The initiation of mammalian reproduction critically depends on two fundamental biological processes: oocyte maturation and oocyte activation. During these transitions, maternal mRNA transcription and translation drive the oocyte-to-embryo transition (OET), marking the onset of embryonic development [1]. Understanding the molecular regulation of the OET is crucial for advancing reproductive biology and addressing challenges in assisted reproduction [2]. Both oocyte maturation and oocyte activation are regulated by intracellular Ca2+ signalling, which initiates Ca2+-dependent temporal events that trigger subsequent embryonic development. Although the Ca2+ signalling patterns differ across the two processes [3],the spatiotemporal dynamics of Ca2+ signals are closely associated with inositol 1,4,5-trisphosphate receptor type 1 (IP3R1).

During the maturation process of porcine oocytes, the Ca2+ reserve of oocytes in the GV phase is located mainly in the endoplasmic reticulum (ER). 2-Aminoethyl diphenylborinate (2-APB), a specific IP3R1 inhibitor, attenuates Ca2+ flux from the ER to mitochondria, thereby mitigating cellular damage induced by Ca2+ overload. The dynamic balance of Ca2+ is regulated by the calcium channel protein IP3R on the mitochondrial-associated endoplasmic reticulum membrane (MAM) [4], and calmodulin kinase (CaMK) is activated, directly or indirectly triggering an increase in the activity of maturation promoting factor (MPF) [5]. With respect to oocyte activation, the key signalling event involves the binding of inositol 1,4,5-trisphosphate (IP3) to IP3R, which is induced by sperm-derived heterologous phospholipase C-ξ (PLC-ξ). This interaction generates continuous and rhythmic "Ca2+ oscillations" [6,7], and initiates sequential Ca2+ -dependent events, such as the release of cortical granules, the loss of activity of cyclin B and its dependent cell cycle kinase (CDK1), and the restoration of the cell cycle of MAPK activity triggering a decrease in MPF activity, and promoting the resumption of meiosis. This results in cell cycle progression to metaphase and the release of the second polar body [8]. The subsequent formation of male and female prokaryotes marks the completion of meiosis [9] and the initiation of the OET [10,11]. In vitro culture is prone to an insufficient supply of endogenous nutrients because of changes in the in vitro microenvironment, which leads to abnormal IP3R/Ca2+ signalling, resulting in the failure of oocyte maturation in vitro and the obstruction of embryo development [12,13]. The specific mechanism by which IP3R1 regulates the OET to initiate cell fate transition remains unclear.

Mitochondria play an important role in both oocyte maturation and early embryonic development. As calcium ion receptors, mitochondria not only play a role in regulating calcium homeostasis, but are also the most important "energy centres" in oocytes. IP3R1 deficiency significantly affects IP3R1-GRP75-VDAC1 protein interactions, presenting a key component of the calcium signalling pathway in MAMs, leading to Ca2+ transport imbalance. This imbalance causes excessive Ca2+ accumulation in mitochondria, ultimately impairing mitochondrial function [14,15]. Mitochondrial function directly affects the success of sperm-induced oocyte activation and subsequent embryonic development [16]. Studies have shown that oocytes contain a significantly greater mitochondrial copy number and mitochondrial DNA (mtDNA) content than other cell types do (approximately 105-106 copies per cell) [[17], [18], [19]]. The mitochondrial mass increases exponentially during late oocyte development to meet the bioenergetic demands of meiosis completion, fertilization, and early embryogenesis. Mitochondrial dysfunction, characterized by reduced adenosine triphosphate (ATP), mtDNA depletion/mutation, disrupted membrane potential, or increased reactive oxygen species (ROS), disrupts chromosome segregation and spindle stability, accelerates telomere shortening, and impairs oocyte quality, ultimately leading to embryonic developmental defects and underscoring the critical role of mitochondrial integrity in reproductive success [20].

Reduced ATP levels, which occur under conditions of nutrient deficiency or mitochondrial dysfunction, lead to an increased AMP/ATP ratio. In response, the evolutionarily conserved adenosine monophosphate-activated protein kinase (AMPK) pathway is swiftly activated, and its key substrates include mitochondrial calcium uniporter (MCU) [21], the crucial enzyme in fatty acid biosynthesis known as acetyl-CoA carboxylase (ACC) [22], and the mammalian target of rapamycin (mTOR) [23]. AMPK phosphorylates a select group of substrates (such as guanosine triphosphate and phosphofructokinase) to biochemically shift metabolism from an anabolic state to a catabolic state. This inhibits the synthesis of the mechanistic target of rapamycin complex 1 (mTORC1), thereby helping to reestablish metabolic balance. The upstream kinases of AMPK (such as CAMKK2) further modulate AMPK activity by sensing calcium signals, thereby enhancing mitochondrial function, catabolic activity, and ATP production to sustain cellular processes [24]. Substantial evidence indicates that the CaMKK2 pathway activates AMPK in response to hormones or growth factors that induce ER Ca2+ release—including those interacting with Gq/G11-coupled G protein-coupled receptors that generate the Ca2+-mobilizing messenger IP3 including IP3R1 [25]. AMPK also facilitates long-term metabolic adaptation through transcriptional regulation, downregulating biosynthetic genes while upregulating those involved in lysosomal and mitochondrial biogenesis. This promotes the clearance of damaged mitochondria and supports cellular energy resilience [25]. AMPK activation increases mitochondrial production, and AMPKγ3 overexpression enhances mitochondrial generation in mice [26]. The regulation of mitochondrial oxidative metabolism by AMPK requires the involvement of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), and AMPK indirectly modulates PGC1α expression via the p38 MAPK and HDAC5 pathways [27]. As a critical regulator of cellular energy metabolism and a major downstream target of AMPK, mTOR interacts with AMPK to maintain the “Yin-Yang” balance, which is essential for sensing nutrient/energy levels and regulating cell growth [28]. This interplay between AMPK and mTORC1 is vital during key mammalian developmental stages, including oocyte maturation, primordial follicle activation, early follicle growth, and follicular antrum formation [29]. Moreover, Ca2+ signalling indirectly regulates cellular energy balance and function through the AMPK and mTORC1 pathways [30].

Calcium ions (Ca2+) serve as critical secondary messengers that directly influence histone methylation dynamics. Recent studies have revealed that AMPK deficiency affects genomic stability through the formation of abnormal R-loop structures, subsequently triggering aberrant epigenetic modifications (such as H3K4me3) and compromising germ cell integrity and DNA damage response mechanisms [[31], [32], [33]]. Moreover, mTOR plays a pivotal role in embryonic development by regulating eukaryotic translation initiation factor 4F (eIF4E). During early mammalian development, eIF4E expression is essential for the maternal-to-embryonic transition. After fertilization, eIF4E is present in the cytoplasm of oocytes and early embryonic cells, supporting the transition from maternal genetic material to embryonic protein synthesis. The inhibition of eIF4E activity using 4EGI-1 leads to developmental arrest at the two-cell stage. Moreover, the mTOR signalling pathway activates the eIF4E translation initiation complex by regulating 4E-BP1 phosphorylation, thereby coordinating protein translation in both the cytoplasm and mitochondria, and subsequently regulating the embryonic development process [34].

To clarify the functional pathway of IP3R1, this study employed Smart-Seq technology to investigate whether IP3R1 regulates mitochondrial function and epigenetic modifications by modulating Ca2+ to activate the AMPK and mTOR pathways, thereby facilitating normal OET initiation.