Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis.

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

Estrogen influences the transzonal projection assembly of cumulus-oocyte complexes through G protein-coupled estrogen receptor during goat follicle development.

Estrogen is an important hormone that plays a role in regulating follicle development and oocyte maturation. Transzonal projections (TZPs) act as communication bridges between follicle somatic cells and oocytes, and their dynamic changes are critical for oocyte development and maturation. However, the roles and mechanisms of estrogen in regulating TZPs during follicular development are not yet understood. We found that the proportion of oocytes spontaneously resuming meiosis increases as the follicle grows, which is accompanied by rising estrogen levels in follicles and decreasing TZPs in cumulus-oocyte complex. To further explore the effect of elevated estrogen levels on TZP assembly, additional estrogen was added to the culture system. The increased estrogen level significantly decreased the mRNA and protein expression levels of TZP assembly-related genes. Subsequent research revealed that TZP regulation by estrogen was mediated by the membrane receptor GPER and downstream ERK1/2 signaling pathway. In summary, our study suggests that estrogen may regulate goat oocyte meiosis arrest by decreasing TZP numbers via estrogen-mediated GPER activation during follicle development.

Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing.

BACKGROUND: Rewriting the genomes of living organisms has been a long-standing aim in the biological sciences. The revelation of the CRISPR/Cas9 technology has revolutionized the entire biological field. Since its emergence, this technology has been widely applied to induce gene knockouts, insertions, deletions, and base substitutions. However, the classical version of this system was imperfect for inducing or correcting desired mutations. A subsequent development generated more advanced classes, including cytosine and adenine base editors, which can be used to achieve single nucleotide substitutions. Nevertheless, these advanced systems still suffer from several limitations, such as the inability to edit loci without a suitable PAM sequence and to induce base transversions. On the other hand, the recently emerged prime editors (PEs) can achieve all possible single nucleotide substitutions as well as targeted insertions and deletions, which show promising potential to alter and correct the genomes of various organisms. Of note, the application of PE to edit livestock genomes has not been reported yet. RESULTS: In this study, using PE, we successfully generated sheep with two agriculturally significant mutations, including the fecundity-related FecBB p.Q249R and the tail length-related TBXT p.G112W. Additionally, we applied PE to generate porcine blastocysts with a biomedically relevant point mutation (KCNJ5 p.G151R) as a porcine model of human primary aldosteronism. CONCLUSIONS: Our study demonstrates the potential of the PE system to edit the genomes of large animals for the induction of economically desired mutations and for modeling human diseases. Although prime-edited sheep and porcine blastocysts could be generated, the editing frequencies are still unsatisfactory, highlighting the need for optimizations in the PE system for efficient generation of large animals with customized traits.

Immunolocalization of antibacterial peptide S100A7 in mastitis goat mammary gland and lipopolysaccharide induces the expression and secretion of S100A7 in goat mammary gland epithelial cells via TLR4/NFκB signal pathway.

The antimicrobial peptide S100A7, with antimicrobial activities for a broad spectrum of bacteria, has attracted more and more attention for the prevention and treatment of mastitis. However, there is little information about the expression and regulation mechanism of S100A7 in mastitis goats. This study revealed that S100A7 was mainly expressed in the stratified squamous epithelium of teat skin and streak canal, and S100A7 was present weakly in the healthy goat alveolus yet densely in the mastitis goat collapsed alveolus. Goat mammary epithelial cells (MECs) were isolated and treated with 2.5, 5, 10 and 20 µg/mL lipopolysaccharide (LPS) respectively for a different time, S100A7 mRNA expression and protein secretion were upregulated significantly with LPS treatment for 3 h, and the secretion level of S100A7 descended after 48 h treatment for all of these four groups. Moreover, after treatment with LPS, the mRNA levels of Toll-like receptor 4 (TLR4) and MyD88 were up-regulated, and the phosphorylation of p65 was up-regulated markedly. However, adding TLR4 inhibitor TAK-242 or/and NF-κB inhibitor QNZ significantly suppressed the phosphorylation of p65, and then inhibited the expression and secretion of S100A7 induced by LPS treatment. In conclusion, LPS induced the expression and secretion of S100A7 in goat MECs via TLR4/NF-κB signaling pathway.

