then treated with various concentrations of metformin (0.001 mM, 0.01 mM, 0.1 mM), dorsomorphin (0.1 mM), or a combination of both (0.1 mM) for 48 hours. Cell number and proliferation were measured using the trypan blue exclusion dye method and MTT assay. Progesterone, estradiol and insulin concentration in culture media were quantified by RIA. The expression of GLUT4, PPARα and SREBP1c was determined by qPCR.

    Result: Metformin enhances the number and proliferation of GC while reducing the concentration of steroid hormones, such as androstenedione, progesterone and estrogen, in a dose-dependent manner. It also decreases the gene expression levels of SREBP1c and upregulates the mRNA expression of PPARα and GLUT4 and dorsomorphin treatment reverses these effects. These results suggest that metformin may modulate ovarian steroidogenesis, metabolism and folliculogenesis, influencing sheep reproductive-performance. However, further studies are required to investigate the detailed mechanism through which metformin exerts its effect.

    INTRODUCTION

    Improving reproductive efficiency is a major focus in farm animals due to its direct contribution to productivity and profitability. Sheep can contribute to food security by maximizing their reproductive traits, such as ovulation rate, fertility and prolificacy. Prolificacy depends on the number of matured oocytes released from ovulatory follicles during each reproductive cycle and is influenced by the interaction of multiple factors, including genetics, nutritional, hormonal and environmental factors (Scaramuzzi et al., 2006). Various approaches have been explored to improve the reproductive performance of sheep, including nutritional (Somchit-Assavacheep  et al., 2013; Viñoles  et al., 2005), immunological (Campbell and Scaramuzzi, 1995; Scaramuzzi et al., 1980) and pharmacological strategies (Kumawat et al., 2024).
           
    Among pharmacological agents recently explored to improve reproductive performance in sheep is metformin (Kumawat  et al., 2023, 2024). Metformin is an insulin-sensitizing drug commonly used in the treatment of type 2 diabetes, cycle disturbance, hyperandrogenism and anovulation in polycystic ovarian syndrome (Palomba et al., 2009; Shpakov, 2021; Velazquez et al., 1997). Recently, metformin treatment for 12 weeks significantly enhanced reproductive performance in ewes, increasing the number of preovulatory follicles, ovulation rate and prolificacy rate with a 2.9-fold rise in multiple births compared to controls. Additionally, metformin treatment also reduces glucose, insulin, total cholesterol, LDL-cholesterol and estradiol concentrations than in the control group (Kumawat et al., 2024). Similarly, in another study on ewes, the combined approach of nutritional flushing with metformin treatment for 6 weeks improved the preovulatory follicular turnover and ovulation rate, leading to increased fetal numbers during early gestation in low body condition score ewes, with a marked reduction in the estradiol and glucose concentration (unpublished data). Both studies indicate that metformin treatment improves both metabolic and ovarian follicular dynamics. Metformin modulates peroxisome proliferator-activated receptors (PPARs), activates the AMPK signalling cascade and inhibits insulin-stimulated cell proliferation (Bai et al., 2019). It improves insulin sensitivity, facilitating glucose uptake, inhibiting the production of androstenedione, estrogen and progesterone by suppressing enzymes such as StAR, 3β-HSD, CYP11A1 and aromatase in human ovarian cells (Palomba et al., 2009). Metformin directly or indirectly inhibits growth, function and cell proliferation in rat (Tosca et al., 2006) and human (Rice et al., 2009), leading to reduced granulosa cell (GC) number, which in turn results in the formation of small follicles that undergo early maturation. However, the precise mechanism through which metformin exerts its effect in ewes remains unclear. Hence, by leveraging an in vitro GC culture model, the present study aimed to investigate the effect of metformin on cell proliferation, steroidogenesis and the expression of metabolic genes and to determine the involvement of AMPK signalling by using dorsomorphin, a pharmacological inhibitor. The present study will enable to determine how metformin influences the follicular development and reproductive performance by linking these cellular effects.

    MATERIALS AND METHODS

    Collection of ovaries
     
    Sheep ovaries (n=60) were collected from the commercial abattoir/ slaughterhouse. The ovaries were placed in chilled Dulbecco’s phosphate-buffered saline (PBS) (HiMedia) containing an antibiotic and antimycotic solution (100 IU/ml of penicillin G sodium, 0.1 mg/ml of streptomycin sulphate, 0.25 μg/ml of amphotericin B, HiMedia) and transported to the laboratory within 2 hours after slaughter (Kumar et al., 2014). Ovaries were washed at least 4-5 times in PBS and processed immediately.
     
