In the aspect of anatomy, physiology, and immunology, pigs demonstrate a higher resemblance to human as compared to model animals like mice [1,2]. The porcine early embryo is the primary stage of embryonic development before implantation; the process of porcine early embryonic development is quite complicated, and an intermolecular regulatory network is critical [[3], [4], [5]]. During early embryonic development, a single fertilized egg undergoes multiple rounds of cell cleavage to form a blastocyst [6]. This procedure involves a series of important events, such as the mitosis, zygotic genome activation and cell fate determination [7,8]. Specialized programs governing gene regulation tightly control the expression of specific genes throughout early embryonic different developmental stages [9,10]. Although, several studies have focused on the development of early embryos in pigs, the regulatory mechanisms that are involved in early embryonic development still require further investigation.

In recent reports, the significance of epigenetic alteration during early embryo growth has gained considerable attention [11,12]. Lysine-specific demethylase 1 (LSD1) was the first H3K4me1/2 and H3K9me1/2 demethylase to be discovered [13], and numerous studies have identified histone and nonhistone targets of LSD1 [13,14]. The findings of these experiments unveiled the engagement of this demethylase in the progression of embryonic development, consequently eliciting the initiation of several pathways implicated in diverse cellular processes [15]. Previous investigations have demonstrated that LSD1 has a significant function during the early embryonic development in mice. Genetic engineering technology has been used to generate oocytes with low or no LSD1 expression, fertilized eggs produced from oocytes completely lacking LSD1 die before or after the first division and cannot complete the transition from mother to zygote. Oocytes with low LSD1 expression result in the birth of pups, but many of these pups die shortly after birth, and pups that survive exhibit abnormal behavior [16]. Further studies showed that a lack of LSD1 expression in mouse embryos led to developmental arrest at the 2-cell stage, which was accompanied by progressive changes in H3K9 and H3K4 methylation patterns. At the transcriptional level, the maternal-to-zygotic transition is not properly induced. Thus, LSD1 likely plays a key role in establishing the correct epigenetic landscape of the zygote after fertilization, maintaining genome integrity, and driving genomic expression patterns during murine early embryonic development [17]. However, no research has reported whether LSD1 is expressed during porcine early embryonic development or whether it regulates early embryonic development in pigs.

Autophagy is a catabolic and recycling process by which eukaryotic cells degrade intracellular material, and it plays an important role in maintaining intracellular homeostasis [18]. Autophagy is also active during early embryo development, and it participates in processes including maternal mRNA degradation [19], cleavage [20] and trophectoderm cell differentiation [21]. For instance, oocyte-specific knockout mice lacking autophagy Atg5 exhibit deficiencies in embryo development beyond the 4- and 8-cell stages upon fertilization with Atg5-null sperm. Interestingly, it is observed that these oocytes can successfully undergo development when fertilized by wild-type sperm [22]. Similarly, autophagy can influence maternal mRNA degradation and apoptosis during porcine early embryonic development. 3-Methyladenine (an autophagy inhibitor) inhibits embryonic autophagy, as shown by the maintenance of significantly high levels of maternal mRNA, reduced developmental rates and increased apoptosis rates [19]. Mitochondrial dysfunction also hinders early embryonic development by promoting autophagy [23]. However, whether and how the LSD1-mediated regulation of autophagy participates in porcine early embryo, development is unclear and require further study and analysis.

Thus, based on previous research, we hypothesized that LSD1 affects porcine early embryonic development and mitochondrial function by regulating autophagy and apoptosis. First, we assessed the levels of LSD1 mRNA and protein starting from the metaphase of the second meiotic division oocytes to the blastocyst stage in pigs. Additionally, we explored the role of LSD1 during porcine embryonic development by utilizing GSK-LSD1, which is a specific inhibitor targeting LSD1.