The influence of follicular fluid on the regulation of apoptosis and related genes during IVM of camel oocytesABSTRACT
Follicular fluid is a haven that provides a microenvironment for the development and maturation of oocytes. It is a medium that provides a means of communication within the follicular cells. The role of follicular fluid in the development and maturation of follicles and oocytes involves the actions of certain genes. Here, we examined the impact of CFF on the gene expression of camel oocytes during (IVM). Hypothetically, IVM medium was enhanced with 2.5% CFF, and we analyzed the expression of several genes including Bax, Bcl-2, P53, Acta2, TGFβ, Cx43, and Tagln. Consequently, the follicular fluid from camels had a substantial effect on the mRNA transcript level of the BCL2 gene, which is related to apoptosis. Acta2 and Tagln genes that relate to cytoskeletal protein “actin” in transforming growth factor beta (TGFβ) pathway were also modulated. However, the follicular fluid increased the expression of TGFβ in camel oocytes. This study showcases the beneficial effects of CFF on camel oocyte Gene Expression and provides a platform for studying the many mechanisms involved in oocyte meiosis, particularly those associated with the TGFβ pathway. We recommend using higher dosage and measuring the expression levels of certain genes involved in programmed cell death and inflammation.
Keywords:
camel oocyte; gene expression; in vitro maturation; follicular fluid
RESUMO
O fluido folicular é um abrigo que proporciona um microambiente para o desenvolvimento e a maturação dos oócitos. É um meio que proporciona um meio de comunicação dentro das células foliculares. A função do fluido folicular no desenvolvimento e na maturação de folículos e oócitos envolve a ação de determinados genes. Aqui, examinamos o impacto da CFF na expressão gênica de oócitos de camelo durante a (IVM). Hipoteticamente, o meio de MIV foi aprimorado com 2,5% de CFF e analisamos a expressão de vários genes, incluindo Bax, Bcl-2, P53, Acta2, TGFβ, Cx43 e Tagln. Consequentemente, o fluido folicular de camelos teve um efeito substancial sobre o nível de transcrição de mRNA do gene BCL2, que está relacionado à apoptose. Os genes Acta2 e Tagln, relacionados à proteína do citoesqueleto “actina” na via do fator de crescimento transformador beta (TGFβ), também foram modulados. No entanto, o fluido folicular aumentou a expressão de TGFβ em oócitos de camelo. Este estudo mostra os efeitos benéficos da CFF na expressão gênica de oócitos de camelo e fornece uma plataforma para o estudo dos muitos mecanismos envolvidos na meiose de oócitos, particularmente aqueles associados à via do TGFβ. Recomendamos o uso de dosagens mais altas e a medição dos níveis de expressão de determinados genes envolvidos na morte celular programada e na inflamação.
Palavras-chave:
oócito de camelo; expressão gênica; maturação in vitro; fluido folicular
INTRODUCTION
Constituents of the follicular fluid (CFF) are an integral factor in the development and maturation of oocytes. CFF is composed of several biological factors that can modulate gene expression related to oocyte development and maturation during the reproductive cycle. The circumstances surrounding the culture can alter gene expressions at the egg stage and blastocyst stage. Therefore, understanding the gene expression profile during the process of oocyte maturation is pertinent to the successful culture (Bartková et al., 2024). For instance, during in vitro oocyte maturation, activation of apoptotic genes can be triggered due to the presence of certain environmental conditions. The balance in the level of expression of pro and anti-apoptotic genes Bax and Bcl2 respectively predetermined the fate of cells that are susceptible to apoptotic stimuli. In essence, Bax and Bcl2 genes have been reported as predictors of oocytes maturation. (Zhang et al., 2023).