    Granulosa cell isolation and culture
     
    Healthy follicles were assessed by clear amber follicular fluid with no debris. The follicular fluid was aspirated from the medium (3.5-5.0 mm) ovarian follicles into chilled media using a 1 ml sterile insulin syringe into a 1.5 ml tube under sterile conditions. After follicular aspiration, the sample was allowed to stand undisturbed to allow thecal cells to settle and GC-rich supernatant was collected and centrifuged at 1200xg for 10 minutes at 4°C (Kumar et al., 2014). The cell pellet was suspended in 200 µl of culture media, DMEM/F12(Gibco) supplemented with 3.3 mg/ml bovine serum albumin (Sigma Aldrich), 1% ITS supplement (insulin, transferrin and sodium selenite, HiMedia), 10-7M Δ4-Androstene-3,17-dione (TCI), 10% fetal bovine serum (FBS) (HiMedia), 0.05 µg/ml  FSH+0.01 µg/ml LH (Stimufol, Reprobiol SPRL), 100 IU/ml of penicillin G sodium, 0.1 mg/ml of streptomycin sulphate, 0.25 μg/ml of amphotericin B (HiMedia). Cell number and viability were estimated/assessed using the trypan blue dye exclusion method in an automatic cell counter, the Countess™ (Invitrogen) (Soni et al., 2018).
           
    The GC were cultured in hanging drops in 24-well culture plates (Nunc™, Thermo Fisher Scientific, Inc.) with a density of 5000 cells in an 8 µl drop supplemented with culture media and cultures were maintained at 37°C for 5% CO2 and 95% RH. After the first 48 hours, cells were either left untreated as a control (CON) or treated with different doses of metformin (MET; 0.001 mM, 0.01 mM, 0.1 mM) (Sigma Aldrich), 0.1 mM dorsomorphin (DM) (Sigma Aldrich), or a combination of both (MET+DM; 0.1 mM) for 48 hours. At the end of the treatment period, hanging drops were collected and processed for RNA isolation, hormonal assay, cell viability and proliferation assay.
     
    Cell viability and proliferation assay
     
    Cell viability was accessed by taking 8 µl cultured drop mixed with trypan blue dye in a ratio of 1:1. Cell proliferation in 3D GC culture was assayed using MTT Cell Proliferation Kit-1 (Roche, Sigma) as per manufacturer protocol. Initially, 8 µl of hanging drop was plated in 96-well plates and then 10 µl of MTT was added. The final volume of one well was maintained at 100 µl with media making final concentration 0.5 mg/ml of MTT. Then 96-well plates containing spheroids were incubated for 4 hours at 37°C. After incubation, purple color formazan crystals were observed dissolved in 100 µl/well of DMSO and the plate was incubated at 37°C overnight. The absorbance was measured at 565nm using Infinite M Nano+ microplate reader (TECAN).
     
    Analysis of hormones by radioimmunoassay
     
    The concentrations of ovarian steroid hormones, particularly progesterone, estradiol and insulin, in the culture medium of GC were measured after 48 hours of treatment to culture by competitive radioimmunoassay (RIA, 125I-labeled) procedure using RIA kits (Immunotech). The analytical sensitivity of progesterone was 0.04 ng/ml and coefficients of intra and inter-assay variation were ≤9.48 and ≤16.85%, respectively, whereas for estradiol was 10.41 pg/ml, ≤10.0 and ≤16.4%, respectively. Insulin concentrations were determined using an immunoradiometric  assay (Immunotech). The kit had an analytical sensitivity of 0.49 µIU/ml and functional sensitivity of 1.35 µIU/ml, while the intra-assay coefficient of variation was ≤3.99%. Two replicates were performed for each sample.
     
    RNA isolation and real-time PCR
     
    Total RNA was extracted using RNAiso plus (Takara) using an optimized protocol to yield high-quality RNA from the cultured GC. In brief, 300 µl RNAiso plus was added to the GC pellet, vortexed briefly, then incubated for 10 min at room temperature. Addition of 400 µl of chloroform to the samples, vortexed for 15-20 seconds and incubated for 15 minutes, followed by centrifugation. The aqueous phase was collected and an equal amount of 100% ethanol was added to it with 2 µl of linear acrylamide (Invitrogen) and kept overnight for incubation at -20°C. The samples were centrifuged and the RNA pellet was washed twice with 75% ethanol, centrifuged, air-dried and dissolved in 20 µl nuclease-free water. RNA was quantified by measuring absorbance using an Infinite M Nano+ microplate reader (TECAN). Samples were stored at -80°C until processed.
           
