RGS12 as a Novel Maternal-Effect Gene Causes Arrest at the Pronuclear Stage of Human Zygote

Tianzhong Ma Guangdong Medical University Chengpeng Zhang Shantou University Medical College Songxia Zhou Shantou University Medical College Xuezhen Xie Shantou University Medical College Jingyao Chen Shantou University Medical College Jing Wang Shantou University Medical College Sini Gao Shantou University Medical College Ruiqin Mai Shantou University Medical College Guohong Zhang (  g_ghzhang@stu.edu.cn ) Shantou University Medical College https://orcid.org/0000-0002-3856-3111


Background
In vitro fertilization (IVF) is now routine for treating infertile women and has brought an estimated at least 8 million babies into the world globally. Indeed, the earliest embryonic development after fertilization is a complex process, including the formation of spermatozoa and oocyte pronuclei (two-pronuclear [2PN] zygote), cytoskeletal rearrangements, pronuclear union, and initiation of cleavage of the zygote. About 5% of fertilized human oocytes present early developmental arrest at the PN stage after IVF trials [1].
However, the crucial gene responsible for PN-arrest zygotes remains largely unknown.

Material And Methods
Ethics approval and consent to participate This study was approved by the Ethics Committee of Guangdong Medical University A liated Hospital (YS2018010) and written informed consent was obtained from participants. We con rmed that patients gave written informed consent for the use of abandoned zygotes and peripheral blood for research on the arrest mechanism of pronuclear (PN) stage, with no monetary payment. All procedures used in the present study were performed in accordance with the relevant guidelines and regulations.

Family recruitment
Families were recruited through the Reproductive Medicine Center at the A liated Hospital of Guangdong Medical University based on the observation of PN arrest during regular in vitro fertilization treatment of two siblings. Eligible families and controls were enrolled after signing a written informed consent.
Peripheral blood samples were taken for DNA extraction.
Patients, ovarian stimulation, oocyte retrieval, and the IVF/ICSI procedure In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were performed according to the laboratory routine insemination procedures on the day of oocyte retrieval (Day 0). The presence of two pronuclei (PN) was observed 16-18 hr after insemination or injection, then the zygotes were cultured in 25 µL pre-equilibrated cleavage medium. The embryos were cultured in incubators at 37 °C under 6% CO 2 .
Embryo morphology was evaluated 42-46 hr (Day 2) and 68-72 hr (Day 3) after insemination. Male and female pronuclei that continued to separate on Day 2 and 3 without fusion were de ned as PN-arrest zygotes.
Whole-exome sequencing and data Germline genomic DNA was subjected to exome capture (60 Mb) with the Agilent SureSelect Human All ExonV6 kit according to the manufacturers' instructions (Agilent, Santa Clara, CA). Paired-end sequencing, resulting in 150 bases from each end of the fragments, was performed with a HiSeq PE150 Genome Analyzer (Illumina) at Novogene Bioinformatics Technology (Beijing). Sequencing reads were mapped to the reference genome (GRCh37, UCSC hg19) by using the Burrow-Wheller Aligner (BWA) and were analyzed by using the Genome Analysis Toolkit (GATK, v3.1) for calling single nucleotide variants, insertions and deletions. The 1000 Genomes, Exome Sequencing Project (ESP6500), Exome Aggregation Consortium (ExAC) and an in-house database were used to annotate the minor allele frequency (MAF) for each variant. In silico analysis, Sort Intolerant from Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), MutationAssessor, and GERP++ (Genomic Evolutionary Rate Pro ling) were used to predict the impact of each nonsynonymous variant.

Variant ltering
The pipeline was designed to lter heterozygous variants: 1) shared by both affected individuals; 2) absent in other unaffected family members; 3) not previously reported or reported to have a frequency < 0.1% in the public databases: 1000 Genomes, ESP6500, ExAC and in-house databases; 4) frameshift, nonsense, splice-site and missense variants predicted to be damaging in at least 3 of the 4 algorithms: SIFT, PolyPhen-2, MutationAssessor, and GERP++. Sanger sequencing validation and segregation analysis for candidate variants The variants were then validated by Sanger sequencing in the affected individuals and other family members. PCR ampli cation and Sanger sequencing were conducted. The RGS12 gene-speci c primers to generate the variation were 5′-CAGGTTCTGGGACCTAAACAAG-3′ (forward) and 5′-GACTGTGCAAGCTGGTGACT-3′ (reverse).
Variants were evaluated for co-segregation based on an autosomal-dominant mode of inheritance.

RNA library preparation and sequencing
The single-cell RNA-seq method used was described previously [4]. Brie y, zygotes were transferred into lysate buffer by using a mouth pipette and a reverse transcription reaction was performed on the wholecell lysate according to the manufacturer's instructions. The terminal deoxynucleotidyl transferase was used to add a poly (A) tail to the 3 end of the rst-strand cDNA, then 20 + 10 cycles of PCR were performed to amplify the single-cell cDNA. The libraries were sequenced on HiSeq PE150 Genome Analyzer platform at Annoroad Gene Technology (Beijing; http://www.annoroad.com).

