Obesity is one common cause of reproductive health problem, which causes infertility issue worldwide though affecting the follicle development and the oocyte maturation quality, while the quality of oocyte maturation is a vital factor for determining subsequent embryonic development. Obesity has a prominent impact on fertility and reproductive health, which is embodied in the hypothalamic-pituitary-ovarian axis, the ovary and oocyte [1]. The common features of obesity are inflammation and oxidative stress, both of them are reported to impair meiotic and cytoplasmic maturation of the oocyte [2]. During meiotic maturation, mammalian oocytes undergo meiosis I and are arrested at metaphase II (MII) stage, which proceeds asymmetric division with producing a polar body. Among this, the multiple cellular elements, such as actin filaments, microtubule and mitochondria, play significant roles during oocyte meiosis.

It has been reported that high fat diet leads to abnormal mitochondrial morphology, an increase in mitochondrial potential and reactive oxygen species (ROS) [3]. In addition, it is showed that mitochondria-associated ER membranes (MAMs) mediated Ca2+ which was related to apoptosis in oocytes from obese mice [4]. And the obese mother can pass on information about mitochondrial and metabolic dysfunction to offspring through oocyte mitochondria [5]. Mitochondria take multiple functions in the oocytes, where it integrates several processes during oocyte maturation, such as energy production, Ca2+ signaling and ROS homeostasis [6]. The dynamics capacity of mitochondria to proceed fission, fusion and transport is a vital factor for controlling mitochondrial quantity and function [7], and several proteins are involved into this regulation, such as mitofusin1/2 (MFN1/2) and optic atrophy 1 (OPA1) for mitochondrial fusion, and Dynamin-related protein 1 (DRP1), mitochondrial fission factor (MFF) for fission. Among the fission factors, DRP1 acts as a major fission protein to ensure a strict control of mitochondrial function [8]. In porcine oocytes, DRP1 deficiency induced mitochondrial dysfunction as well as oxidative stress and apoptosis [9]. During embryo development in mice, fission-deficient oocytes caused by DRP1 deletion appeared a high failure rate in peri- and post-implantation development [10]. Mitochondria remodeling by fission and fusion, and repositioning themselves are both related to cytoskeleton dynamics [11]. For example, in AcCoAhi (acetyl-CoA) RA T cells, tubulin acetylation stabilized the microtubule cytoskeleton and positioned mitochondria in a perinuclear location [12]. Deficiency of cofilin 1, which was required for actin dynamics in mouse embryonic fibroblasts (MEFs), caused DRP1 accumulation and mitochondria fragmentation [13].

Previous studies showed that obesity disrupted meiotic spindle morphology and F-actin assembly in oocytes [[14], [15], [16]]. Similarly, disarrayed meiotic spindles with disordered chromosomes were also observed in obese oocytes [17], indicating the negative effects of obesity on cytoskeleton dynamics in oocytes. Besides their roles on mitochondrial fission and transport, actin filaments and microtubules are also involved into multiple cell processes in oocytes. Actin filaments are shown to regulate chromosome movement, cortical spindle anchorage, and first polar body emission. During this, metaphase I (MI) chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by several actin nucleators such as ER-bound formin-2 (Fmn2) and Arp2/3 complex [18,19]. In addition, motor protein Myosin II and the actin binding proteins Spire 1/2 also involved into this process for coordination [20]. While microtubules are required to form the meiotic spindle and chromosome segregation, and also as a track for kinesin and dynein to the transport of organelles and proteins. Acetylation in alpha-tubulin is the exclusive posttranslational modification to mark the lumina surface of microtubules [21], and tubulin acetylation was reported to be associated with microtubule stability in several models. In the neuron of fly model, the acetylation-blocking mutation in α-tubulin reduces microtubule stability [22]. In mouse oocytes, the decreased level of acetylated tubulin led to the weakened resistance ability of microtubule stability [23]. And tubulin acetylation regulates microtubule function mainly governing by opposing actions of α-tubulin acetyltransferase 1 (α-TAT1) and HDAC6 [24].

Formins are defined by the presence of a catalytic formin homology 2 (FH2), which function in cellular processes such as cytokinesis and cell polarization [25]. Due to the modular domain, formins includes several members such as FH1/FH2 domain containing protein 3 (FHOD3), Formin-1 (FMN1), Formin-like protein 1 (FMNL1) and Delphilin [26]. During mouse oocyte meiosis, FHOD1 and FMNL3 are reported to mediate cytoplasm actin assembly and spindle migration [27,28]. Moreover, FMNL2 is proved to be essential for meiotic cytoskeleton and regulate ER/mitochondria functions in mammalian oocytes [29]. Inverted-formin 2 (INF2) was unique for its FH1 and FH2 domains placing at the N-terminus. It was found to localize at the contact of ER and mitochondria [30], and it played an important role of regulating actin filament polymerization [31]. In addition, INF2 was also reported that it had the unique ability to accelerate both actin polymerization and depolymerization [32]. Deficit of INF2 gives rise to dysfunction of mitochondria and/or actin, which were associated with dozens of diseases, such as prostate cancer [33], kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth disease (CMTD) [34,35]. During the reproduction process of C. elegans, loss of EXC-6, a homolog of INF2, which was highly expressed in the spermatheca, led to approximately half of ovulations extreme defects due to an increase in the population of junction-associated actin filaments [36,37]. Considering the INF2 in modulating mitochondrial function and actin dynamics, we hypothesized that it might be intricately linked to obesity, which was closely associated with mitochondrial dysfunction.

In this study, we showed that INF2 expressed during mouse oocyte maturation and INF2 decreased in the oocytes of obese mice. We used knockdown approach to stimulate the INF2 deficiency under obesity to investigate the functions of INF2 in obese mouse oocyte maturation. We demonstrated that INF2 was involved in mitochondrial fission through actin filaments aggregating for DRP1 recruitment to mitochondria. We also showed that INF2 associated HDAC6 to manage the microtubule stability for mitochondrial transport. Our study provided the evidence for the roles of INF2 to mitochondrial fission and transport, which contribute to the declined oocyte quality in obese mice.