A good number of chemicals have been tested to improve the growth condition in an in-vitro maturation (IVM). In cattle, there are reports of studies that have shown that the use of extra follicular fluid dramatically affects the ability of oocytes in cattle to undergo nuclear and cytoplasmic maturation, fertilization, and embryonic development. The supplement of follicular fluid to the medium used in IVM is an innovation that may enhance the effectiveness of the process. Follicular fluid is a rich source of steroid hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth factors, cytokines, glucose, cholesterol, triglycerides, albumin, globulin, electrolytes, enzymes, and nutrients. It also includes a high concentration of growth factors (Da Broi et al., 2018; Collodel et al., 2023). In addition, the inclusion of follicular fluid to the in vitro culture medium in its natural form would enable imitation of an in-vivo environment. (Al-Mutary et al., 2019). In essence, the culture conditions would have the potential to influence the expression of genes throughout the oocyte and blastocyst stages. Acquisition of information related to the gene expression during the maturation of oocytes is necessary in order to improve the development of embryos (Tripathi et al., 2023). Hence, the present study seeks to explore the influence of CFF on the modulation of gene expression patterns within the camel oocyte. Understanding the gene expression pattern can give more insights into the underlying molecular mechanism of successful oocyte maturation and enhance the optimization strategies for assisted reproductive technologies in camels.
MATERIALS AND METHODS
If not stated otherwise, all chemicals used in this project were obtained from Sigma.
Camel ovaries were obtained from a slaughterhouse (location) between October 2023 to December 2023. The ovaries were transported to the laboratory within a timeframe of 3-6 hours, immersed in normal saline solution and then maintained at a temperature ranging from 30-33℃. 18-gauge needles connected to a10ml syringes were used to extract the contents of antral follicles, which were varied in size from 2 to 8mm in diameter. The cumulus-oocyte complexes displayed a uniformly granulated cytoplasm. Subsequently, the specimens underwent three rounds of washing using tissue culture medium-199 (TCM-199) enriched with 2mM sodium hydroxide, 25mM hydrogen peroxide, 0.1% bovine serum albumin (BSA), and 5 μg/ml gentamycin sulfate. The IVM medium consisted of TCM-199, which was supplemented with specific amounts of follicle stimulating hormone, luteinizing hormone, 17β-estradiol, epidermal growth factor, insulintransferrin-selenium, cysteamine, L-glutamine, fetal bovine serum (FBS), and gentamycin sulfate (Ammari et al., 2022, 2023, 2024).
The extraction of RNA was done based on the protocol in PureLinkTM RNA micro kit (Catalog number 12183-018A, manufactured by Thermo Fisher Scientific in Waltham, Massachusetts, United States of America). Briefly, 0.4mL lysis solution was added to each sample, vortexed, precipitated, and washed and a pure RNA was eluted with RNAse free water. The RNA was quantified with Nanodrop (manufacturer) and later used in reverse transcription process. A high-capacity complementary DNA reverse transcription kit (from Thermo Fisher Scientific Catalogue No. 4368814) was used for the conversion. Briefly, the cDNA synthesis was carried out by combining 10 microliters of RNA, 2 microliters of 10XRT buffer, 2 microliters of random primers, 0.8 microliters of dNTP mix (100mM), 1 microliter of multiscribe reverse transcriptase, and 4.2 microliters of nuclease-free water in a PCR tube with a capacity of 200 microliters. Samples were allowed to run in a thermocycler in accordance with these parameters: incubation at 250C for ten minutes, then incubation at 37 degrees Celsius for one hundred twenty minutes, and lastly, incubation at 85 degrees Celsius for five minutes. The cDNA samples were stored at -200C and later used for the PCR.
Also, SYBR green master mix (Thermo Fisher Scientific) was used for the gene amplification process. BAX, BCL-2, P53, TCTA2, TGFB1, CX43, and TAGLN were all examined for their expression level in comparison to the housekeeping gene GAPDH. Real time quantitative PCR was carried out using Applied Biosystems ViiA 7 real-time PCR equipment manufactured by Thermo Fisher Scientific. A reaction volume of 12.5μL was used for the amplification. This mixture included 6.25μl of SYBR Green, 0.25μL of each forward and reverse primer, 2μL of the cDNA template, and 3.75 μL of nuclease-free water. The process of RT-PCR was carried out in accordance with Amran et al. (2024). The procedure that is detailed below: The temperature was maintained at 500C for a period of 2 minutes, and then it was raised to 950C for a period of 10 minutes. After this, there were forty cycles of denaturation at 950C for 15 seconds, annealing at 600C for 1 minute, and extension at 950C for 15 seconds. The last extension was at a temperature of 600C for 1 minute. Table 1 shows the forward and reverse primers, as well as the predicted sizes of the amplified fragments for each transcript, an examination of the genes of interest was carried out in triplicate on clear 96-well plates.
