Identification of variant in RGS12 responsible for phenotype of PN arrest of human zygote
To identify novel PN-zygote arrest-specific genes, we recruited a family with multiple infertile individuals who presented recurrent visible PN zygotes with second polar-body emission that failed to complete PN fusion after 24 to 68 hr during IVF trials (Fig. 1a and Supplementary Table S1). We used whole-exome sequencing (WES) with 2 individuals, an unaffected sibling, and their parents (Fig. 1b). Given the pedigree structure, we used an autosomal-dominant inheritance pattern and identified heterozygous, rare, potential pathogenic variants co-segregated with PN zygote arrest. Initially, 13 candidate genes were filtered by WES (Supplementary Table S2), and only the missense variant c.C1630T (minor allele frequency [MAF] = 0.00018 in ExAC database) resulting in a p.R544W of regulator of G protein signaling-12 (RGS12) was confirmed by Sanger sequencing results available for relatives and was characterized by paternal transmission (Fig. 1d).
RGS12 is the largest protein in the regulators of the G-protein signaling (RGS) family, and is a negative regulator of specific G-protein–coupled receptor (GPCR) signals . It has the highest expressive levels in testis and ovary . RGS12 has PDZ, PTB, RGS, RBD domains and GoLoco motifs (Gαi) . It interacts with G protein via the RGS domain to inhibit cyclical activation of PLCζ-IP3 and intracellular Ca2+ release from endoplasmic reticulum and with the tyrosine-phosphorylated N-type Ca2+ channel by binding its PTB domain to regulate extracellular Ca2+ influx . RGS12 p.R544W is located between the RGS and PTB domains and presents a non-conserved pattern between human and mouse (Fig. 1c), so it might have species-specific functional effect.
Identification of the molecular landscape underlying PN arrest zygote caused by RGS12 mutation
To describe the molecular landscape underlying the PN-arrest zygote (Fig. 2a), we performed single-cell RNA sequencing of PN-arrest zygotes (n = 3) from patient III-3 to explore the transcriptional profiles of PN-arrest zygotes by comparison with normal PN zygotes (n = 22, GSE65481). We found significant upregulation of 1415 genes (fold change > 2, P < 0.001) and downregulation of 1545 genes (Fig. 2b and Supplementary Table S3). Differentially expressed genes (DEGs) were enriched in the Gene Ontology (GO) terms (biological processes) RNA processing (false discovery rate [FDR] = 1.13 × 10− 21), translational elongation (FDR = 1.25 × 10− 16), intracellular transport (FDR = 1.63 × 10− 15), and cell cycle (FDR = 4.11 × 10− 13), which indicates that the oocyte-specific transcription and translation machinery is not complete established (Fig. 2c-d). Pathway enrichment analysis revealed that DEGs in PN-arrest zygotes were also mainly involved in RNA processing and translation, such as ribosome (FDR = 6.40 × 10− 22) and spliceosome (FDR = 1.52 × 10− 5) (Fig. 2f). The switch from oocyte to embryo transition is driven by a maternal stockpile of mRNA and translational machinery that are “packed” into the oocyte. Furthermore, oocyte activation after fertilization includes changes to oocyte coverings to prevent polyspermy, release of oocyte meiotic arrest, generation of haploid female and male pronuclei, changes in maternal mRNA and protein populations, and cytoskeletal rearrangements. Our transcriptional prolife results implied that the PN-arrested zygotes had properties of failure of complete oocyte activation after fertilization.
Mutant RGS12 affects Ca2+ oscillations during oocyte activation after fertilization
Oocyte activation events present different Ca2+ requirements: 1) for cortical granules and blocking polyspermy; 2) inducing the resumption of meiosis including second meiotic polar body extrusion and initiating recruitment of maternal mRNAs; and 3) promoting pronuclear formation and initiation of embryonic mitosis. On oocyte activation, after PN formation, Ca2+ signalling continues to play a role in PN fusion and DNA synthesis for initiation of embryonic mitosis during the oocyte-to-embryo transition.
RGS12 suppresses Ca2+ oscillations by inhibiting the activity of G proteins. RGS2-depleted oocytes were found to undergo spontaneous Ca2+ release, causing slight and consistent first Ca2+ oscillations after fertilization . Therefore, we suspected that a loss-of-function effect of RGS12 p.R544W caused spontaneous and abnormal Ca2+ oscillations after fertilization as well.