    Total RNA samples were reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Real-time PCR was run using SYBR Green Master mix (Thermo Fisher Scientific). Each reaction was done in duplicate. The mRNA levels of GLUT4, PPARα and SREBP1c were analysed by the ΔCt (threshold cycle) method, where ΔCt was calculated by subtracting the Ct value of the internal control gene (RPL19) (Schulze et al., 2017) from the Ct value of target genes. Relative gene expression levels were then determined using the 2(-ΔΔCT) method (Livak and Schmittgen, 2001). The primer sequence is summarized in Table 1.

    Table 1: Sequence of theprimers used in real-time PCR.



    Statistical analysis
     
    All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data were expressed as median ± interquartile range (IQR) due to non-normal distribution. The Kruskal-Wallis test, a non-parametric alternative to one-way ANOVA, was used to compare differences among groups, followed by Dunn’s post hoc test to compare differences between the groups. A p-value less than 0.05 was considered statistically significant.

    RESULTS AND DISCUSSION

    Cell proliferation and viability
     
    GC of sheep were incubated with different concentrations of MET (0.001, 0.01, or 0.1 mM), DM and a combination of both MET+DM for 48 hrs and a dose-dependent increase in cell numbers was observed with metformin treatments (Fig 1A). Statistical analysis revealed there was a significant difference between groups (p = 0.03). The cell number tended to be greater at 0.1mM MET (p = 0.09) than that of DM. Similarly, the proliferation % of cells showed a significant difference among the groups (p<0.01). Post hoc analysis between the groups showed a higher proliferation at 0.1 mM MET than that of DM (p = 0.02) and MET+DM (p = 0.09) (Fig 1B).

    Fig 1: Effect of different concentrations of metformin and dorsomorphin on cell number (A) and cell proliferation in ovine granulosa cells.


           
    The increased MET-induced cell number and proliferation results in our study align with the findings of Sonntag et al., (2005), who reported that metformin preincubation of primary GC significantly increases the basal cell viability as compared to the control and hypothesized that metformin modulates the insulin receptor, which leads to activation of AKT preferentially as compared with the MAPK pathway. Also, metformin promoted cell viability and proliferation in mouse female germline stem cells by enhancing histone 3 lysine 27 modification at the Traf2 to regulate MAPK signalling, contributing to increased cell proliferation (Chen et al., 2023). Both MAPK/AKT signalling pathways are closely related to cell proliferation. The reduced cell number and proliferation following dorsomorphin treatment in our study are consistent with Garulli et al., (2014), where dorsomorphin inhibits BMP signalling, resulting in reduced cell proliferation. However, in contrast to our results, metformin treatment has also been reported to reduce cell proliferation and protein synthesis in bovine GC (Tosca et al.,  2006, 2007, 2010).
     
    Steroidogenesis
     
    The effect of treatment on the concentration of progesterone showed a significant difference among groups (p<0.01). Post hoc analysis indicated that its concentration was significantly lower with 0.01 mM MET (p = 0.04) and 0.1 mM MET (p = 0.02) treatments than that of DM (Fig 2A). A significant effect of treatments was observed in estrogen levels (p = 0.02) between different groups. The MET treatment caused a dose-dependent decrease in the estradiol concentration in the culture media, the concentrations being lower (p = 0.09) with 0.1 mM MET than that of CON. Notably, the estradiol concentration was significantly lower (p = 0.03) with DM than that of CON (Fig 2B). The treatments had no significant effect on insulin levels in GC culture medium (Fig 2C).

    ​

    Fig 2: Effect of different concentrations of metformin and dorsomorphin on progesterone (A), estrogen (B) and insulin (C) levels in ovine granulosa cells.


           
    Metformin has been shown to reduce estradiol synthesis by inhibiting aromatase expression through MAPK signalling pathways in human GC (Rice et al., 2009; Fuhrmeister et al., 2014). Similar to our study, metformin treatments showed a dose-dependent inhibitory effect on estrogen and progesterone secretion in rat GC, possibly through inhibiting some steroidogenic factors (Tosca et al.,  2006, 2007) and a decrease in the abundance of pERK and pAkt in addition to downregulation of FSHR, StAR, CYP11A1, HSD3B steroidogenic genes (Weaver and Ramachandran, 2023). Our findings indicated stable insulin levels across treatment groups, implying a lack of insulin uptake or metabolism by GC under the given culture conditions. Although insulin was not supplemented exogenously to the culture, the presence of insulin in our culture may have been through serum-derived components, such as FBS and ITS supplement. These findings suggest that metformin modulates insulin signalling pathways in GC independent of the changes in extracellular insulin.
     
