The Effect of Insulin Like Growth Factor-1 (IGF-1) Supplementation in Maturation Media on Transforming Growth Factor-Beta (TGF-β) and Growth Differentiation Factor-9 (GDF-9) Expression in Kacang Goat Oocytes

The Kacang goat is an indigenous Indonesian ruminant widely reared by rural communities, serving as an important source of animal protein and supporting food security (Wungo et al., 2023). Economically, it provides additional income and improves dietary protein intake. However, its population growth and genetic quality remain suboptimal (Susilowati et al., 2021). The genetic quality of this native breed is threatened by frequent crossbreeding, which reduces its purity and highlights the urgent need for conservation efforts (Mustofa et al., 2023). To address this issue, reproductive technologies have been developed. Technologies such as artificial insemination, embryo transfer, in vitro fertilization (IVF), cryopreservation, and genetic engineering can improve genetic quality in Kacang goats (Suprayogi et al., 2022). IVF, in particular, offers potential to increase productivity (Najiati et al., 2022), but its success is often limited by low oocyte quality and availability (Widjiati et al., 2020).

In vitro maturation (IVM) is a critical stage in the IVF process, largely determined by the maturation medium composition (Widjiati et al., 2022). IVM outcomes are influenced by medium constituents, hormonal stimulation, species-specific traits, exposure time, and intrinsic oocyte quality, particularly morphology (Ilmi et al., 2023; Nursadida et al., 2024; Coticchio et al., 2015). Environmental factors such as contamination, temperature, pH, and osmolality also affect maturation (Herta et al., 2018). Supplementation with growth factors, hormones, or antioxidants has been shown to enhance maturation efficiency and oocyte quality (Soto-Heras et al., 2019).

Insulin-like growth factor-1 (IGF-1), produced by almost all tissues, acts via IGF1R receptors to activate MAPK and PI3K pathways, promoting cumulus expansion and oocyte maturation (Miescher et al., 2023). During maturation, oocytes and cumulus cells secrete Transforming growth factor-β (TGF-β) and growth differentiation factor-9 (GDF-9), both detected in Kacang goat oocytes (Widjiati et al., 2010, 2011). TGF-β regulates follicular cell proliferation, meiosis, apoptosis, and cumulus expansion (Widjiati et al., 2012; Yang et al., 2019; Hao et al., 2022), while GDF-9 supports follicular growth, oocyte maturation, and embryo quality (Gode et al., 2011; Faizah and Aswin, 2021).

Given this background, further research is warranted to investigate the effects of IGF-1 supplementation on TGF-β and GDF-9 expression during Kacang goat oocyte maturation, contributing to improved reproductive performance and genetic preservation of this native breed.

The Animal Care and Use Committee of the Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, authorized the experimental procedure. The study was performed in compliance with established protocols and adhered to animal welfare and medical ethical standards. The ethical clearance number is 1.KEH.77.05.2025.

The materials used in this study included physiological NaCl (0.95%), Phosphate-Buffered Saline (PBS), Gentamicin (PT. Indofarma), mineral oil (Vitrolife, Sweden), G-MOPS (Vitrolife®), G-IVF (Vitrolife®), Recombinant Goat IGF-1 (MedikBio®, Catalogue number: MDB-EP01108600), Poly-L lysine-coated slides, vaseline, 3% hydrogen peroxide, Growth Differentiation Factor-9 (GDF-9) Polyclonal Antibody (Bioss), TGF-β Antibody (TGF-β 1 [3C11]: sc-130348, Santa Cruz Biotechnology), 70% alcohol, methanol, 1% aceto-orcein acid, 0.025% trypsin, Biotinylated Link (Yellow) drops, Streptavidin (Red) drops, 3,3-Diaminobenzidine tetrahydrochloride (DAB) chromogen, and methylene green.