Mature oocytes await fertilization while arrested at MII, which is maintained by maturation promoting factor (MPF) consisting of cyclin B1/CDK1 subunits. Cytostatic factor (CSF) mediates MPF stabilization by inhibiting anaphase-promoting complex (APC), which would otherwise destroy cyclin B. Fertilization breaks the MII arrest via cytoplasmic Ca2+ oscillation and triggers the APC, which mediates the degradation of cyclin B and thus inactivation of MPF. To confirm the spontaneous and abnormal Ca2+ oscillations, we traced the transcriptional change in genes that participate in vital processes from Ca2+ oscillation, CSF and APC (Fig. 2e). Results are as follows:
Sperm-induced Ca2+ oscillations prevent subsequent fertilization by inducing cortical granule release, which modifies the zona pellucida. The sperm binds to ZP3, which is consistent with the G-protein activation, and causes transient Ca2+ influx . PN-arrest zygotes showed 9.54-fold increased ZP3 expression, which indicates sustained ZP3-evoked Ca2+ entry by Ca2+ influx and activation of G protein. Furthermore, the plasma membrane Ca2+ channel ORAI1 mediates Ca2+ influx of oocytes after fertilization. In PN-arrest zygotes, ORAI1 showed 7.2-fold upregulation, which further confirmed the Ca2+ influx. The frequency and duration of Ca2+ oscillations are strictly temporal and spatial; otherwise, the inordinate Ca2+ oscillation during oocyte activation usually leads to impaired oocyte-to-embryo transition . Especially, high-frequency Ca2+ oscillations via increased PLCζ cause efficient oocyte activation (pronuclei formation) and cleavage stage arrest . In PN-arrest zygotes, the expression change of sperm-specific PLCζ was not observed, whereas upregulation of PLCD3 and PLCH2 and downregulation of PLCβ1 was identified. Lack of PLCβ1 disrupted amplitude Ca2+ oscillation with normal duration and frequency after fertilization . These results indicate a spontaneous and abnormal Ca2+ oscillation in PN-arrest zygotes.
Next, we explored further evidence to support spontaneous and abnormal Ca2+ oscillation and its effects. CAMKII (CAMK2A) links Ca2+ oscillations and inactivates the MPF as well as translation and degradation of maternal mRNAs. Emission of the second polar body is driven largely by the early CaMKII-driven meiosis-resumption events . We found upregulation of CAMK2A in PN-arrest zygotes, which implies the existence of prolonged Ca2+ oscillations. CaMKII activation by Ca2+ oscillations leads to activation of the APC via inhibition of CSF activity, which suppresses APC via EMI1 working with MOS. As a consequence of abnormal Ca2+ oscillations, EMI1 (FBXO5) was significantly downregulated (12.25-fold) in PN-arrest zygotes, which indicates the lower CSF level and possible high APC level.
The activation of APC is regulated by two activators, CDC20 and CDH1. CDC20 and CDH1 expression was 2.7- and 10.3-fold increased, respectively, in PN-arrest zygotes. CDC20 and CDH1 bind to APC7, whose level was also increased in PN-arrest zygotes. The mitotic checkpoint complex (MCC), composed of CDC20, MAD2, BUBR1, CENPE and BUB3, acts as an APC inhibitor , but we found downregulated BUB3 and CENPE in PN-arrest zygotes. Downregulation of EMI1 and BUB3 and upregulation of CDC20, CDH1, and APC7 implied continually increased APC level in PN-arrest zygotes.
Activation of APC/CDC20 and APC/CDH1 mediates cyclin-A and -B1 destruction and alters the substrate specificity. The catalytic center of APC is formed by APC11 and APC2 along with APC10 and the co-activators CDC20 or CDH1 for substrate recognition. APC10 is crucial for cyclin-B1 substrate recognition but not cyclin-A destruction . Cyclin A is an extremely efficient APC substrate, requiring minimal amounts of CDC20 for its destruction . We found the expression of APC10 and cyclin A2 (CCNA2) downregulated, with no change in CCNB1 expression. CCNA2 is required for timely nuclear-envelope breakdown (NEBD) . We also found downregulation of GMNN (geminin), an APC substrate and essential for regulation of DNA replication for zygotes.
Each pronucleus undergoes DNA replication and NEBD before their chromosomes eventually intermingle and enter the first mitosis. CDC7/DBF4 initiate DNA replication, whereas geminin is essential to prevent DNA re-replication . CDC7/DBF4 and geminin are substrates of APC/CDC20 and APC/CDH1 . Furthermore, depletion of EMI1 leads to geminin and cyclin A degradation due to unopposed APC/C activity. In PN-arrest zygotes, CDC7/DBF4 expression was decreased ~ five-fold. Therefore, continuous APC disrupted NEBD and DNA replication in PN-arrest zygotes.
