NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte‐to‐Embryo Transition

Abstract Sperm‐induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2‐cell stage. The impaired developmental potential of Nlrp14‐deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14‐deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin‐like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14‐deficient oocytes, and Uhrf1‐deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+/Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27‐linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.


Introduction
The initiation events of early embryonic development in mammals are mainly controlled by maternal effectors which are encoded by maternal effect genes and accumulated during oogenesis.However, our understanding of the function of maternal effect genes in early embryo development is very limited.Maternal mRNAs and proteins are accumulated during oocyte growth, which is required for subsequent fertilization and early embryo development. [1]A growing body of evidence has supported a role for maternal factors, such as MATER (NLRP5), PADI6, and STELLA (also named DPPA3 or PGC7), in early embryonic development. [2]The NLRP (Nucleotidebinding oligomerization domain, Leucinerich Repeat, and Pyrin domain containing) family is composed of 14 members containing similar structures.Among this family, NLRP1 and NLRP3 are involved in apoptosis and inflammation.However, several members (including NLRP2, 4, 5, 6, 9, 14) are reproduction-related.

www.advancedscience.com
2a] Decreased expression of Mater and Nlrp14 has been observed in oocytes during maternal aging. [3]The phylogenetic tree of NLR LRR domains analysis has shown that NLRP14 and MATER are orthologues within a subgroup. [4]However, the functions of maternal NLRP14 in early development remain unknown.
Sperm-induced oocyte activation is a critical starting step for a new life, and oocyte activation inefficiency is the most challenging problem for failed fertilization and embryonic development.Upon fertilization, Ca 2+ is released from the intracellular Ca 2+ stores, followed by a series of repetitive Ca 2+ transients (hereafter, Ca 2+ oscillations) that persist for several hours in oocytes. [5]Mitochondria in oocytes provide ATP to participate in the continuous transport of Ca 2+ into the ER, maintain low cytoplasmic Ca 2+ concentration ([Ca 2+ ]i), and maintain long-term [Ca 2+ ]i oscillations after oocyte activation. [6]At the same time, mitochondria also uptake cytoplasmic Ca 2+ , which regulates the metabolism of mitochondria. [7]Mitochondrial Ca 2+ homeostasis regulates oxidative phosphorylation by affecting cell energy supply. [7]However, little is known about how Ca 2+ oscillations are regulated and maintained during oocyte activation until now.
In somatic cells, nuclear localization of UHRF1 recognizes hemimethylated DNA and then recruits DNMT1 to the replication foci to maintain DNA methylation. [8]Li et al. have demonstrated that STELLA prevented DNA methylation mediated by DNMT1 by regulating the localization of UHRF1 in oocytes. [9]ery recently, it has been showed that DNMT1 and its cofactor UHRF1 changed from cytoplasmic localization to nuclear localization in zygotes in the absence of Nlrp14, which impaired pas-sive DNA demethylation and zygotic genome activation. [10]However, UHRF1 showed almost entirely cytoplasmic localization in oocytes, [9,11] and the exclusive function of cytoplasmic NLRP14 in oocytes remains unknown.
In the present study, we generated Nlrp14 knockout mice using CRISPR/Cas9 and found that Nlrp14-null oocytes showed development arrest.Through systematically analyzing the quality indicators of oocytes and early embryos, proteomic analysis, spindle transfer, and a series of biochemical experiments, we showed that infertility and early embryonic development failure of Nlrp14 mNull female mice are mainly due to impaired cytoplasmic Ca 2+ homeostasis.NLRP14 interacts with NCLX to regulate NCLX stability via K27-linked ubiquitination, revealing the novel function of maternal NLRP14 in maintaining calcium homeostasis in early development.

Nlrp14 is a Maternal Effect Gene Required for Early Embryonic Development in Mice
We first analyzed the expression profile of Nlrp14.Western blots showed that NLRP14 was predominantly expressed in mouse ovaries, but not in other tissues in female mice (Figure 1A).Moreover, the NLRP14 protein was primarily detected in oocytes rather than in granulosa cells (Figure 1B).In mouse oocytes and preimplantation embryos, NLRP14 protein remained at high levels until the blastocyst stage (Figure 1C,D).Quantitative RT-PCR (qRT-PCR) analysis showed that Nlrp14 mRNA was highly expressed in oocytes and persisted until 2-cell stage embryos but significantly decreased after this stage (Figure S1A, Supporting Information).Thus, the expression Nlrp14 showed a typical pattern of maternal effect genes.In order to accurately detect the localization of NLRP14 in oocytes and early embryos, we constructed Nlrp14-3xflag knock-in mice (Figure 1E and Figure S1B, Supporting Information).Immunofluorescence showed that NLRP14 localized exclusively to the cytoplasm in oocytes and preimplantation embryos (Figure 1F and Figure S1C,D, Supporting Information).These findings indicate that Nlrp14 is a maternal effect gene.
To investigate the physiological function of Nlrp14, we established a line of Nlrp14 knockout mice using the CRISPR/Cas9 system.Mice carrying frameshift mutations (inserted two nucleotides in exon3) in Nlrp14 allele were obtained (Figure 2A,B).The genotypes of mice including Nlrp14 +/+ , Nlrp14 +/− and Nlrp14 −/− were confirmed by DNA sequencing.Subsequent germ-line transmission was obtained and null mice were generated within a mixed genetic CD1 background.Western blots showed that NLRP14 protein completely disappeared in Nlrp14 −/− (hereafter, Nlrp14 mNull ) oocytes (Figure 2C).
Nlrp14 −/− mice appeared normal at birth and had no discernible histologic abnormalities.By natural mating, we found that the Nlrp14 mNull female mice were infertile, while the Nlrp14 +/+ , Nlrp14 +/− (hereafter, control) mice showed normal fertility (Figure 2D).To find the reason for female infertility, we analyzed the uterus at post-implantation stages and found no embryos (Figure 2E).These results clearly showed that loss of the maternal NLRP14 caused early embryonic lethality.At E3.5, blastocysts were flushed from the uterus of control mice but not from Nlrp14 mNull mice (Figure 2F).Then, we isolated zygotes from control and Nlrp14 mNull females after mating with normal males.The infertility of Nlrp14 mNull female mice appeared not to be related to ovulation since approximately the same number of zygotes could be obtained from Nlrp14 mNull mice when compared with control mice in natural ovulation assays.However, when culturing these embryos in vitro, few embryos derived from Nlrp14 mNull mice developed beyond the 2-cell stage while control embryos developed into morula and blastocyst at E2.5 and E3.5, respectively (Figure 2G).Interestingly, some oocytes that appeared to be unfertilized are actually penetrated by sperm, but the chromatin of sperm and oocytes were unable to complete de-condensation to form a pronucleus (Figure S1E, Supporting Information).In addition, the Nlrp14 mNull mice were still infertile, even within a mixed genetic CD1 background, implying an indispensable function of NLRP14.Collectively, the above analysis shows that maternal NLRP14 is required for fertilization and early embryonic development.