Sulforaphane Suppresses H2O2-Induced Oxidative Stress and Apoptosis via the Activation of AMPK/NFE2L2 Signaling Pathway in Goat Mammary Epithelial Cells.

Oxidative stress in high-yielding dairy goats adversely affects lactation length, milk quality, and the economics of dairy products. During the lactation period, goat mammary epithelial cells (GMECs) are often in a state of disordered metabolic homeostasis primarily caused by the overproduction of reactive oxygen species (ROS). Sulforaphane (SFN), an electrophilic compound that is enriched in broccoli, is a promising antioxidant agent for future potential clinical applications. The objective of the present study was to investigate the function of SFN on hydrogen peroxide (H2O2)-induced oxidative damage in primary GMECs and the underlying molecular mechanisms. Isolated GMECs in triplicate were pretreated with SFN (1.25, 2.5, and 5 µM) for 24 h in the absence or presence of H2O2 (400 µM) for 24 h. The results showed that SFN effectively enhanced superoxide dismutase (SOD) activity, elevated the ratio of glutathione (GSH)/glutathione oxidized (GSSG), and reduced H2O2-induced ROS and malondialdehyde (MDA) production and cell apoptosis. Mechanically, SFN-induced nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) translocation to the nucleus through the activation of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway coupled with inhibition of the caspase apoptotic pathway. In addition, GMECs were transfected with NFE2L2 small interfering RNA (NFE2L2 siRNA) for 48 h and/or treated with SFN (5 µM) for 24 h before being exposed to H2O2 (400 µM) for 24 h. We found that knockdown of NFE2L2 by siRNA abrogated the preventive effect of SFN on H2O2-induced ROS overproduction and apoptosis. Taken together, sulforaphane suppressed H2O2-induced oxidative stress and apoptosis via the activation of the AMPK/NFE2L2 signaling pathway in primary GMECs.

Rab23/Kif17 regulate meiotic progression in oocytes by modulating tubulin acetylation and actin dynamics.

Cytoskeletal dynamics are involved in multiple cellular processes during oocyte meiosis, including spindle organization, actin-based spindle migration and polar body extrusion. Here, we report that the vesicle trafficking protein Rab23, a GTPase, drives the motor protein Kif17, and that this is important for spindle organization and actin dynamics during mouse oocyte meiosis. GTP-bound Rab23 accumulated at the spindle and promoted migration of Kif17 to the spindle poles. Depletion of Rab23 or Kif17 caused polar body extrusion failure. Further analysis showed that depletion of Rab23/Kif17 perturbed spindle formation and chromosome alignment, possibly by affecting tubulin acetylation. Kif17 regulated tubulin acetylation by associating with αTAT and Sirt2, and depletion of Kif17 altered expression of these proteins. Moreover, depletion of Kif17 decreased the level of cytoplasmic actin, which abrogated spindle migration to the cortex. The tail domain of Kif17 associated with constituents of the RhoA-ROCK-LIMK-cofilin pathway to modulate assembly of actin filaments. Taken together, our results demonstrate that the Rab23-Kif17-cargo complex regulates tubulin acetylation for spindle organization and drives actin-mediated spindle migration during meiosis.

Single-cell transcriptome analysis reveals that maternal obesity affects DNA repair, histone methylation, and autophagy level in mouse embryos.

Obesity causes many reproductive dysfunctions such as reduced conception, infertility, and early pregnancy loss, and this is largely due to the negative effects of obesity on oocyte and embryo quality. In the present study, we employed single-cell RNA transcriptome sequencing to investigate the potential causes for the maternal obesity effects on mouse embryos. Our results showed that the 4-cell and morula/blastocyst rates were all significantly decreased during embryo development in obese mice. Genome-wide analysis indicated that obesity altered the expression of more than 1100 genes in 2-cell embryos, including the genes which were related to the p53 signaling pathway and apoptosis. Further analysis showed that the expression of 47 genes related to DNA damage was changed, and a positive γH2A signal and the altered expression of Rad51 and Tex15 were observed in the obese embryos. Obesity also affected histone methylation, shown by the decrease of the H3K4-me2 level. Besides this, we observed the occurrence of autophagy and apoptosis in the embryos of obese mice. There were 42 genes that were related to autophagy/apoptosis that showed aberrant expression, and the positive LC3 signal and the decrease of Clec16a, Rraga, and Atg10 level were also observed. In summary, our study suggested that obesity affected early embryonic development by inducing DNA damage, aberrant histone methylation, and autophagy levels in mice.