The statistical analysis was conducted utilizing the Mine Tab INSTAT software. The means of relative gene expression were compared across all groups using one-way analysis of variance (ANOVA) and post-LSD Dunnett's test, respectively. For each gene, three replicates of gene expression were utilized.
RESULTS AND DISCUSSION
In developing oocytes, the constituent of the follicular fluids has a crucial role in regulating the process of folliculogenesis. The homeostatic balancing in the number of surviving and apoptotic cells is key to the success of the process, hence the balance between pro-apoptotic and anti-apoptotic gene expression (Kala et al., 2017). Within the follicular microenvironment, CFF influences follicle development and oocyte quality (Da Broi et al., 2018). In our studies, the comparison of the fold of expression of Bax gene between treated and untreated samples shows an upregulation of pro-apoptotic gene in CFF supplemented sample. Meanwhile, no fold expression of Bax gene in the untretead sample (Fig. 1a). In contrast, the anti-apoptotic gene Bcl2 was found to be downregulated in CFF supplemented IVM media (Fig. 1b).
The balance between the expression of these two genes suggests that CFF can regulate apoptosis during IVM. In addition, the role of p53 gene in apoptosis regulation, oxidative stress response, DNA damage repair, and senescence regulation may play a significant role in modulating ovarian function and follicle development (Asselin et al., 2000). CFF significantly upregulated p53 gene when compared to the control sample (Fig. 1c).
Fold expression of apoptosis regulation gene of camel oocytes in CFF supplemented IVM media. A= Bax gene, B = Bcl2 gene C = P53 gene, Sample 1 = IVM media without CFF; sample 2 = CFF supplement IVM media.
Moreso, the TGF-β1 pathway is important to ovarian function in that it regulates the granulosa cell proliferation, oocyte maturation, follicular growth and as well as influencing the remodeling of extracellular matrix within the follicle, which is essential for proper oocyte development. Two important genes Acta2 and Tagln are associated to TGF-β1 pathway (Gao et al., 2015). Our result shows that CFF regulated the expression of Acta2 gene while the Tagln gene was upregulated. Tagln and Acta2 are genes involved in actin dynamics and are involved in cell motility and transformation, hence, the high expression of Tagln gene suggests the influence of CFF supplemented IVM media in camel oocyte maturation.
Expression levels of TGF-β1 gene and its associated genes from camel oocytes. A = TGF-β1 gene, B = Acta2 gene and C = Tagln gene , Sample 1 = IVM media without CFF; sample 2 = CFF supplement IVM media.
In a similar note, Connexin 43 (Cx43) is also connected to oocyte maturation. Cx43 is a crucial gene in gap junction, it plays a communication role between granulosa cells, theca cells, it plays a role in regulating follicular growth, steroidogenesis, oocyte maturation and follicle survival. The expression of CX43 from this present study shows that CFF upregulated CX43 expression compared to the control sample 1 (Figure 3). The high fold expression in CX43 can be attributed to the CFF supplementation.
The expression of Cx43 gene from camel oocyte. Sample 1 = IVM media without CFF; sample 2 = CFF supplement IVM media.
CONCLUSION
The composition of follicular fluid, including growth factors, cytokines, and hormones, can influence the expression of these genes. Studies have shown that factors like insulin-like growth factor (IGF), transforming growth factor-beta (TGF-β), and follicle-stimulating hormone (FSH) present in follicular fluid can regulate the expression of apoptotic genes. An imbalance in the expression of pro and antiapoptotic gene might implicate follicle development and oocyte quality. An increase apoptosis induction can lead to follicular atresia while subjugated apoptosis might impair oocyte development.
ACKNOWLEDGMENTS
The authors sincerely acknowledge the Researcher Support Project (RSP-2024/R232) for funding this work at King Saud University, Riyadh, Saudi Arabia.
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