Maternal NLRP14 Regulates the Stability and Subcellular Distribution of UHRF1
To find the causes for embryonic development arrest in Nlrp14 mNull mice, we collected 50 control MII oocytes and 50 Nlrp14 mNull MII oocytes (three biological replicates in each group) for quantitative proteomic analyses using an advanced trapped-ion mobility selecting (timsTOF Pro) mass spectrometer (Figure 3A, Table S1, Supporting Information).Subsequent quantitative proteomic analysis showed that 3509 proteins were quantified and that a total of 112 proteins were differently expressed, including 65 upregulated proteins and 47 downregulated proteins in Nlrp14 mNull oocytes compared with control oocytes (Figure 3B).Strikingly, we found that the UHRF1 was significantly decreased in the Nlrp14 mNull group as well as the DNMT1.Western blot and immunofluorescent detection also confirmed this result (Figure 3C,D).It should be pointed out that embryos derived from Dnmt1-deficient oocytes showed normal preimplantation development. [12]11b,13] Interestingly, the STELLA expression level even increased partially in Nlrp14 mNull oocytes.However, there was no significant difference in the Uhrf1 mRNA levels between Nlrp14 mNull oocytes and control oocytes (Figure 3E).To unequivocally examine the proteins directly affected by maternal NLRP14, we simultaneously conducted immunoprecipitation coupled to mass spectrometry (IP-MS) using Nlrp14-3xflag knock-in mice.We identified 267 proteins that specifically interact with NLRP14 within mouse oocytes (Figure 3F and Table S2, Supporting Information).Among the proteins identified in IP-MS, only UHRF1 and NLRP14 itself were observed in downregulated protein sets in Nlrp14 mNull oocytes (Figure 3G).Interaction between NLRP14 and UHRF1 was also confirmed by immunoprecipitation, indicating that NLRP14 and UHRF1 could form heteromeric complexes (Figure 3H and Figure S2, Supporting Information).Significantly, the NLRP14 was obviously decreased in oocytes lacking maternal UHRF1 (Figure 3I), suggesting the stability of one protein is dependent on the presence of the other.Interestingly, unlike in Stella mNull fully grown oocytes (FGOs) where a large proportion of UHRF1 became cytoplasmic distribution, Nlrp14 mNull ; Stella mNull FGOs showed exclusive nuclear distribution of residual UHRF1 (Figure 3J).

Abnormal Calcium Homeostasis in Nlrp14 mNull Oocytes
To clarify the reason for early embryonic development arrest, we systematically analyzed the quality indicators of oocytes and early embryos.Loss of maternal NLRP14 had no obvious influence on meiotic maturation as the Nlrp14 mNull oocytes showed normal polar body extrusion (PBE) rates and normal spindle morphology (Figure S3A, Supporting Information).To analyze aneuploidy in metaphase-II oocytes, we employed chromosome spreading of metaphase-II oocytes and counted chromosomes (Figure S3B, Supporting Information).There was no difference in the number of chromosomes between control oocytes and Nlrp14 mNull oocytes.Cortical granules (CGs) are membrane-bound secretory organelles located at the cortex of MII oocytes, and formation of the cortical granule-free domain (CGFD) is not only a criterion of cytoplasmic maturation but also a feature of oocyte polarization.Similar to the control MII oocytes, obvious CGFD formed in Nlrp14 mNull oocytes (Figure S3C, Supporting Information).Then we performed RNA sequencing (RNA-seq) with MII oocytes to investigate the effects of maternal Nlrp14 deletion on the transcriptome profile in oocytes and found that there were very high correlation coefficients in expression profiles between Nlrp14 mNull oocytes and control oocytes (Figure S4A, Supporting Information).Moreover, we analyzed the decay of representative maternal mRNA in control and Nlrp14 mNull oocytes during meiotic maturation and after fertilization by qRT-PCR.These maternal mRNAs were degraded in MII oocytes and 2-cell embryos in Nlrp14 mNull mice, similar to the control (Figure S4B, Supporting Information).A subset of "maternal effect genes", namely Mater (Nlrp5), Tle6, Ooep, Filia (Khdc3), and Padi6, encode proteins of the subcortical maternal complex (SCMC).Recently, a subcortical maternal complex (SCMC) was identified to be essential for mouse preimplantation development, which was composed of multiple proteins encoded by several maternal effect genes (Mater, Tle6, Ooep, Filia, and Padi6).Thus, we asked whether maternally expressed NLRP14 linked SCMC to function in early embryonic development.However, there was no difference in the expression levels of SCMC proteins and mRNA between control and Nlrp14 mNull oocytes (Figure S4C,D, Supporting Information).
Next, to determine whether Nlrp14 mNull embryonic arrest is caused by cytoplasmic defects, we reciprocally exchanged the spindle-chromosome complex between control and Nlrp14 mNull MII oocytes, then carried out parthenogenetic activation (PA) and further culture of the reconstructed oocytes in KSOM medium (Figure 4A).Obviously, the reconstructed MII oocytes composed of control cytoplasm and Nlrp14 mNull chromosomes (termed WT cyto +KO sp ) could develop beyond the 2-cell stage, similar to WT cyto +WT sp MII oocytes upon parthenogenetic activation.On the contrary, the reconstructed oocytes composed of Nlrp14 mNull cytoplasm and control chromatin (termed KO cyto +WT sp ) showed activation inefficiency after parthenogenetic activation (Figure 4B).Meanwhile, the WT cyto +KO sp MII oocytes showed a relatively normal calcium oscillation pattern, while KO cyto +WT sp MII oocytes displayed a consistently high intracellular Ca 2+ concentration upon PA (Figure S5 and Videos S1 and S2, Supporting Information).We next examined the developmental potential of Nlrp14 mNull oocytes after parthenogenetic activation.Surprisingly, the Nlrp14 mNull oocytes rarely formed a pronucleus after parthenogenetic activation, showing severe parthenogenetic activation defects, indicated by MII phase exit failure and chromatin de-condensation failure (Figure 4C,D).In addition, when parthenogenetically activated Nlrp14 mNull oocytes were cultured in KSOM until the next day, few reached the 2-cell stage, accompanied by a large percentage of deaths (Figure 4E).We then examined cytoplasmic Ca 2+ concentrations in control oocytes and Nlrp14 mNull oocytes during parthenogenetic activation.We used Fluo-4-AM, an improved version of the calcium indicator, to measure the intracellular calcium level.The control oocytes showed normal Ca 2+ oscillation patterns; the initial Ca 2+ wave was followed by repetitive [Ca 2+ ]i transients.However, the Nlrp14 mNull oocytes maintained a high cytoplasmic Ca 2+ concentration and there were no [Ca 2+ ]i oscillations, implying that Ca 2+ homeostasis was defective in oocytes lacking maternal NLRP14 (Figure 4F,G and Videos S3 and S4, Supporting Information).In addition, AMP-activated protein kinase (AMPK) is an important energy sensor to sense energy deficiency, which could be activated in a CAMKK2-dependent manner.Western blotting showed that the activity of AMPK (Thr172) was significantly reduced in Nlrp14 mNull oocytes, implying that prolonged continuous (non-oscillatory) elevation of [Ca 2+ ]i reduced the CaMKK2 activity (Figure 4H).Collectively, these results show that the low quality of Nlrp14 mNull oocytes is mainly due to cytoplasmic defects and disrupted calcium homeostasis.