The ovaries were rinsed with 0.9% NaCl with 100 µL gentamicin. Preantral follicles measuring 2–6 mm were aspirated with a 10 mL syringe fitted with an 18G needle containing medium, followed by three washes in G-MOPS and two washes in G-IVF maturation media. Oocytes were classified morphologically as follows: Grade 1, characterized by homogeneous cytoplasm with three or more cumulus layers; Grade 2, exhibiting one to three layers and homogeneous cytoplasm; Grade 3, presenting few or no cumulus layers and heterogeneous cytoplasm; Grade 4, displaying abnormal morphology with detached cumulus and heterogeneous cytoplasm, indicative of apoptosis (Widjiati et al., 2020, 2024). Figure 1 illustrates oocyte grading.

Maturation process of the oocytes was conducted using G-IVF medium as the maturation substrate. The medium was supplemented with IGF-1 at concentrations of 0 (control), 50, 100, and 150 ng/mL, following the protocols of Javvaji et al. (2020) and Jimenez et al. (2016). For each experimental group, Petri dishes were prepared with four aliquots of the maturation medium (50 µg/mL), each covered with mineral oil to prevent evaporation and maintain osmolarity. The oocytes were then incubated under controlled conditions of 5% CO₂, incubation temperature at 38.5 °C, with approximate relative humidity by 95%, and for a duration of 20–22 hours (Kusindarta, 2009; Widjiati et al., 2011).

Matured oocytes were placed on Poly-L-lysine-coated slides, loosely covered with a cover glass sealed with vaseline, and fixed for 24 hours. They were then treated with 3% hydrogen peroxide for 5–10 minutes, followed by two PBS washes (pH 7.4) for 5 minutes each. Oocytes were incubated with 0.025% trypsin at 37°C for 15 minutes and rewashed with PBS.Ultra V block was applied for 5 minutes, followed by incubation with either anti-TGF-β or anti-GDF-9 antibodies for 60 minutes at room temperature. Biotinylated Link (Yellow) was added for 30 minutes, then washed with PBS, followed by Streptavidin (Red) for 30 minutes. DAB chromogen was applied for 6–10 minutes, followed by PBS and aquadest washes. The final steps included rinsing with distilled water (aquadest) for 5 minutes, staining with methylene green for 5–10 minutes, and gently blotting excess stain to reduce moisture (Kasman et al., 2020). The oocytes then observed under an Olympus® CX-41 microscope at 400× magnification, Expression assessment was performed using the Remmele scale, known as the Immunoreactive Score (IRS). The outcome were obtained by the multiplication of the positive cells percentage with the staining intensity score. The scoring system for the percentage are shown in Table 1 (Widjiati et al., 2022).

Table 1: The index remmele scale.

The data were analyzed using the Kruskal–Wallis test, and when a significant difference was detected (p < 0.05), pairwise comparisons were carried out with the Mann–Whitney U test. Correlation analyses were conducted using two-tailed Kendall’s tau and Spearman’s rho coefficients. The analyses assumed that the population under study was normally distributed, homogeneous, and composed of independent samples. All statistical procedures were performed using the Statistical Package for the Social Sciences (SPSS).

The expressions of TGF-β and GDF-9 were identified using the immunohistochemical staining method. All slides were examined microscopically and assessed semi-quantitatively using the Remmele method. The immunocytochemical staining results are shown in Figures 2 and 3. The expressions of both markers are indicated by brown coloration in the oocytes. In contrast, oocytes that did not express both markers appeared green, reflecting the methylene green counterstain. The brown coloration signifies the presence of antigen–antibody–chromogen binding, indicating positive expression. Conversely, green coloration in oocytes or cumulus cells indicates the absence of this binding and, therefore, no expression by the two protein. These staining differences served as the basis for determining the expression scores.