FASCIN regulates actin assembly for spindle movement and polar body extrusion in mouse oocyte meiosis.

During mouse oocyte meiotic maturation, actin filaments play multiple roles in meiosis such as spindle migration and cytokinesis. FASCIN is shown to be an actin-binding and bundling protein, making actin filaments tightly packed and parallel-aligned, and FASCIN is involved in several cellular processes like adhesion and migration. FASCIN is also a potential prognostic biomarker and therapeutic target for the treatment of metastatic disease. However, little is known about the functions of FASCIN in oocyte meiosis. In the present study, we knocked down the expression of FASCIN, and our results showed that FASCIN was essential for oocyte maturation. FASCIN was all expressed in the different stages of oocyte meiosis, and it mainly localized at the cortex of oocytes from the GV stage to the MII stage and showed a similar localization pattern with actin and DAAM1. Depletion of FASCIN affected the extrusion of the first polar body, and we also observed that some oocytes extruded from the large polar bodies. This might have resulted from the defects of actin assembly, which further affected the meiotic spindle positioning. In addition, we showed that inhibition of PKC activity decreased FASCIN expression, indicating that FASCIN might be regulated by PKC. Taken together, our results provided evidence for the important role of FASCIN on actin filaments for spindle migration and polar body extrusion in mouse oocyte meiosis.

Mps1 controls spindle assembly, SAC, and DNA repair in the first cleavage of mouse early embryos.

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development.

RAB7 GTPase regulates actin dynamics for DRP1-mediated mitochondria function and spindle migration in mouse oocyte meiosis.

RAB7 is a small GTPase that belongs to the Rab family, and as a vesicle trafficking factor it is shown to regulate the transport to late endocytic compartments, autophagosome maturation and organelle function. In present study, we showed the critical roles of RAB7 GTPase on actin dynamics and mitochondria function in oocyte meiosis. RAB7 mainly accumulated at cortex and spindle periphery during oocyte maturation. RAB7 depletion caused the failure of polar body extrusion and asymmetric division, and Rab7 exogenous mRNA supplement could rescue the defects caused by RAB7 RNAi. Based on mass spectrometry analysis, we found that RAB7 associated with several actin nucleation factors and mitochondria-related proteins in oocytes. The depletion of RAB7 caused the decrease of actin dynamics, which further affected meiotic spindle migration to the oocyte cortex. In addition, we found that RAB7 could maintain mitochondrial membrane potential and the mitochondrial distribution in mouse oocytes, and this might be due to its effects on the phosphorylation of DRP1 at Ser616 domain. Taken together, our data indicated that RAB7 transported actin nucleation factor for actin polarization, which further affected the phosphorylation of DRP1 for mitochondria dynamics and the meiotic spindle migration in mouse oocytes.

Loss of Arf Guanine Nucleotide Exchange Factor GBF1 Activity Disturbs Organelle Dynamics in Mouse Oocytes.

GBF1 [Golgi brefeldin A (BFA) resistance factor 1] is a member of the guanine nucleotide exchange factors Arf family. GBF1 localizes at the cis-Golgi and endoplasmic reticulum (ER)-Golgi intermediate compartment where it participates in ER-Golgi traffic by assisting in the recruitment of the coat protein COPI. However, the roles of GBF1 in oocyte meiotic maturation are still unknown. In the present study, we investigated the regulatory functions of GBF1 in mouse oocyte organelle dynamics. In our results, GBF1 was stably expressed during oocyte maturation, and GBF1 localized at the spindle periphery during metaphase I. Inhibiting GBF1 activity led to aberrant accumulation of the Golgi apparatus around the spindle. This may be due to the effects of GBF1 on the localization of GM130, as GBF1 co-localized with GM130 and inhibiting GBF1 induced condensation of GM130. Moreover, the loss of GBF1 activity affected the ER distribution and induced ER stress, as shown by increased GRP78 expression. Mitochondrial localization and functions were affected, as the mitochondrial membrane potential was altered. Taken together, these results suggest that GBF1 has wide-ranging effects on the distribution and functions of Golgi apparatus, ER, and mitochondria as well as normal polar body formation in mouse oocytes.