Loss of NLRP14 Impairs Mitochondria Dynamics and Functions
We next sought to find the cause of disrupted calcium homeostasis.We examined organelle distribution in control oocytes and Nlrp14 mNull oocytes using ER-Tracker and Mito-Tracker to label the ER and mitochondria, respectively (Figure 5A).Strikingly, altered mitochondrial morphology was observed in Nlrp14 mNull oocytes.The major axis of mitochondria in Nlrp14 mNull oocytes became longer compared to the mitochondria in the control oocytes.Electron microscopy (EM) observation also showed that mitochondria in Nlrp14 mNull oocytes were elongated, suggesting that loss of maternal NLRP14 severely affected mitochondrial morphology (Figure 5B and Figure S6, Supporting Information).One particular note is that mitochondria are numerous, small, and round in appearance in oocytes when compared to somatic cells.Mitochondrial dynamics are essential for mitochondrial energy metabolism and stress response.Inconsistent with the radial mitochondria distribution around the nucleus in control oocytes before GVBD, the mitochondria were always concentrated in the subcortical region in Nlrp14 mNull oocytes, implying defects in mitochondria dynamics (Videos S5-S8, Supporting Information).Meanwhile, normal mitochondrial membrane potential (MMP) is a prerequisite for mitochondria to carry out respiratory activity and produce ATP, which is necessary for the maintenance of mitochondrial function.Mitochondrial coupled Mass Spectrometry analysis for NLRP14 interacting proteins.IP samples from three independent experiments were used for Mass spectrometry analysis.G) Venn diagram depicting common proteins identified from down-regulated proteins in Nlrp14 mNull oocytes and NLRP14 interacting proteins.H) Interaction between NLRP14 and UHRF1 was confirmed by immunoprecipitation.HA-tag, HA-tagged mouse UHRF1 and myc-tagged mouse NLRP14 were expressed in HEK293T cells as indicated for 48 h, and then Co-IP (HA-MYC) and Western blot analysis for UHRF1 and NLRP14.I) Immunoblotting analyses of the control and Nlrp14 mNull MII oocytes were performed using antibodies against the indicated proteins.J) The signal of UHRF1 in control, Nlrp14 mNull and Nlrp14 mNull ;Stella mNull oocytes, respectively.Scale bar, 20 μm.membrane potential (Δ) was assessed by JC-1 staining.Surprisingly, compared to the rounded mitochondria in control oocytes, the elongated mitochondria in Nlrp14 mNull oocytes showed remarkably reduced MMP, suggesting compromised mitochondrial activity (Figure 5C,E).The cellular reactive oxygen species (ROS) level is proportional to the activity of mitochondrial electron transport.Consistent with the compromised mitochondrial activity, we found that the ROS level was obviously reduced in Nlrp14 mNull oocytes (Figure 5D,F).Similarly, ATP content analyzed by luminometric analysis was significantly decreased in Nlrp14 mNull oocytes (Figure 5G).Subsequently, we tested whether the mtDNA copy number was affected in Nlrp14 mNull oocytes using RT-qPCR, and showed that there was no difference in the mtDNA copy number between Nlrp14 mNull oocytes and control oocytes (Figure 5H).Taken together, the above findings indicated that maternal NLRP14 is essential for mitochondrial energy metabolism, which is closely associated with Ca 2+ homeostasis in mouse oocytes.

Decreased UHRF1 Expression in Nlrp14 mNull Oocytes is also a cause for Disrupted Calcium Homeostasis
Given that NLRP14 and UHRF1 formed heteromeric complexes and were highly expressed in mouse oocytes, and that deletion of NLRP14 decreased UHRF1 expression, we hypothesized that loss of maternal UHRF1 also affected mitochondrial function and Ca 2+ homeostasis.The Uhrf1-deficient oocytes showed a lower MMP in contrast to control oocytes, demonstrating that UHRF1 played a vital role in maintaining normal mitochondrial function (Figure 6A,B).Consistent with in Nlrp14 mNull oocytes, the ATP content was significantly decreased in Uhrf1 fl/△ ;SKO oocytes (Figure 6C).Furthermore, Uhrf1-deficient oocytes displayed aberrant [Ca 2+ ]i oscillations, which maintained a high intracellular Ca 2+ concentration after Ca 2+ release from intracellular stores (Figure 6D and Videos S9 and S11, Supporting Information) and showed severe parthenogenetic activation defects (Figure 6E).Taken together, our results indicate that maternal UHRF1 also plays a vital role in safeguarding the Ca 2+ homeostasis in mouse oocytes rather than only causing the aberrant DNA methylome in oocytes lacking maternal STELLA.