TGF-β expression was significantly highest in the 100 ng/mL IGF-1 treatment group, with a mean rank of 20.50 as shown in Table 2. This group exhibited significantly higher TGF-β expression (p < 0.05) compared to the control group (6.67), the 50 ng/mL IGF-1 group (9.92), and the 150 ng/mL IGF-1 group (12.92). These findings are illustrated in Figure 4. Similarly, GDF-9 expression was also significantly highest in the 100 ng/mL IGF-1 treatment group, with a mean rank of 18.83 as shown in Table 3. The control group had a mean rank of 6.50, which was significantly lower than both the 100 ng/mL (18.83) and 150 ng/mL (17.50) groups, but not significantly different from the 50 ng/mL group. Both the 100 ng/mL (18.83) and 150 ng/mL (17.50) IGF-1 groups showed significantly higher GDF-9 expression compared to the control (6.50) and 50 ng/mL (7.17) groups (p < 0.05). The results are also depicted in Figure 5.

Table 2: Mean rank of TGF-β expression.

Table 3: Mean rank of GDF-9 expression.

The correlation significance results are presented in Table 4, where the data were tested using two-tailed Kendall’s tau and Spearman’s rho statistical methods to assess the relationship between the expressions of TGF-β and GDF-9 during oocyte maturation with IGF-1 supplementation. The analysis showed a statistically significant correlation (p<0.05) between the two variables, with a p-value of 0.013. Table 5 displays the correlation coefficients, with Kendall’s tau (τ) and Spearman’s rho (r) values of 0.424 and 0.499, respectively. The positive values in both tests indicate a positive relationship between the two variables. This suggests that an increase in TGF-β expression is likely accompanied by an increase in GDF-9 expression, indicating a potential significant positive association between these two proteins in supporting the in vitro maturation of Kacang goat oocytes.

Table 4: Significance analysis of the correlation between TGF-β and GDF-9 expression.

Table 5: Correlation coefficient analysis between TGF-β and GDF-9 expression.

IGF-1 is a growth factor produced by almost all tissues, playing an essential role in cell survival, proliferation, and differentiation, including oocytes (Hakuno and Takahashi, 2018; Miescher et al., 2023). By binding to the IGF-1 receptor (IGF1R), it activates intracellular signaling cascades that support oocyte maturation, notably the PI3K/Akt, ERK/MAPK, and phospholipase C pathways (Miescher et al., 2023). In the present study, supplementation with 100 ng/mL IGF-1 in the maturation medium resulted in the highest expression of TGF-β and GDF-9, with significantly higher levels (p < 0.05) than the control and other treatment groups. IGF-1 appears to enhance TGF-β via the PI3K/Akt pathway, involving Akt phosphorylation and NF-κB activation (Rameshwar et al., 2000; Bai et al., 2009), which in turn upregulates EGF-like growth factors—AREG, EREG, and BTC that promote oocyte maturation (Fang et al., 2014). The ERK/MAPK pathway further supports maturation by phosphorylating Connexin 43 (CX43), diminishing gap junction communication and facilitating meiotic resumption (Fang et al., 2023; Li et al., 2024).

IRS, Grb2, Sos, and Raf, which activates USF-1, a transcription factor binding E-box motifs within the GDF-9 promoter (Lavoie and Therrien, 2015; Datta et al., 2015; Jiang et al., 2020). GDF-9 enhances cumulus expansion via Smad2/3 activation upon binding ALK5 and BMPR2, leading to transcription of HAS2, GREM1, and PTGS2 (Fang et al., 2014; Faizal et al., 2024).

Both TGF-β and GDF-9 contribute to in vitro maturation by lowering cAMP levels, activating ERK/MAPK in follicular cells, and closing gap junctions (Cx43 and Cx37), thereby restricting cGMP transfer and permitting meiotic progression (Widjiati et al., 2010, 2011, 2012; Hough et al., 2012; Das and Arur, 2022). Meiotic arrest is maintained by elevated cAMP and PKA activity, supported by GPR3 and cGMP-mediated PDE3 inhibition (del Llano et al., 2022). ERK-mediated gap junction closure permits PDE3 activation, reducing cAMP and PKA, thereby activating MPF (CDK1–Cyclin B), inducing GVBD and meiotic resumption (Kalous et al., 2018; Daldello et al., 2019).