Transcript alignment and assembly
Overall read quality was checked by using FASTQC v.0.11.5. The raw sequence data, in the form of FASTQ les, were aligned to the human genome (GRCh38, Ensembl Homo_sapiens) by using HISAT2 (v. for each sample were generated by using HTSeq v0.6.0.
Functional enrichment analysis RNA-seq normalized data (FPKM) were subjected to principal component analysis (PCA) by using an unsupervised approach to observe the whole clustering pro le. Gene Ontology (GO, biological processes) and pathway enrichment was performed by using DAVID (http://david.abcc.ncifcrf.gov/) with the Benjamini and Hochberg FDR to adjust the P value. The signi cantly enriched GO categories were visualized by using REVIGO [5] (http://revigo.irb.hr/). The network of enriched terms was evaluated by using Metascape [6] (http://metascape.org/). To infer the transcription factor regulatory network of this study, we used all 1,665 human transcription factors in the human TFDB 3.0 (http://bioinfo.life.hust.edu.cn/AnimalTFDB#!/).

RGS12 -knockout mice
RGS12-knockout mice on a C57BL/6J background were from Cyagen Biosciences (Guangzhou, China). All animal studies were approved by Institutional Animal Care and Use Committee of Shantou University Medical College.

Data availability
All RNA-seq data sets that were generated in this study have been deposited in Gene Expression Omnibus. Human oocyte, preimplantation embryo RNA-seq data were obtained from GSE44183 [7]. The RNA-seq data for normal PN zygotes (n = 22) were downloaded from GSE6548 [8]. The list of differentially expressed genes from RNA-seq data for validation were downloaded from a previous publication [9].

Identi cation of variant in RGS12 responsible for phenotype of PN arrest of human zygote
To identify novel PN-zygote arrest-speci c 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 identi ed heterozygous, rare, potential pathogenic variants co-segregated with PN zygote arrest. Initially, 13 candidate genes were ltered 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 con rmed 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 speci c G-protein-coupled receptor (GPCR) signals [10]. It has the highest expressive levels in testis and ovary [11]. RGS12 has PDZ, PTB, RGS, RBD domains and GoLoco motifs (Gαi) [12]. It interacts with G protein via the RGS domain to inhibit cyclical activation of PLCζ-IP3 and intracellular Ca 2+ release from endoplasmic reticulum and with the tyrosine-phosphorylated N-type Ca 2+ channel by binding its PTB domain to regulate extracellular Ca 2+ in ux [12]. 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-speci c functional effect.
Identi cation 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 pro les of PNarrest zygotes by comparison with normal PN zygotes (n = 22, GSE65481) [8]. We found signi cant 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-speci c 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 Ca 2+ oscillations during oocyte activation after fertilization Oocyte activation events present different Ca 2+ 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, Ca 2+ 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 Ca 2+ oscillations by inhibiting the activity of G proteins. RGS2-depleted oocytes were found to undergo spontaneous Ca 2+ release, causing slight and consistent rst Ca 2+ oscillations after fertilization [13]. Therefore, we suspected that a loss-of-function effect of RGS12 p.R544W caused spontaneous and abnormal Ca 2+ 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 Ca 2+ oscillation and triggers the APC, which mediates the degradation of cyclin B and thus inactivation of MPF. To con rm the spontaneous and abnormal Ca 2+ oscillations, we traced the transcriptional change in genes that participate in vital processes from Ca 2+ oscillation, CSF and APC (Fig. 2e). Results are as follows: Sperm-induced Ca 2+ oscillations prevent subsequent fertilization by inducing cortical granule release, which modi es the zona pellucida. The sperm binds to ZP3, which is consistent with the G-protein activation, and causes transient Ca 2+ in ux [14]. PN-arrest zygotes showed 9.54-fold increased ZP3 expression, which indicates sustained ZP3-evoked Ca 2+ entry by Ca 2+ in ux and activation of G protein.
Furthermore, the plasma membrane Ca 2+ channel ORAI1 mediates Ca 2+ in ux of oocytes after fertilization. In PN-arrest zygotes, ORAI1 showed 7.2-fold upregulation, which further con rmed the Ca 2+ in ux. The frequency and duration of Ca 2+ oscillations are strictly temporal and spatial; otherwise, the inordinate Ca 2+ oscillation during oocyte activation usually leads to impaired oocyte-to-embryo transition [15]. Especially, high-frequency Ca 2+ oscillations via increased PLCζ cause e cient oocyte activation (pronuclei formation) and cleavage stage arrest [16]. In PN-arrest zygotes, the expression change of sperm-speci c PLCζ was not observed, whereas upregulation of PLCD3 and PLCH2 and downregulation of PLCβ1 was identi ed. Lack of PLCβ1 disrupted amplitude Ca 2+ oscillation with normal duration and frequency after fertilization [17]. These results indicate a spontaneous and abnormal Ca 2+ oscillation in PN-arrest zygotes.
Next, we explored further evidence to support spontaneous and abnormal Ca 2+ oscillation and its effects. CAMKII (CAMK2A) links Ca 2+ 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 [18]. We found upregulation of CAMK2A in PN-arrest zygotes, which implies the existence of prolonged Ca 2+ oscillations. CaMKII activation by Ca 2+ 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 Ca 2+ oscillations, EMI1 (FBXO5) was signi cantly 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 [19], 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 speci city [20]. The catalytic center of APC is formed by APC11 and APC2 along with APC10 and the coactivators CDC20 or CDH1 for substrate recognition. APC10 is crucial for cyclin-B1 substrate recognition but not cyclin-A destruction [21]. Cyclin A is an extremely e cient APC substrate, requiring minimal amounts of CDC20 for its destruction [20]. 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) [22]. 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 rst mitosis. CDC7/DBF4 initiate DNA replication, whereas geminin is essential to prevent DNA re-replication [23]. CDC7/DBF4 and geminin are substrates of APC/CDC20 and APC/CDH1 [24]. 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 ~ ve-fold. Therefore, continuous APC disrupted NEBD and DNA replication in PN-arrest zygotes.
Normally, su cient Ca 2+ oscillations promote APC to prevent MPF activation, which continues for about 4 hr, thus allowing the oocyte to enter interphase (marked by PN formation) [25]. In turn, the pronucleus results in cessation of Ca 2+ oscillations to trigger the process of NEBD [26]. The partially activated oocyte does not progress further and is arrested again in the PN stage, described as a new metaphase-III arrest [27], caused by CaMKII activation by Ca 2+ oscillations [28] . Here we show that spontaneous and abnormal Ca 2+ 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 [29]. Ca 2+ 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 Ca 2+ oscillation causing prolonged APC activation. Hence, a precise pattern of Ca 2+ oscillations after fertilization should be evaluated for further treating optimal oocyte activation.