Maternal Depletion of NLRP14 Leads to Loss of NCLX and Impairs Mitochondrial Ca 2+ Dynamics
Mitochondria sequester and release of Ca 2+ affects the pattern of [Ca 2+ ]i in the cytosol, which acts as Ca 2+ buffer.Mitochondrial Ca 2+ activates key enzymes involved in ATP synthesis at the electron transport chain (ETC).To monitor mitochondrial Ca 2+ concentration ([Ca 2+ ]m) dynamic changes during PA, we employed a genetically-encoded Ca 2+ indicator Mt-GCaMP6s (Ca 2+ sensors GCaMP6s was fused with mitochondrial localization signal peptide cloned from Trmt10c gene) (Figure 7A).As shown in Figure 7B, Mt-GCaMP6s was colocalized with mitochondria.Strikingly, [Ca 2+ ]m dynamic changes completely disappeared in Nlrp14 mNull oocytes, implying impaired [Ca 2+ ]m homeostasis in oocytes lacking maternal NLRP14 (Figure 7C and Videos S12 and S13, Supporting Information).[Ca 2+ ]m homeostasis is mediated by controlling the expression and activity of mitochondrial Ca 2+ channels and transporters, which are required for the uptake and extrusion of mitochondrial Ca 2+ .Naturally, we asked which Ca 2+ channels and transporters were affected in Nlrp14 mNull oocytes.Control oocytes and Nlrp14 mNull oocytes were collected for Western blot analysis.Surprisingly, despite no difference in the mitochondrial content, the expression level of NCLX (the mitochondrial Na + /Ca 2+ /Li + exchanger, also known as SLC8B1) was severely reduced in mitochondria-associated Ca 2+ channels in Nlrp14 mNull oocytes compared with control oocytes (Figure 7G,H).In addition, the elongated mitochondria in Nlrp14 mNull oocytes might not be caused by changes in mitochondrial fusion-related proteins MFN1 and MFN2 (Figure 7D).Also, the protein level of mitochondrial fission-related protein DRP1 was slightly decreased in Nlrp14 mNull oocytes (Figure 7E,F).Then we asked whether supplementation of exogenous Nclx mRNA could rescue the phenotype of parthenogenetic activation failure of Nlrp14 mNull oocytes.Although the Nlrp14 mNull oocytes supplemented with exogenous Nclx mRNA still could not reach the 2-cell stage, the embryonic mortality rate was significantly decreased for Nlrp14 mNull oocytes microinjected with Nclx mRNA (Figure 7I,J).Similarly, treatment of control oocytes with NCLX inhibitor, CGP37157, during parthenogenetic activation led to oocyte death (Figure S7, Supporting Information).The above results suggest that maternal NLRP14 is required for [Ca 2+ ]m homeostasis by regulating NCLX level.

NLRP14 Maintains the Stability of NCLX by Regulating its K27 Ubiquitination
The results so far indicated that NLRP14 was required for NCLX stability.We next set out to investigate a possible interplay between NLRP14 and NCLX proteins.To investigate this question, we constructed plasmids expressing GFP-NCLX, Myc-NCLX, and a series of truncated NCLX mutants.We detected the interaction between NLRP14 and NCLX proteins by Coimmunoprecipitation (Co-IP) in cells co-expressing the two proteins (myc-NLRP14 and GFP-NCLX), confirming that NLRP14 does interact with NCLX (Figure 8A).Then we asked which MII oocytes were parthenogenetically activated in an activation medium for 6 h, then cultured in KSOM.Representative images of parthenogenetic activated embryos with the indicated genotypes at day 1 and day 2, respectively.Red arrowheads show visible pronuclei.D) Representative images of immunostaining for DNA (blue) and -tubulin (green) showing the MII exit and pronuclei formation in the parthenogenetic-activated embryos.Scale bar, 20 μm.E) Bar charts showing percentages of parthenogenetic activation with indicated genotypes.***p <0.001.F) Fluo-4-AM staining of oocytes showed the intracellular Ca 2+ concentration dynamics at different stages of two continuous [Ca 2+ ]i oscillations during PA.The oocytes are indicated with genotypes.Scale bar, 20 μm.G) [Ca 2+ ]i oscillation patterns after parthenogenetic activation of oocytes indicated with genotypes, respectively.H, Western blot analysis of the energy sensor AMPK using the indicated antibodies in control and Nlrp14 mNull oocytes.AMPK is composed of three subunits, the  subunit has catalytic activity (including two or three isoforms (1 and 2)), its Thr172 phosphorylation is the target for regulating the catalytic activity of the enzyme, while the  and  subunits are regulatory subunits.The experiment was repeated three times independently.domain of NCLX was required for interaction with NLRP14.Through Co-IP experiments, we found that truncated NCLX including NCLX △103-246 , NCLX △247-420, and NCLX △421-574 could still interplay with NLRP14 proteins (Figure 8B,C).However, IDRs mutants of NCLX in N-terminal and C-terminal were not expressed, which indicated that the IDRs domain is important to NCLX stability (Figure 8C).In addition, in order to further analyze whether the intrinsically disordered regions (IDRs) domain was necessary for the interaction between NLRP14 and NCLX, we constructed plasmids expressing GFP-NCLX-IDR1 (the Nterminal) and GFP-NCLX-IDR2 (the C-terminal).Notably, Co-IP analyses showed that both IDR1 and IDR2 could directly interact with NLRP14 (Figure 8D).This result indicated the importance of IDRs domain for the interaction of NCLX to NLRP14.Intriguingly, we noticed that co-expression of NLRP14 with NCLX often led to increased protein levels of NCLX.We asked whether NLRP14 could increase NCLX levels in a dose-dependent manner.As shown in Figure 8E, co-expressed Myc-NCLX with an increasing amount of GFP-NLRP14 in HEK293T cells indeed led to the increase of NCLX level.To exclude the possibility that the loss in NCLX protein was caused by reduced expression of the Nclx mRNA, we used RT-PCR to analyze the Nclx mRNA and found that the abundance of Nclx mRNA was absent until late 2-cell after zygotic gene activation (Figure 8F).This result indicated that the expression of NCLX might be mainly regulated by posttranslational modifications.Then we co-expressed GFP-NLRP14 and the Myc-NCLX with HA-ubiquitin (HA-Ub) to determine whether NLRP14 stabilized NCLX by regulating its ubiquitination modification.GFP-NLRP14 overexpression significantly increased Myc-NCLX ubiquitination compared with the GFP-NLRP14 absent group (Figure 8G).We co-expressed HA-tagged ubiquitin mutants with Myc-NCLX in the absence or presence of GFP-NLRP14 to characterize the possible linkage of ubiquitin chains on NCLX.Meanwhile, immunoprecipitation assays showed that NLRP14 predominantly mediated the K27linked ubiquitination (all others were replaced with arginine), but there was no obvious change at levels with ubiquitin containing K48, K63, K6, K11, K29, or K33 alone (Figure 8G).Notably, K27linked chain has been shown to slow down degradation by the proteasome. [14]Taken together, these data show that the IDRs do-main of NCLX were sufficient for its interaction with NLRP14 and subsequently modified with K27-linked ubiquitination.