Normally, sufficient Ca2+ oscillations promote APC to prevent MPF activation, which continues for about 4 hr, thus allowing the oocyte to enter interphase (marked by PN formation) . In turn, the pronucleus results in cessation of Ca2+ oscillations to trigger the process of NEBD . The partially activated oocyte does not progress further and is arrested again in the PN stage, described as a new metaphase-III arrest , caused by CaMKII activation by Ca2+ oscillations. Here we show that spontaneous and abnormal Ca2+ oscillation increased APC level by the mutant RGS12, leading to defective NEBD and DNA replication after 24 to 68 hr in IVF trials. The trigger for the oocyte-to-embryo transition is oocyte activation. APC also contributes to the change from meiosis in the oocyte to mitosis in the embryo . Ca2+ ionophores such as A23187 improve embryonic development of fertilized human oocytes with PN arrest, but the success rates are still poor. Therefore, our evidence supports that the PN arrest is due to spontaneous and abnormal Ca2+ oscillation causing prolonged APC activation. Hence, a precise pattern of Ca2+ oscillations after fertilization should be evaluated for further treating optimal oocyte activation.
Validation of Ca2+ oscillation–CSF–APC signaling in PN arrest zygotes
To validate the CSF, APC and MPF levels and their key genes in PN-arrest zygotes, we integrated the zygote transcriptome data from this study with previous work by Suo et al. We identified 589 common genes enriched in the GO terms translational elongation (FDR = 5.88 × 10− 34) and translation (FDR = 2.45 × 10− 17) and confirmed the incomplete oocyte activation. The key components of Ca2+ oscillation, CSF and APC signaling, EMI1, CCNA2, CDC7/DBF4, and GMNN were also identified (Fig. 3a-d). To investigate the master regulators and construct the transcriptional regulatory network in the PN-arrest zygotes, we used the ARACNe method to analyze transcription factors. Only the transcription factor MAX was upregulated (Fig. 3e), which indicates that MYC-MAX may play a critical role in the cell cycle entry of PN-arrest zygotes.
We observed upregulation of 2- to 4-cell arrest-specific genes TLE6 and PATL2, which indicates that the RGS12 mutation caused an earlier embryonic development arrest (Fig. 3e). Furthermore, we found downregulation of SYCP3 and TUBB8, which indicates defective cytoskeletal rearrangements. We did not find a disruption of zygotic arrest 1 (ZAR1), a oocyte-specific maternal-effect gene for mouse oocyte-to-embryo transition.
Loss of RGS12 causes minor defects in PN arrest and is compatible with mouse development
To determine whether RGS12 deficiency caused PN arrest, RGS12-deficient (RGS12−/−) mice containing both RGS and RBD1 domains of Rgs12 were generated. Like RGS2−/− females, RGS12−/− mice are viable and fertile and have high blood pressure due to increased Ca2+ release in response to vasoconstrictors, which act through GPCR (data not shown). The first cleavage occurred in 24/104 (23%) in vitro-fertilized embryos from RGS12−/− females as compared with Rgs12+/− mice (30/100, 30%) versus two-cell embryos observed in 105/139 (76%) of embryos from RGS12 wild-type females (Supplementary Fig. 1). Ca2+ oscillations are largely species-specific, with different species possessing specific patterns of amplitude, duration and frequency over time. Our data indicate that RGS12 has only a minor role in PN and that loss of RGS12 is compatible with mouse embryonic development. Discussion
Maternal genes play a critical effect in the earliest stages of embryonic development. Although Zar1 was first identified as oocyte-specific maternal-effect gene that functions at the oocyte-to-embryo transition in mice, it could not be utilized in human PN-arrest zygotes. Regarding to TUBB8 mutations cause female infertility by oocyte meiotic spindle assembly and maturation, homozygous mutation c.322G > A (p.Glu108Lys) of TUBB8 had been observed in a sporadic patient with phenotype of PN-arrest zygote after IVF by Sanger sequencing strategy. However, nothing is known about the genetic cause of phenotype of human PN-arrest zygotes.
To the best of our knowledge, this is the first report to describe variant in the RGS12 responsible for female infertility characterized by arrest at the PN stage during multiple IVF. The genetic basis for infertility characterized by abnormalities in human oocyte development and early embryogenesis (2- to 4-cell) had been described[2, 3, 33]. Variant in RGS12 extends the genetic causes of infertility. We have proposed that the data presented here provide the basis for developing diagnostic strategy that use the Sanger sequencing for the identification of women with mutations in RGS12.
RGS12 controls Ca2+ oscillations, which provides an important spatially restricted Ca2+ signal required for complete oocyte activation after fertilization, and triggers the CSF-APC signaling to switched from meiotic to mitotic process. Our single-cell transcriptome sequencing data revealed unique features in translation, RNA processing and cell cycle impairments of failure of complete oocyte activation, and uncovered Ca2+ oscillation–CSF–APC signaling pathway that mutant RGS12 exerted its maternal effect on PN-arrest. The genes involved PN-arrest of Ca2+ oscillation–CSF–APC signaling pathway were validated in the other data. The partially activated oocyte do not progress further and get arrested again in PN stage had been described as a new metaphase-III arrest. The key genes underling PN-arrest zygote (Fig. 4) improve the understanding of why and how RGS12 mutation causes the phenotype.