IGF-1 circulates as a ternary complex with IGFBP and ALS, with IGFBP regulating its bioavailability (Silva et al., 2009; Susilowati et al., 2021). Notably, the 150 ng/mL IGF-1 dose was less effective than 100 ng/mL, possibly due to receptor saturation or negative feedback mechanisms that impair maturation or trigger apoptosis (Jimenez et al., 2016).

Both TGF-β and GDF-9 belong to the TGF-β superfamily, and their expression has been shown to be enhanced by IGF-1. The upregulation of these markers may reflect a synergistic relationship in regulating oocyte maturation, as evidenced by the significant positive correlation between TGF-β and GDF-9 expression observed in this study (p < 0.05; Kendall’s tau = 0.424; Spearman’s rho = 0.499). Although TGF-β was originally thought to be expressed exclusively by follicular cells, it is also produced by oocytes, acting via the Smad2/3 pathway to modulate gene expression within the follicular environment, whereas GDF-9, secreted by oocytes, regulates cumulus cell function (Widjiati et al., 2010).

Before the identification of GDF-9 and BMP-15, recombinant TGF-β1 and TGF-β2 were commonly applied to improve in vitro maturation, with these factors exhibiting positive feedback through the Smad2/3 pathway (Gilchrist et al., 2008; Yan et al., 2018). Notably, GDF-9 has also been shown to enhance TGF-β expression by activating the TGF signaling pathway through binding to the BMPR2–TGFBR1 complex, which initiates a Smad2/3-mediated phosphorylation cascade leading to increased TGF-β transcription (Vitt et al., 2002; Huang et al., 2024). In contrast to TGF-β, however, GDF-9 does not appear to regulate its own expression, relying instead on USF-1-mediated transcription (Datta et al., 2015), and primarily acts in a paracrine or autocrine manner on somatic follicular cells, with no evidence of autoregulation within oocytes (Ramirez et al., 2020; Fountas et al., 2024).

This study recommends considering IGF-1 supplementation at a dosage of 100 ng/ml in the in vitro maturation medium for Kacang goat oocytes, as it significantly increases the expression of TGF-β and GDF-9, essential for oocyte development in Kacang goats. The findings underscore the potential of IGF-1 supplementation to improve oocyte maturation in Kacang goats, hence enhancing the genetic quality of this significant indigenous breed. Additional research is required to examine the pathways connecting biomarker levels to their influence on enhancing oocyte quality during in vitro maturation.

The authors extend their heartfelt appreciation to the Faculty of Veterinary Medicine, Universitas Airlangga, for affording the essential facilities and support for the completion of this research. The authors aspire that the findings in this publication will enhance understanding and facilitate future research in in vitro maturation (IVM) and in vitro embryo production (IVEP).

This study is unique in recommending IGF-1 supplementation in the in vitro maturation medium for Kacang goat oocytes to increase the expression of TGF-β and GDF-9, essential for oocyte development in Kacang goats. The findings underscore the potential of IGF-1 supplementation to improve oocyte maturation in Kacang goats.

Mohamad Raviansyah Jawindra: Responsible for conceptualization, provision of resources, and drafting the original manuscript. Widjiati Widjiati: Responsible for data verification, data evaluation, supervision, critical revision of the manuscript, and securing funding. Rimayanti Rimayanti: Contributed to data verification, data interpretation, supervision, and critical review and editing of the manuscript. Suherni Susilowati: Contributed to data verification, data assessment, and overall supervision of the study. Epy Muhammad Luqman: Contributed to data verification, data examination, and supervision of the research process. Mirni Lamid: involved in data verification, data review, and supervisory roles. Viski Fitri Hendrawan: Contributed to writing, critical review, and editing of the manuscript.

The authors declare that no Genrative AI was used in the creation of this manuscript.

The authors hereby declare that no actual or potential conflicts of interest exist with respect to the research, authorship, and/or publication of this article.