FMNL3 regulates FASCIN for actin-mediated spindle migration and cytokinesis in mouse oocytes†.

Formin-like 3 (FMNL3) is a member of the formin-likes (FMNLs), which belong to the formin family. As an F-actin nucleator, FMNL3 is essential for several cellular functions, such as polarity control, invasion, and migration. However, the roles of FMNL3 during oocytes meiosis remain unclear. In this study, we investigated the functions of FMNL3 during mouse oocyte maturation. Our results showed that FMNL3 mainly concentrated in the oocyte cortex and spindle periphery. Depleting FMNL3 led to the failure of polar body extrusion, and we also found large polar bodies in the FMNL3-deleted oocytes, indicating the occurrence of symmetric meiotic division. There was no effect of FMNL3 on spindle organization; however, we observed spindle migration defects at late metaphase I, which might be due to the decreased cytoplasmic actin. Microinjecting Fmnl3-EGFP mRNA into Fmnl3-depleted oocytes significantly rescued these defects. In addition, the results of co-immunoprecipitation and the perturbation of protein expression experiments suggested that FMNL3 interacted with the actin-binding protein FASCIN for the regulation of actin filaments in oocytes. Thus, our results provide the evidence that FMNL3 regulates FASCIN for actin-mediated spindle migration and cytokinesis during mouse oocyte meiosis.

Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis.

Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization.

RAB8A GTPase regulates spindle migration and Golgi apparatus distribution via ROCK-mediated actin assembly in mouse oocyte meiosis†.

Actin filaments are widely involved in multiple cellular processes in oocyte meiosis, such as spindle migration and polar body extrusion. The actin nucleators like Arp2/3 complex and formins are the most recognized molecules for actin assembly in oocytes. In the present study, we report that the vesicle trafficking factor, RAB8A GTPase, is a new regulator critical for actin assembly in meiosis. Our results showed that RAB8A was localized at both the spindle periphery and cortex in mouse oocytes, which was similar to the localization patterns of actin filaments. RAB8A depletion caused spindle migration defects and the failure of polar body extrusion, which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes. Based on mass spectrometry analysis, we showed that RAB8A promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway. Moreover, we demonstrated that RAB8A colocalized and interacted with GM130 at the spindle periphery and that RAB8A depletion caused the disruption of GM130-docked Golgi distribution. Taken together, our data indicated that RAB8A was required for Golgi distribution, spindle migration, and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.

RAB35 depletion affects spindle formation and actin-based spindle migration in mouse oocyte meiosis.

Mammalian oocyte maturation involves a unique asymmetric cell division, in which meiotic spindle formation and actin filament-mediated spindle migration to the oocyte cortex are key processes. Here, we report that the vesicle trafficking regulator, RAB35 GTPase, is involved in regulating cytoskeleton dynamics in mouse oocytes. RAB35 GTPase mainly accumulated at the meiotic spindle periphery and cortex during oocyte meiosis. Depletion of RAB35 by morpholino microinjection led to aberrant polar body extrusion and asymmetric division defects in almost half the treated oocytes. We also found that RAB35 affected SIRT2 and αTAT for tubulin acetylation, which further modulated microtubule stability and meiotic spindle formation. Additionally, we found that RAB35 associated with RHOA in oocytes and modulated the ROCK-cofilin pathway for actin assembly, which further facilitated spindle migration for oocyte asymmetric division. Importantly, microinjection of Myc-Rab35 cRNA into RAB35-depleted oocytes could significantly rescue these defects. In summary, our results suggest that RAB35 GTPase has multiple roles in spindle stability and actin-mediated spindle migration in mouse oocyte meiosis.

FHOD1 regulates cytoplasmic actin-based spindle migration for mouse oocyte asymmetric cell division.

