Ovaries were obtained from local slaughterhouses, and granulosa cells were aspirated from small growing follicles and cultured in well plates as previously described [ 16 ]. Sub-confluent cells were co-transfected with either the wild type construct or the mutant construct in the presence or absence of a bta-miR mimic using the Lipofectamine transfection reagent Invitrogen, Darmstadt, Germany.
Data were calculated as the ratio of firefly luciferase activity to Renilla luciferase activity and compared with the group transfected with empty vector alone.
Statistical analyses were performed using the SAS 9. ANOVA was performed to assess statistically significant differences, respecting normality and homoscedasticity premises. One hundred seventy-four oocytes were collected and individually activated. Among these oocytes, Bars depict means and error bars depict standard errors of the means. We evaluated the levels of the phosphorylated Akt protein p-Akt in FCs within the distinct oocyte quality groups to determine the mechanism by which the PI3K-Akt pathway influences oocyte competence.
We investigated the levels of bta-miR and bta-miRa, two validated regulators of PTEN in humans and bovine, respectively Liu et al. Additionally, bta-miRa was recently validated as a regulator of PTEN in bovine granulosa cells [ 13 ]. The PI3K-Akt signaling pathway regulates different cellular effects, including changes in protein synthesis, DNA repair, cell cycle progression, apoptotic signaling etc. We chose to analyze downstream genes related to different cellular effects using RT-PCR to identify the mechanisms by which PI3K-Akt signaling regulates oocyte competence. We dissected ovarian follicles, stored FCs, and individually activated oocytes to evaluate their developmental competences and determine whether components of the PI3K-Akt pathway serve as predictors of oocyte quality.
In vitro parthenogenetic development allowed us to pool FCs into different groups according to oocyte competences using a retrospective method for subsequent RNA and protein assays. That the main components of the PI3K-Akt signaling pathway displayed different levels of mRNA or protein expression, depending on oocyte quality. In addition, we investigated genes regulated by PI3K-Akt signaling and observed an increase in FOXO3a levels, suggesting the existence of mechanisms governing the modulation of cell-cycle progression, protection against oxidative stress, and the anti-apoptotic response.
Oocyte growth and follicle development rely on the regulation of pathways involved in cell proliferation and survival, such as the PI3K-Akt signaling pathway [ 17 ]. The recruitment of primordial follicles is affected by the proliferation of granulosa cells and the regulation of the interaction between the Notch and PI3K-Akt pathways [ 17 , 18 ]. Follicle loss, maintenance, and growth are strictly controlled by complex molecular interactions, including PI3K-Akt signaling.
Stimulation of PI3K promotes Akt phosphorylation, resulting in follicle survival and the induction of growth. Alternatively, this pathway is suppressed by the action of PTEN [ 18 ]. The inhibition of PTEN in human ovary in vitro results in the increased activation of primordial follicles, but compromises the development of growing follicles [ 18 ]. In the murine ovary, the deletion of PTEN in mouse oocytes resulted in pan-ovarian follicle activation and premature oocyte depletion, whereas granulosa cell-specific disruption of PTEN did not affect the initiation of follicle growth, but increased GC proliferation and enhanced ovulation [ 9 , 20 ].
Similar analysis were performed for bta-miRa, which was validated as a regulator of PTEN in granulosa cells [ 13 ].
Thus, o PTEN levels are regulated by the increase in miR and miRa expression in follicles harboring competent oocytes. Additionally, multiple miRNAs regulating the same mRNA target are necessary required to induce a physiological response, such as upregulation of Akt, which was observed in cells obtained from follicles associated with higher quality oocytes. Follicular cells from follicles harboring incompetent oocytes have a molecular profile marked by an increase in PTEN expression and lower expression of bta-miR and bta-miR20a, which indicates lower activity of the PI3K-Akt pathway.
In contrast, the competent group display different levels of these indicators, confirming the increased activity of the PI3K-Akt pathway in this group. These two molecular profiles are associated with oocyte competence, and based on previously described functions of the PI3K-Akt pathway in follicular environments, we postulate that these differences in oocyte competence are related to the regulation of cell proliferation, protection from oxidative stress, and inhibition of apoptosis in the FCs, as discussed below.
In addition to its involvement in cell proliferation and survival, the PI3K-Akt signaling pathway plays roles in apoptosis, protein synthesis, the cell cycle, angiogenesis, DNA repair, and metabolic pathways, as reviewed in a previous publication [ 22 ].aclexpert.com/wp-content/proposals/batyb-plenty-of.php
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We observed differences in the levels of the FOXO3a and BAX transcripts between FC groups, which were associated with different oocyte capacities to develop into blastocysts. The transcription factor FOXO3a plays an important role in ovarian activity [ 23 ], and, when activated, it has broad anti-proliferative and pro-apoptotic cellular effects.
