Oocyte in vitro maturation (IVM) technology is fundamental for elite seed propagation in modern animal husbandry, the conservation of rare germplasm resources, and advancements in embryonic biotechnology [1]. The quality of mature oocytes is directly linked to their fertilization capacity and subsequent embryonic developmental competence, ultimately influencing the lifelong fate of newborn individuals [2]. In contrast to oocytes matured in vivo, those cultured in vitro are removed from the intrafollicular microenvironment. Consequently, they often experience inadequate cytoplasmic maturation, which includes mitochondrial dysfunction, endoplasmic reticulum stress [3], and erroneous epigenetic modifications, stemming from nutrient sensing dysregulation and metabolic stress [4]. This situation results in diminished oocyte maturation quality and reduced embryonic developmental potential [5]. Therefore, elucidating the mechanisms of oocyte nutrient sensing in vitro and reconstructing a nutrient microenvironment that mimics the in vivo conditions are essential for overcoming the challenges associated with IVM technology.

Amino acids are vital energy providers for cellular growth and development, serving not only as substrates for protein synthesis but also functioning as signaling molecules, antioxidants, and energy sources [3]. Among these, Glycine, the simplest non-essential amino acid, is present in the highest concentration in the fluids of the female reproductive tract, including oviductal, uterine, and follicular fluids [6]. Additionally, the metabolic activity of Glycine increases progressively during the maturation of mammalian oocytes, transitioning from the germinal vesicle (GV) stage to metaphase II (MII) of the second meiosis, which indicates a specific requirement for Glycine during oocyte maturation [7]. However, during in vitro production (IVP) of gametes, the endogenous supply of Glycine is inadequate due to alterations in the external microenvironment, leading to a “functional deficiency” that restricts oocyte developmental potential. Our previous studies have shown that exogenous Glycine supplementation can significantly enhance mitochondrial function and promote the synchronous maturation of the nucleus and cytoplasm in porcine oocytes [8]. Nonetheless, the mechanisms by which oocytes nutritionally sense Glycine to exert its biological functions remain unclear.

Nutrient sensing is pivotal in regulating cellular metabolism, enabling organisms to detect fluctuations in environmental or intracellular nutrient levels and to adjust their metabolic processes accordingly [9]. This complex mechanism involves monitoring both extracellular and intracellular nutrient availability and energy status through an intricate molecular network. Within this network, AMPK and mTORC1 serve as two fundamental regulatory hubs, establishing a “Yin-Yang” balance in cellular metabolism [10,11]. As an “energy sensor,” AMPK is activated during glucose deprivation and energy deficiency, characterized by an elevated AMP/ATP ratio. It facilitates catabolic processes such as fatty acid oxidation, glycolysis, and autophagy to produce ATP, while concurrently inhibiting energy-intensive processes like protein and lipid synthesis [12]. Conversely, mTORC1 functions as a “nutrient and growth sensor,” responding to amino acids and growth factors; its activation promotes protein synthesis and ribosome biogenesis, thereby supporting cell growth and development [13].

Research indicates that AMPK directly inhibits mTORC1 by phosphorylating TSC2 and Raptor, thereby halting energy-consuming growth under conditions of energy deficiency [14]. Additional studies have demonstrated that AMPK activation enhances the GAP activity of the TSC complex through TSC2 phosphorylation, facilitating the conversion of RHEB from its GTP-bound (active) form to the GDP-bound (inactive) form. Inactive RHEB is unable to activate mTOR, which indirectly obstructs the positive regulatory signal of RHEB on mTOR. Furthermore, mitochondria can autonomously sense antioxidant demand by regulating internal glutathione (GSH) levels [15]. Collectively, these mechanisms form a complex nutrient-sensing network [16].

The regulation of embryonic development by nutrient sensing is primarily mediated through the precise control of translational processes. Under nutrient-sufficient conditions, mTOR phosphorylates 4E-BP1, resulting in its dissociation from eIF4E. The liberated eIF4E initiates cap-dependent translation, effectively synthesizing proteins essential for cell proliferation and differentiation, such as PRC2 and SET2 family kinases, which provide a critical material basis for embryonic development [17]. Conversely, during nutrient deprivation, the AMPK pathway is activated, and eIF4E activation is inhibited by blocking the RHEB-mTOR signaling pathway. This leads to the sustained binding of 4E-BP1 to eIF4E, thereby downregulating global translational efficiency and reducing the synthesis of non-essential proteins while prioritizing the expression of core developmental genes [18]. Simultaneously, H3K27me3 and H3K36me3 synergistically regulate gene expression through epigenetic reprogramming, further ensuring the orderly progression of embryonic development. During critical developmental stages, H3K27me3 precisely modulates the spatiotemporal expression of essential developmental genes through its inhibitory effect, maintaining genomic imprint stability and parent-of-origin-specific gene expression, which establishes the foundation for normal embryonic implantation and development [19]. In contrast, H3K36me3 promotes the accuracy of gene transcription through its activating effect, specifically enhancing the expression of genes associated with cell proliferation and differentiation [20]. This mechanism complements the protein synthesis regulated by nutrient-sensing pathways, providing adequate energy and material support for development. Additionally, it participates in the regulation of DNA damage repair and transcriptional fidelity, thereby reducing the risk of gene mutations during embryonic development [21].

Glycine is sensed to influence the in vitro development of porcine oocytes, thereby providing sufficient energy, such as ATP production, for early embryos. Our previous studies have demonstrated that Glycine regulates Ca2+ levels through IP3R, a critical interaction factor on the mitochondrial-associated endoplasmic reticulum membrane (MAM). This regulation maintains calcium homeostasis under in vitro stress conditions, protects normal mitochondrial function, and ultimately promotes the in vitro development of porcine oocytes [22]. Is this process linked to nutrient-sensing? Research involving patients with Alzheimer's disease (AD) has shown that increased intracellular calcium [23], induced by NMDA receptor activation, activates AMPK in a CAMKK2-dependent manner. Furthermore, studies on neural cells indicate that Ca2+ signals can modulate the interaction between AMPK and mTOR, thereby influencing cell survival and metabolic balance [24].

We hypothesize that Glycine plays a role in regulating Ca2+levels. This study aims to investigate how porcine oocytes sense Glycine nutritionally, thereby facilitating nutritional compensation under low glucose stress to sustain normal biological functions during in vitro development. A thorough examination of the nutrient-sensing mechanisms in oocytes is crucial for elucidating the regulatory network that governs oocyte growth and metabolism. By uncovering the signaling pathways, key regulatory factors, and their interactions involved in nutrient sensing, particularly regarding amino acids, this research lays the groundwork for developing more comprehensive in vitro cellular metabolism models. This endeavor will enhance our understanding of how cells detect and respond to nutrient signals to perform specific biological functions.