FHOD1 is a member of Diaphanous-related formins (DRFs) which belongs to the Formin family. Previous studies have shown that the DFRs might affect several cellular functions such as morphogenesis, cytokinesis, cell polarity, and embryonic differentiation. However, there is no evidence showing the functions of FHOD1 during oocyte meiosis. This study is aimed at exploring the roles of FHOD1 during the mammalian oocyte maturation. Immunofluorescent staining showed that FHOD1 was restricted to the nucleus in germinal vesicle (GV) stage of the oocytes, after the GV breakdown FHOD1 was primarily located at two poles of the spindle at both metaphases I and II stages. Knockdown of FHOD1 by siRNA injection did not affect polar body extrusion but generated the large polar bodies. In addition, we observed the spindle migration failure in metaphase I oocytes, with a large number of meiotic spindles anchoring in the center of cytoplasm. The expression level of cytoplasmic actin but not cortex actin was significantly reduced, indicating that FHOD1 regulates cytoplasmic actin distribution for the spindle movement. Furthermore, we found that the disruption of ROCK (the Rho-dependent protein kinase) with inhibitor Y-27632 caused the decreased FHOD1 protein expression. Therefore, our data indicate that FHOD1 is regulated by ROCK for cytoplasm actin assembly and spindle migration during mouse oocyte meiosis.

Melatonin protects against defects induced by deoxynivalenol during mouse oocyte maturation.

Deoxynivalenol (DON) is one of the most prevalent fusarium mycotoxins in feedstuff and food. DON causes detrimental effects on human and animal reproductive systems by inducing oxidative stress and apoptosis. However, melatonin is a multifunctional endogenous hormone that plays crucial roles in the development of animal germ cells and embryos as a robust deoxidizer. In this study, we explored the effects of melatonin on the DON exposure mouse oocytes. Our in vitro and in vivo results showed that DON adversely affected mouse oocyte maturation and early embryo cleavage, while melatonin administration ameliorated the toxic effects of DON. DON exposure disrupted the meiotic spindle formation and kinetochore-microtubule attachment, which induced aneuploidy in oocytes. This might be through DON effects on the acetylated tubulin level. Moreover, we found that DON exposure caused the alteration of DNA and histone methylation level, which might affect early embryo cleavage. The toxic effects of DON on oocytes might be through its induction of oxidative stress-mediated early apoptosis, while the treatment with melatonin significantly ameliorated these phenotypes in DON-exposed mouse oocytes. Collectively, our results indicated the protection effects of melatonin against defects induced by DON during mouse oocyte meiotic maturation.

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells.

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor ß (Esr2/ERß) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERß and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERß and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERß and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERß and GPER in cumulus cells.

Intermittent fasting improves the oocyte quality of obese mice through the regulation of maternal mRNA storage and translation by LSM14B.

Obesity has significant repercussions for female reproductive health, including adverse effects on oocyte quality, fertility, embryo development and offspring health. Here, we showed that intermittent fasting (IF) has several notable effects on follicular development, oocyte development and maturation and offspring health in obese mice. IF treatment prevents obesity-associated germline-soma communication defects, mitochondrial dysfunction, oxidative damage, apoptosis, and spindle/chromosomal disruption. RNA-sequencing analysis of oocytes from normal diet (ND), high-fat diet (HFD), and HFD + IF mice indicated that IF treatment improved mitochondrial oxidative phosphorylation function and mRNA storage and translation, which was potentially mediated by the Smith-like family member 14 B (LSM14B). Knockdown of LSM14B by siRNA injection in oocytes from ND mice recapitulates all the translation, mitochondrial dysfunction and meiotic defect phenotypes of oocytes from HFD mice. Remarkably, the injection of Lsm14b mRNA into oocytes from HFD mice rescued the translation, mitochondrial dysfunction and meiotic defect phenotypes. These results demonstrated that dysfunction in the oocyte translation program is associated with obesity-induced meiotic defects, while IF treatment increased LSM14B expression and maternal mRNA translation and restored oocyte quality. This research has important implications for understanding the effects of obesity on female reproductive health and offers a potential nonpharmacological intervention to improve oocyte quality and fertility in obese individuals.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.