Discussion
The early events of embryonic development in mammals are mainly controlled by maternal effectors, which are encoded by maternal effect genes, accumulated, and stored during oogenesis.The importance of maternal factors for quality control of mammalian oocytes is self-evident.However, our understanding of the maternal effect genes involved in female fertility and early embryo development is very limited in mammals.In this study, we demonstrate that NLRP14 acts as a safeguard of Ca 2+ homeostasis by interacting with NCLX IDRs domain and regulating its K27-linked ubiquitination.
NLRP14 female KO mice were completely sterile and exhibited 2-cell developmental arrest.Yan et al. recently reported that UHRF1 and DNMT1 shifted from cytoplasmic localization to nuclear localization in zygotes that lacked maternal NLRP14, which hindered the occurrence of passive DNA demethylation associated with DNA replication and resulted in significant higher DNA methylation levels in the paternal genome than control embryos. [10]Notably, DNA methylation can be normally established in Nlrp14-deficient oocytes.However, through a spindle  transfer assay, we found that Nlrp14 mNull embryo development arrest was mainly caused by cytoplasmic defects, suggesting that NLRP14 plays a more important function in the cytoplasm.The protein level of UHRF1 is significantly decreased (nearly loss) in oocytes lacking NLRP14.Through systematic screening of cytoplasm-related indicators, we found that the ATP content in the cytoplasm was significantly reduced, and the mitochondrial morphology and dynamics were severely damaged in Nlrp14-deficient oocytes.Of note is the failure of parthenogenetic activation in Nlrp14-deficient oocytes, with severe abnormalities in calcium oscillation patterns and very high cytoplasmic calcium concentrations.Intriguingly, loss of maternal UHRF1 caused the obvious decrease of NLRP14 protein level.In addition, NLRP14 interacted with UHRF1.Surprisingly, oocytes lacking maternal UHRF1 also maintained a high intracellular Ca 2+ concentration and displayed an aberrant [Ca 2+ ]i oscillations pattern upon parthenogenetic activation.The present study reveals a novel function of UHRF1 in calcium homeostasis beyond a DNA methylation regulator.The major and most surprising finding in our study is that loss of NLRP14 or UHRF1 would disrupt calcium homeostasis and thus oocyte activation and early embryonic development failure.It will be very interesting to determine whether other proteins are involved in the formation of this complex, providing detailed molecular and functional insights into this maternal module and how it orchestrates calcium homeostasis in mouse oocytes.
Another interesting aspect of our study is that NCLX is the only mitochondrial Ca 2+ transport channel significantly affected in Nlrp14 mNull oocytes.Nclx transcript was undetectable in oocytes, which suggests that NCLX function in oocytes is mainly regulated at the protein level.Our data revealed that loss of maternal NLRP14 caused a striking decrease of NCLX, which disrupts the [Ca 2+ ]i and [Ca 2+ ]m in oocytes.In addition, our study showed that NLRP14 regulates NCLX K27-linked ubiquitination through an unknown E3 ligase.Future studies need to identify the underlying mechanism by which NLRP14 regulated K27-linked ubiquitination of NCLX.
Ca 2+ is of central importance for a series of cellular processes including energy generation, chromosome segregation, pronucleus formation, and gene expression.It has been demonstrated that calcium homeostasis is essential for oocyte activation and embryonic development. [15]Once the sperm and oocyte are fused, the cytosolic Ca 2+ rises, which is a prerequisite for successful fertilization and oocyte-to-embryo transition.[Ca 2+ ]i oscillations last for several hours, which is essential for embryo development after fertilization.The regulatory mechanism of long-lasting series of repetitive [Ca 2+ ]i oscillations during oocyte activation is still a mystery. [5]Moreover, mitochondria possess a series of Ca 2+ transport influx and efflux channels to buffer Ca 2+ in the cytoplasm. [7]It is worth noting that [Ca 2+ ]m must be finely regulated because excessive Ca 2+ could disturb oxida-tive phosphorylation (OXPHOS) in mitochondria, namely, mitochondrial Ca 2+ uptake and release must be balanced in the homeostatic condition. [7]6b,17] Furthermore, mitochondrial dynamics, including fission, fusion, motility, and morphology, play an even more critical role in the functional status of mitochondria and eukaryotic cells. [18]In fact, female mice with oocytes deficient for mitochondrial fission are infertile. [19]Mitochondrial architecture is strikingly different in oocytes compared with somatic cells, characterized by its smaller and rounder appearance in oocytes. [20]Consistent with previous studies that calcium directly or indirectly regulates mitochondrial dynamics, [21] Nlrp14 mNull oocytes that are deficient in calcium homeostasis showed significant mitochondrial dysfunction such as being more prone to fusion than fission, restricted mitochondrial movement and lower mitochondrial oxidative capacity.Given that many mutations in genes encoding cytoplasmically localized proteins often cause abnormal oocyte-to-embryo transition, we can screen the Nlrp14 mutations in patients whose embryos are arrested in early cleavage, so as to provide possible guidance for diagnosis and treatment of such clinical cases.
Taken together, this study has systematically revealed, for the first time, that NLRP14 is an essential maternal factor that regulates oocyte Ca 2+ homeostasis by interacting with NCLX IDRs domain and regulating its K27-linked ubiquitination and that UHRF1 cooperates with NLRP14 to ensure the normal calcium homeostasis in mouse oocytes.Our study answered a fundamental and unanswered question on how maternal factors regulate calcium homeostasis in oocytes, especially during calcium oscillations.