    Gene expression
     
    A significant difference in GLUT4 mRNA expression was observed between the treatment groups (p<0.01). The expression was highest in the 0.1 mM MET group, which was significantly higher (p = 0.01) than that in DM, while numerically (p = 0.07) elevated compared to the MET+DM group (Fig 3A). The gene expression of PPARα also significantly differed among the groups (P<0.01). Multiple comparison tests showed a significant upregulation of PPARα mRNA expression in 0.1mM MET (p = 0.01) and 0.01 mM MET (p = 0.05) groups as compared to the DM group (Fig 3B). MET treatments resulted in a dose-dependent downregulation in SREBP1c mRNA expression, which was reduced in 0.01 mM MET (p = 0.09) and 0.1 mM MET (p = 0.02) groups as compared to the DM group. Additionally, a numerical reduction was observed in the 0.1 mM MET group compared to the MET+DM group (p = 0.09) (Fig 3C).

    ​

    Fig 3: Effect of different concentrations of metformin and dorsomorphin on GLUT4 (A), PPARá (B) and SREBP1c (C) gene expression in sheep granulosa cells.


           
    GLUT 4, a member of the glucose transporter family, is expressed in the GC of sheep (Williams et al., 2001). In insulin-resistant conditions, expression and translocation of GLUT4 are often impaired; resulting in decreased cellular glucose uptake and insulin-sensitizing agents can enhance GLUT4 expression (Carvajal et al., 2013; Ferreira et al., 2014; Rice et al., 2011; Zhai et al., 2012). In GC, metformin increases insulin-stimulated GLUT4 translocation to the plasma membrane through PI3K-mediated Akt activation (Rice et al., 2011). The downregulation in GLUT4 expression in the D treatment group in our study is in line with findings of Kou et al., (2021) wherein GLUT4 expression in 3T3-L1 cells following dorsomorphin treatment was suppressed through the involvement of the AMPK signalling pathway. Dorsomorphin treatment reverses the effect of AMPK activator SN-4 on GLUT4 translocation (Toma et al., 2024) suggesting that the reverse effect on GLUT4 in our study following dorsomorphin treatment may be through the AMPK pathway.
           
    Previous studies reported that the insulin-sensitising drugs upregulate the PPARα mRNA expression (Arruda et al., 2020; Lee et al., 2006; Sozio et al., 2011). Insulin enhances both phosphorylation and transcriptional activity of PPARá and suggests that this might be modulated by insulin-mediated phosphorylation (Shalev et al., 1996). Similar to our study, metformin treatment of H4IIEC3 cells could activate AMPK to inhibit the activity of PPARα and this effect could be reversed by the treatment of compound C (Sozio et al., 2011). Further, Kang et al., (2016) reported an increase in PPARα mRNA levels in metformin-administered mice and this effect was markedly diminished by treatment with compound C in primary hepatocytes.
           
    Insulin signalling is closely related to lipid metabolism and lipids are essential for the development and maintenance of oocytes. Metformin is involved in the regulation of lipogenesis by downregulating SREBP1c gene expression (Zhou et al., 2001). Metformin increases AMPK phosphorylation, which in turn enhances the phosphorylation of SREBP1c. This phosphorylation inhibits the nuclear translocation of SREBP1c, thereby reducing lipid synthesis (Li et al., 2011). Similar to our study, Zhang et al., (2018) observed that the effect of metformin on SREBP2 expression is not completely inhibited by compound C.

    CONCLUSION

    In conclusion, our findings suggest that metformin treatment in sheep GC results in an increase in cell proliferation with reduced steroidogenic activity in a dose-dependent manner and this effect is reversed by dorsomorphin. The metformin treatment downregulated the lipogenic gene SREBP1c expression while upregulating the metabolic genes GLUT4 and PPARα. However, the exact pathways through which metformin regulates gene expression in our study need further investigation.

    ACKNOWLEDGEMENT

    The authors acknowledge the Director, ICAR-Central Sheep and Wool Research Institute, Avikanagar, Tonk, Rajasthan, India, for providing the necessary permissions, funds and facilities to conduct this research and the administration of Rajasthan University of Veterinary and Animal Sciences, Jobner, Jaipur, Rajasthan, India, for the necessary approvals.
     
    Disclaimers
     
    All views and findings discussed in this article reflect the authors’ interpretations only and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Funding
     
    This research work was supported by the Indian Council of Agricultural Research under the institute of ICAR-Central Sheep and Wool Research Institute, Avikanagar, Tonk, Rajasthan, India (PHY/01/02/20-25).

    CONFLICT OF INTEREST

    None of the authors had any conflict of interest to declare.

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