In addition to these effects, FOXO3a protects quiescent cells from oxidative stress [ 24 ]. Moreover, FOXO3a has also been reported to protect mouse oocytes from oxidative stress [ 25 ]. The effects induced by increased FOXO3a expression may also vary, depending on the signal integration and associated factors, and may range from triggering cell death to promoting survival by inducing the expression of oxidative stress resistance factors [ 26 ]. However, since different levels of FOXO3a were detected in FCs from different oocyte competence groups, we propose that the Akt-FOXO3a interaction functions to adjust the regulation of the oxidative stress response and cell proliferation and leads to apoptosis by either favoring or preventing the acquisition of competence.
Moreover, Akt, which inhibits FOXO3a, allows a final proliferative and anti-apoptotic response in high competence follicles. Another gene that displayed differences in expression levels among the developmental competence groups was BAX , which was increased in the higher quality group. BCL2 and BAX are anti-apoptotic and pro-apoptotic genes, respectively, and serve as markers of apoptosis in ovarian tissues [ 27 ].
Oocyte competence may be related to events linked to apoptosis, and early apoptosis signals in the oocyte [ 28 ] or in GCs [ 29 ] are positively correlated with oocyte competence, suggesting the involvement of apoptotic processes in follicular and oocyte development. The homodimerization of BAX and its translocation to the mitochondria to trigger apoptosis is directly regulated by the p-Akt protein [ 33 ].
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The p-Akt protein phosphorylates the BAX protein and regulates its activity; thus, BAX phosphorylation prevents its translocation to the mitochondria and induces its maintenance in the cytoplasm as an heterodimer with BCL-2, preventing apoptosis. In the results described in this study, the BL group, in addition to displaying higher BAX levels, also displayed elevated p-Akt level. Therefore, in these cells, the elevation of the BAX mRNA did not lead to a higher incidence of apoptosis, due to its low activity resulting from the phosphorylation induced by p-Akt.
Finally, p-Akt also helps control apoptosis by regulating BCL-2 availability. Phosphorylation of BAD by p-Akt leads to the isolation of BAD by the protein family and a subsequent anti-apoptotic effect [ 34 ]. Therefore, p-Akt, acting by either phosphorylating and sequestering BAD or by phosphorylating BAX and inducing its localization in the cytoplasm, triggers the anti-apoptotic response within cells. The mechanisms involved in the acquisition of oocyte competence have not yet been completely elucidated.
The acquisition of oocyte competence is a multifactorial process, and although some basic processes are well known, such as the progression of meiosis and migration of cortical granules, some intracellular processes and molecular events that lead to the formation of an oocyte capable of supporting development are still unclear. Several factors influence oocyte quality and regulate competence acquisition. However, an adequate follicular microenvironment is a key factor in successful follicle growth and oocyte competence acquisition.
In conclusion, using a retrospective developmental competence model, we investigated whether the PI3K-Akt pathway was associated with oocyte quality.
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This model allowed us to compare the molecular profiles of components of the PI3K-Akt signaling pathway in FCs with oocyte competence levels ranging from development to the blastocyst stage. This molecular landscape is compatible with cellular responses related to protection against oxidative stress, cell proliferation, and inhibition of apoptosis, suggesting that these PI3K-Akt-induced cellular responses contribute to oocyte competence acquisition during oogenesis.
Pentagons indicate whether a given component of the pathway was upregulated upward arrow or downregulated downward arrow in FCs from the BL and NC groups. Lines ending in arrowheads indicate stimulation, whereas lines ending in bars indicate blockage. The authors like to thank the staff and students at the Laboratory of Molecular Morphophysiology and Development LMMD for all their help with the sample collections and laboratory procedures.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. About this book Oocyte Physiology and Development in Domestic Animals reviews the most recent advances in the research of physiological and biochemical mechanisms underlying oocyte growth and development, providing readers with the fundamental understanding of these key processes and summarizing this important field of research.
The book covers multiple molecular and physiological mechanisms including initiation of oocyte growth during folliculogenesis and in vitro follicle culture to support oocyte competence, that are critical to health and quality. Physiological process ranging from gene expression to metabolism will be covered with an eye toward using these factors to uncover biomarkers that will further advance the field.
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In addition, the text looks at the effects of in vitro maturation environments on oocyte quality and developmental outcome. Author Bios Rebecca L. Free Access.
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Summary PDF Request permissions. Accurate patient selection can improve IVM clinical outcome. IVM of oocytes cryopreserved may assist urgent fertility preservation in cancer patients. However, there is insufficient data regarding this outcome. Improving the culture conditions may increase the maturation rates and the potential of IVM oocytes.
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Besides that, in mouse oocytes, I-Carnitine LC supplementation during vitrification of germinal vesicle GV and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. However, no data to date has proven this benefit in fetal development and birth of healthy offspring after embryo transfer to surrogate females. However, this protocol could potentially improve the quality of vitrified human oocytes and embryos during IVM.
From Wikipedia, the free encyclopedia. Journal of Experimental Medicine. Fertility and Sterility. Scandinavian Journal of Clinical and Laboratory Investigation. Current Opinion in Obstetrics and Gynecology.
Animal Development I: Fertilization & Cleavage
British Medical Bulletin. Clinical and Experimental Reproductive Medicine. In Vitro Maturation. Retrieved Human Reproduction Update. A; Wani, G. M; Khan, M.