Experimental Section
Mice: Oligos encoding a single guide RNA (sgRNA) that targets the exon 3 of Nlrp14 were inserted into px330 plasmid.Unique sgRNA sequence was chosen on the basis of the Genetic Perturbation Platform from the Broad Institute website.An Nlrp14 targeting vector was constructed and microinjected with Cas9 mRNA and sgRNA into zygotes of C57BL/6 mice.The sgRNA was designed to target exon 3 of the endogenous mouse Nlrp14 gene.The genotypes of mice including Nlrp14 +/+ , Nlrp14 +/− and Nlrp14 −/− were confirmed by DNA sequencing.Nlrp14 +/+ and Nlrp14 +/− female mice were designated as "control".Nlrp14 −/− female mice were designated as "Nlrp14 mNull ".The mutant mouse line was maintained on a mixed genetic background of C57BL/6 and CD1.
Generation of mice expressing 3xflag tagged NLRP14 was performed by Beijing Biocytogen, China.Briefly, the tags including 3xflag, GFP, and iCreERT2 were inserted before stop codon of Nlrp14 CDS using Crispr/Cas9.P2A and IRES sequence allow for co-expression of Nlrp14-3xflag, GFP, and iCreERT2 from a single transcript and independent translation.Cas9 mRNAs, gRNAs, and dsDNA donors were mixed for was confirmed by immunoprecipitation.GFP-tagged mouse NCLX-IDR and myc-tagged mouse NLRP14 were expressed in HEK293T cells as indicated for 48 h, and then co-IP and Western blot analysis for NCLX and NLRP14.E. GFP-tagged mouse NLRP14 and myc-tagged mouse NCLX were expressed in HEK293T cells as indicated for 48 h.The amount of GFP-tagged mouse NLRP14 plasmid is gradient increased as indicated.F. qRT-PCR showing the relative levels of Nclx transcripts in GV oocytes, MII oocytes, and zygotes.G. GFP-tagged mouse NLRP14, myc-tagged mouse NCLX and HA-tagged ubiquitin or ubiquitin mutants were expressed in HEK293T as indicated for 48 h, and then co-IP and western blot analysis for the ubiquitination of myc-tagged mouse NCLX.incubated for 1 h at room temperature.Then the zygotes were washed three times (5 min each time) in PBS/BSA, and incubated with corresponding fluorescent secondary antibodies for 1 h at room temperature, followed by incubation with Hoechst 33342 for 20 min.These cells were mounted on glass slides and analyzed using a laser-scanning confocal microscope (Zeiss LSM 880).
Transmission Electron Microscopy: The overall processes of biological sample preparation for transmission electron microscopy were as follows.About 60 oocytes were placed on a dish filled with pre-cooled glutaraldehyde; the samples were embedded in agar and cut into small pieces of ≈1 mm 3 with a double-sided blade, and then rinsed with 0.1 m PBS for three times, 15 min/time.1% osmium acid was added for fixation for 1-1.5 h, and then the samples were rinsed with 0.1 m PBS for three times, 15 min/time.The samples were dehydrated with gradient acetone of 30%, 50%, 70%, 80%, 90%, 95%, and 100%, 10-15 min each, and then replaced with 100% acetone twice, 10 −15 min/time.Then the samples were soaked with gradient acetone: resin, and finally embedded in pure resin and polymerized for 12 h at 37 °C, 12 h at 45 °C, 24 h at 60 °C, or directly polymerized at 60 °C for 48 h.Ultra-thin sections at 60 nm were prepared and stained with uranyl acetate-lead citrate double dyeing method.Finally, electron microscope observation was performed with an F20 Field Emission Gun Transmission Electron Microscope.
Spindle Transfer: MII oocytes super-ovulated from BDF1 strain female mice (or Nlrp14 mNull female mice) at the age of two months were used as spindle-chromosome complex and cytoplast donors.The transfer of the spindle-chromosome complex was performed using a piezo-driven micromanipulator.Enucleation and transplantation of spindle-chromosome complexes were performed in manipulation drops of M2 medium.Then the transferred spindle-chromosome complexes were exposed to the hemagglutinating virus of Japan envelope (Cosmo Bio, ISK-CF-001-EX) and transplanted into the sub-zona pellucida space of enucleated recipient oocytes.About 30 min later, these reconstructed oocytes were parthenogenetically activated.
Real-Time Quantitative Polymerase Chain Reaction (qPCR) Analysis of Mitochondrial DNA (mtDNA) Content in Oocytes: The total amount of mtDNA copy per oocyte was determined using quantitative real-time PCR (qPCR) procedure according to a previous study. [25]The mouse mtDNAspecific primers were: mtB6 forward: AACCTGGCACTGAGTCACCA and mtB6 reverse: GGGTCTGAGTGTATATATCATGAAGAGAAT. [26]10-fold serial dilutions of purified plasmid standard DNA ranging from 10 copies to 100 000 000 copies of mtDNA per 1 μL were used to generate the standard curve.The amplification efficiency of the standard curve was calculated using the qPCR efficiency calculator from Roche.Briefly, a single oocyte was loaded in a PCR tube with 10 μL lysis buffer containing 0.12 mg mL −1 proteinase K and incubated at 55 °C for 2 h, then 95 °C for 10 min, and then 2 μL of sample was taken directly for qPCR analysis.The cycling conditions were as follows: an initial phase of 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.The melting temperature was 72 °C for 1 min.Linear regression analysis of all standard curves for samples with copy numbers between 10 3 and 10 8 showed a correlation coefficient higher than 0.97.The standards of known mtDNA copy number (described above) were included during each reaction.Each oocyte measurement was performed in triplicates at least.At least fifteen MII oocytes from three mice were analyzed.
Immunoprecipitation Assay: HEK293T cells were cultured in DMEM (C11330500BT, Gibco) supplemented with 10 FBS (ST30-3302, PAN).Sixwell cultures were incubated at 37 °C, 5% CO 2 in a humidified incubator.For co-IP of exogenous proteins, HEK293T cells were transfected with the indicated plasmid (s).The cells were collected 48 h after transfection and lysed in IP Lysis buffer (Beyotime Biotechnology, P0013) containing 1× protease inhibitor cocktail for 30 min at 4 °C with occasional vortexing.The lysates were cleared by centrifugation at maximum speed for 20 min at 4 °C.Then the supernatant was transferred to a new tube and incubated with 1 μL corresponding primary antibody plus 20 μL protein A-agarose beads as indicated.Incubated tubes were placed on a rotator at 4 °C for 4 h to overnight.Then the beads were washed six times in lysis buffer.The protein-beads complexes were boiled with 1X SDS-PAGE buffer at 95 °C for 5 min, and analyzed by SDS-PAGE.