Validation of Ca 2+ 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. [9] We identi ed 589 common genes enriched in the GO terms translational elongation (FDR = 5.88 × 10 − 34 ) and translation (FDR = 2.45 × 10 − 17 ) and con rmed the incomplete oocyte activation. The key components of Ca 2+ oscillation, CSF and APC signaling, EMI1, CCNA2, CDC7/DBF4, and GMNN were also identi ed (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 PNarrest zygotes.
We observed upregulation of 2-to 4-cell arrest-speci c 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 nd a disruption of zygotic arrest 1 (ZAR1), a oocyte-speci c maternal-effect gene for mouse oocyte-toembryo transition [30].
Loss of RGS12 causes minor defects in PN arrest and is compatible with mouse development To determine whether RGS12 de ciency caused PN arrest, RGS12-de cient (RGS12 −/− ) mice containing both RGS and RBD1 domains of Rgs12 were generated. Like RGS2 −/− females [31], RGS12 −/− mice are viable and fertile and have high blood pressure due to increased Ca 2+ release in response to vasoconstrictors, which act through GPCR (data not shown). The rst 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). Ca 2+ oscillations are largely species-speci c, with different species possessing speci c patterns of amplitude, duration and frequency over time [16]. 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 rst identi ed as oocyte-speci c maternal-effect gene that functions at the oocyte-to-embryo transition in mice [30], 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 [32]. However, nothing is known about the genetic cause of phenotype of human PN-arrest zygotes.
To the best of our knowledge, this is the rst 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 4cell) 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 identi cation of women with mutations in RGS12.
RGS12 controls Ca 2+ oscillations, which provides an important spatially restricted Ca 2+ 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 Ca 2+ oscillation-CSF-APC signaling pathway that mutant RGS12 exerted its maternal effect on PN-arrest. The genes involved PN-arrest of Ca 2+ oscillation-CSF-APC signaling pathway were validated in the other data [9]. The partially activated oocyte do not progress further and get arrested again in PN stage had been described as a new metaphase-III arrest [27]. The key genes underling PNarrest zygote (Fig. 4) improve the understanding of why and how RGS12 mutation causes the phenotype.

Conclusions
In conclusion, we have identi ed an RGS12 variant as the potential cause of female infertility characterized by arrest at the PN stage during multiple IVF. This gene should be further screened in individuals with infertility caused by arrest at the PN stage during IVF. These ndings expand our knowledge of the genetic basis of human early embryonic arrest and provide the basis for genetic diagnoses of clinically infertile individuals with this phenotype.

Consent for publication
Not applicable.

Availability of supporting data
All data generated through this study are included in this article.

Competing interests
The authors declare no competing nancial interests.   Validation of transcriptome pro le in other PN-arrest zygotes. a, Venn diagram shows overlapped differentially expressed genes between previous study by Suo et al [9] (PN arrest I) and our study (PN arrest II) for PN arrest groups each compared with their control group. b, REVIGO scatterplot summarizes the overrepresented GO terms (biological processes) for representative subsets of terms. c, Metascape enrichment network of the intra-cluster and inter-cluster similarities of enriched GO terms. d, Metascape