LC-MS/MS Analysis Of Mouse Oocytes:
For each sample, 50 MII oocytes were collected into a 1.5 ml EP tube.50 μL 0.2% sodium deoxycholate was added to the oocytes for lysis on ice for 30 min.Subsequently, samples were reduced by 5 mM DL-Dithiothreitol (Sigma, D9779) and alkylated with 10 mM iodoacetamide (Sigma, I1149).After digestion by Trypsin/Lys-c Mix (Promega, V5071) overnight, the peptide solution was desalted using SOLAμ HRP SPE spin plates (Thermo Scientific, 60209-001).After desalting, the eluent was dried in a vacuum evaporator.Samples were redissolved into 0.1% formic acid before use.
Samples were performed on a timsTOF Pro mass spectrometer (Bruker Daltonics) in dda-PASEF mode with a 120-min nonlinear LC gradient.MS and MS/MS spectra were recorded from m/z 100 to 1700 with 1/K0 range from 0.6 V•s cm −2 to 1.6 V•s cm −2 .The ramp time was set to 100 ms while keeping the duty cycle fixed at 100%.A total cycle time of 1.16 s contained one MS1 scan and ten PASEF MSMS scans.MS/MS spectra were searched against the UniProtKB/Swiss-Prot mouse database using Peaks Online X.The precursor tolerance was limited to 15 ppm and the fragment tolerance was set as 0.05 Da.
IP-MS: For each sample, fourteen ovaries were lysed in IP Lysis buffer (Beyotime Biotechnology, P0013) (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 1 × Roche complete mini protease inhibitor cocktail, and 1 × Pierce phosphatase-inhibitor cocktail) for 30 min at 4 °C with occasional vortexing.The lysates were cleared by centrifugation at maximum speed for 20 min at 4 °C.Then the supernatant was transferred to a new tube and incubated with anti-FLAG M2 Magnetic Beads (Sigma, M8823) for 6 h to overnight.After extensive washing with IP lysis buffer, the protein-beads complexes were transferred to a new 1.5 ml EP tube.These samples were eluted with 100 μL of glycine HCl (pH 2.5-3.0).The eluent was precipitated with 600 μL ice-cold acetone for 6 h at −20 °C and then centrifuged at 16 000×g for 30 min.The precipitate was denatured with 8 m urea, reduced, and alkylated with 5 mM Tris(2-carboxyethyl) phosphine hydrochloride (Thermo Scientific, PG82080) and 10 mM iodoacetamide.Digestion was performed with Trypsin/Lys-c Mix.The resulting peptide was desalted and then dried in a vacuum evaporator.Samples were redissolved into 0.1% formic acid before use.
Statistical Analysis: For each experiment, at least three replicates were performed.GraphPad Prism 8.02 (GraphPad Software) was employed to perform statistical analysis.The differences were assessed by unpaired Student's t-test.Statistical significance was ascribed to p< 0.05.Graphs were presented as the mean standard error of the mean ± SEM.

Figure 1 .
Figure 1.Developmental expression of NLRP14 in mouse.A) Western blot analysis showed that NLRP14 is only expressed in the ovaries rather than other tissues including heart, liver, kidney, thymus, and uterus in female mice.B) Western blot showed that NLRP14 was only expressed in oocytes rather than in granulosa cells (GCs).C) The expression pattern of NLRP14 during oocyte maturation.D) The expression pattern of NLRP14 during early embryonic development.E) Establishment of Nlrp14-3xflag knock-in mouse model, the tags including 3xflag were inserted before stop codon of Nlrp14 CDS using Crispr/Cas9.F) Representative images of subcellular localization of NLRP14 during oocyte maturation and early embryonic development.The oocytes and embryos, derived from Nlrp14-3xflag knock-in female mice, were immunolabeled with FLAG antibody (pink) and counterstained with DAPI (blue).Scale bar, 20 μm.

Figure 2 .
Figure 2. Nlrp14 is a maternal effect gene required for early embryonic development in mice.A,B) Establishment of Nlrp14 knockout mouse model carrying frameshift mutations (inserted two nucleotides in exon3).C) Western blot analysis of protein level in Nlrp14 +/− and Nlrp14 −/− oocytes.Level of -actin was used as an internal control.D) Breeding assays showed complete infertility of the Nlrp14 −/− female mice.Continuous breeding assessment showed the cumulative number of progeny per female Nlrp14 +/− and Nlrp14 −/− mouse for 6 months.At least six mice of each genotype were used.Data are the mean ± SEM (n = 6).E) Representative uterus and number of implantation sites at E6.5 in Nlrp14 +/− and Nlrp14 −/− mice.F) Both Nlrp14 +/− and Nlrp14 −/− female mice underwent natural ovulation after mating with WT male mice; embryo development was examined in the uterus at day E3.5.G) Representative images of embryos from Nlrp14 +/− and Nlrp14 −/− females cultured in KSOM medium at Day 2, Day 3, Day 4, and Day 5, respectively.Scale bar, 100 μm.

Figure 3 .
Figure 3. Maternal NLRP14 and UHRF1 form heteromeric complexes A) Schematic of MII oocytes collection and timTOF Pro MS analysis.MS samples from three independent experiments were used for Mass spectrometry analysis.B) Significantly upregulated (red) and downregulated (blue) proteins in Nlrp14 mNull oocytes.C) Immunoblotting analyses of the control and Nlrp14 mNull MII oocytes were performed using antibodies against the indicated proteins.D) The signal of UHRF1 in control and Nlrp14 mNull oocytes.Scale bar, 20 μm.E) Average expression of Uhrf1 mRNA during oocyte maturation and early embryonic development.Analysis is based on our RNA-seq data.Data are the mean ± SEM (n = 3).F) Schematic of immunoprecipitation

Figure 4 .
Figure 4. Ablation of maternal NLRP14 caused the failure of the [Ca 2+ ]i induced by parthenogenetic activation.A) A schematic illustration of the spindle transfer assay between control and Nlrp14 mNull MII oocytes.WT indicates control (Nlrp14 +/− ), KO indicates Nlrp14 mNull , PA indicates parthenogenetic activation.The hybrid oocytes produced by spindle exchange were parthenogenetically activated in an activation medium for 6 h, then cultured in KSOM.Representative images of parthenogenetically activated embryos with the indicated genotypes at day 2, day 3, and day 5. B) Bar charts showing percentages of parthenogenetic activation with indicated genotypes and treatments.Data are the mean ± SEM (n = 3).***p <0.001.C) control and Nlrp14 mNull

Figure 5 .
Figure 5. Abnormal mitochondrial morphology and mitochondrial activity in Nlrp14 mNull oocytes.A) ERs and mitochondria were labeled with ER-Tracker (blue) and MitoTracker (red) in control and Nlrp14 mNull oocytes.Scale bar, 20 μm.B) Electron micrographs of 6-week-old control and Nlrp14 mNull oocytes.White arrows indicate the mitochondria in oocytes with the indicated genotypes.Scale bar, 500 nm.C) Distribution of mitochondria with high membrane potential (red) and low membrane potential (green) in oocytes with the indicated genotypes, respectively.Scale bar, 20 μm.D) Representative images of ROS fluorescence of MII oocytes with the indicated genotypes, respectively.Images were analyzed by confocal microscopy with identical fluorescence parameters.Scale bar, 20 μm.E), Relative fluorescence intensity of ratio of red/green fluorescence analysis for each oocyte was conducted using Image J software.Significant difference between control and Nlrp14 mNull oocytes was observed.Data are expressed as mean±SEM of at least three independent experiments.**p <0.01.Data are the mean ± SEM (n = 20).F) Quantitative analysis of ROS fluorescence intensity.The fluorescence intensity analysis for each oocyte was conducted using Image J software.Data are expressed as mean±SEM of at least three independent experiments.***p <0.001.Data are the mean ± SEM (n = 20).G) The adenosine triphosphate (ATP) content of mouse oocytes with the indicated genotypes, respectively.Data are the mean ± SEM (n = 60).ATP was measured using a Berthold Lumat LB 9501 luminometer and a commercial assay kit.Data are expressed as mean±SEM of at least three independent experiments.**p <0.01.H, mtDNA copy numbers of MII oocytes in all three groups were analyzed by RT-qPCR.Data from more than 20 MII oocytes were analyzed for each group.Data are expressed as mean±SEM of at least three independent experiments (n = 3).n.s.represents the non-significant difference.

Figure 6 .
Figure 6.UHRF1 is essential for maintaining calcium homeostasis in oocytes.A) Distribution of mitochondria with high membrane potential (red) and low membrane potential (green) in oocytes with the indicated genotypes, respectively.Scale bar, 20 μm.B) Relative fluorescence intensity of ratio of red/green fluorescence analysis for each oocyte was conducted using Image J software.Significant difference between control and Uhrf1 fl/△ ;SKO oocytes was observed.Data are expressed asmean±SEM of at least three independent experiments (n = 3).**p <0.001.C) The adenosine triphosphate (ATP) content of mouse oocytes with the indicated genotypes, respectively.ATP was measured using a Berthold Lumat LB 9501 luminometer and a commercial assay kit.Data are expressed as mean±SEM of at least three independent experiments(n = 3).**p <0.01.D) [Ca 2+ ]i oscillation patterns after parthenogenetic activation of oocytes with indicated genotypes, respectively.E) control and Uhr f1fl/△ ;SKO MII oocytes were parthenogenetically activated in an activation medium for 6 h, then cultured in KSOM.Representative images of parthenogenetic activated embryos with the indicated genotypes at day 2, respectively.

Figure 7 .
Figure 7. Maternal NLRP14 mainly affected Ca 2+ homeostasis by regulating the stability of NCLX in mouse oocytes.A) Schematic representation of Mt-GCaMP6s.Ca 2+ sensors GCaMP6s were fused with mitochondrial localization signal peptide under the control of T7 promoter.B) Representative images of control and Nlrp14 mNull MII oocytes, which were microinjected with Mt-GCaMP6s mRNA (green) and labeled with MitoTracker (red).Mt-GCaMP6s was colocalized with MitoTracker.Scale bar, 20 μm.C) [Ca 2+ ]m oscillation patterns after parthenogenetic activation of MII oocytes with indicated genotypes, respectively.D) Capillary-based immunoassays for indicated proteins in oocytes with indicated genotypes, respectively.Loading control, GAPDH.E) Immunoblotting analyses of the control and Nlrp14 mNull MII oocytes were performed using antibodies against the indicated proteins.F) Bar charts showing level of DRP1 in MII oocytes with indicated genotypes.G) Mitochondrial content was evaluated by COX IV.Western blots showing similar mitochondrial components between control and Nlrp14 mNull MII oocytes.H) Immunoblotting analyses of the control and Nlrp14 mNull MII oocytes were performed using antibodies against the indicated proteins.I) Both control and Nlrp14 mNull MII oocytes were microinjected with Nclx mRNA, respectively.After culturing for 2 h, these oocytes were parthenogenetically activated in an activation medium for 6 h, then cultured in KSOM.Representative images of parthenogenetic activated embryos with indicated genotypes at day 2, respectively.Scale bar, 100 μm.J), Bar charts showing percentages of parthenogenetic-activated embryonic mortality with indicated genotypes and treatments.Data are the mean ± SEM (n = 3).

Figure 8 .
Figure 8. NLRP14 maintained the stability of NCLX by regulating its K27 ubiquitination.A) Interaction between NLRP14 and NCLX was confirmed by immunoprecipitation. myc-tag, myc-tagged mouse NCLX, and GFP-tagged mouse NLRP14 were expressed in HEK293T cells as indicated for 48 h, and then co-IP (myc-GFP) and Western blot analysis for NCLX and NLRP14.B) Schematic of mouse NCLX truncation mutants.C) Interaction between NLRP14 and truncated NCLX was confirmed by immunoprecipitation.GFP-tagged truncated mouse NCLX and myc-tagged mouse NLRP14 were expressed in HEK293T cells as indicated for 48 h, and then co-IP and Western blot analysis for NCLX and NLRP14.D. Interaction between NLRP14 and NCLX-IDR