Background Abnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified that are involved in female infertility caused by abnormalities in oocyte development, fertilisation and embryonic development. However, the genetic factors responsible for multiple pronuclei formation during fertilisation and early embryonic arrest remain largely unknown.
Objective We aim to identify genetic factors responsible for multiple pronuclei formation during fertilisation or early embryonic arrest.
Methods Whole-exome sequencing was performed in a cohort of 580 patients with abnormal fertilisation and early embryonic arrest. Effects of mutations were investigated in HEK293T cells by western blotting and immunoprecipitation, as well as minigene assay.
Results We identified a novel homozygous missense mutation (c.397T>G, p.C133G) and a novel homozygous donor splice-site mutation (c.546+5G>A) in the meiotic gene REC114. REC114 is involved in the formation of double strand breaks (DSBs), which initiate homologous chromosome recombination. We demonstrated that the splice-site mutation affected the normal alternative splicing of REC114, while the missense mutation reduced the protein level of REC114 in vitro and resulted in the loss of its function to protect its partner protein MEI4 from degradation.
Conclusions Our study has identified mutations in REC114 responsible for human multiple pronuclei formation and early embryonic arrest, and these findings expand our knowledge of genetic factors that are responsible for normal human female meiosis and fertility.
- Female infertility
- Early embryonic arrest
- Mendelian disease
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It has been estimated that 10.7%–15.5% of couples are affected by infertility.1 Normal oocyte fertilisation and embryonic cleavage are key procedures for successful human reproduction, and defects in these procedures will cause female infertility. With the prevalence of in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI),2 it has become easier to evaluate phenotypic changes in the processes of oocyte development, fertilisation and early embryonic development and to investigate the genetic basis of female infertility.
Pronuclei formation is a critical process during fertilisation. Normally, there are two pronuclei (2PN), including the paternal pronucleus and the maternal pronucleus, in the zygote after fertilisation.3 It is generally accepted that multiple pronuclei (MPN) formation is due to the abnormal extrusion of the second polar body or to abnormal fertilisation with multiple sperm.4 The MPN therefore can cause infertility and recurrent failure IVF/ICSI. In 2017, transcription levels of several genes in oocytes have been identified to alter by analysing differentially expressed gene profiles between normal and MPN zygotes.5 However, no genes have been reported to cause infertility characterised by MPN.
Early embryonic arrest is referred to as preimplantation embryo lethality and the arrested cleaved embryos that could not form blastocysts. The subcortical maternal complex (consisting of PADI6, TLE6, KHDC3L, OOEP, NLRP2 and NLRP5) has been reported to play important roles in embryogenesis, and we previously identified mutations in some of these proteins (PADI6, TLE6, KHDC3L, NLRP2 and NLRP5) as being responsible for early embryonic arrest.6–8 In addition, some rare variants in TUBB8 and PATL2 have also been found to cause low-quality embryos.9–13 Nevertheless, the genetic factors behind the majority of patients with early embryonic arrest remain to be discovered.
In our study, we have identified a homozygous missense and a homozygous splicing mutation in REC114 in female patients with MPN zygotes and early embryonic arrest from two independent consanguineous families. REC114 plays important roles in the formation of DSBs during meiosis.14 15 We also investigated the effects of these two mutations in vitro.
Materials and methods
Patients from families and controls were recruited from the Shanghai Ji Ai Genetics and IVF Institute and Shanghai Ninth Hospital affiliated to Shanghai Jiao Tong University. Our study was approved by the Ethics Committee of the Medical College of Fudan University and the Reproductive Study Ethics Committee of the hospital.
Peripheral blood was collected from the patients after obtaining informed consent. The QIAamp DNA Blood Mini Kit (Qiagen) was used to isolate genomic DNA from the blood, and the exomes of the samples were sequenced using an Illumina HiSeq 3000 platform. We performed whole-exome sequencing for each patient in a cohort of 580 patients with abnormal fertilisation and early embryonic arrest. In the consanguineous state of the families, homozygosity mapping was carried out with HomozygosityMapper after obtaining the exome data. We screened for candidate genes in family 1 within and around the homozygous regions according to the following stringent criteria: (1) a minor allele frequency of the variants to be less than 1% in the Exome Aggregation Consortium (ExAC, V.0.3.1) database and the genome Aggregation Database (gnomAD), as well as in our in-house exome database, (2) inheritance of the homozygous variants from both the father and mother when the blood of parents was available and (3) in silico prediction to be damaging according to the PolyPhen-2 and SIFT databases. The candidate genes were confirmed by Sanger sequencing of the affected probands as well as their parents and unaffected siblings using the sequencing primers shown in online supplementary table S1.
Plasmid construction and expression in HEK293T cells
The full-length REC114 human coding sequence (Gen Bank: NM_001042367.2) was amplified from MI oocyte cDNA and inserted into the pCMV6 expression vector with Myc and Flag tags at the C-terminus using a homologous recombination kit (Novoprotein). Point mutagenesis was carried out with the KOD-Plus mutagenesis kit (SMK-101, TOYOBO). The full-length MEI4 human coding sequence was synthesised by Shanghai Boshang Biotechnology Limited Company and cloned into the pCMV6 expression vector with an HA tag at the C-terminus.
The human embryonic kidney 293T (HEK293T) cells were cultured in (Dulbecco's modified Eagle’s medium) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco) in an atmosphere of 5% CO2 at 37°C. The constructs were transfected into HEK293T cells using the PolyJet In Vitro DNA Transfection Reagent (SignaGen) according to the manufacturer’s protocol.
Western blotting and cycloheximide (CHX) chase assay
HEK293T cells were seeded in a 6-well plate 1 day prior to transfection and then transfected with 1 µg of the desired constructs. After 36 hours, cell lysates were acquired in RIPA lysis buffer (Shanghai Wei AO Biological Technology) with 1% protease inhibitor cocktail (Bimake). Equal amounts of protein were separated using SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membrane. The membrane was blocked with 5% non-fat milk diluted in phosphate-buffered saline with 0.1% Tween 20, followed by incubating with antibodies against Flag (1:1000 dilution, Sigma-Aldrich, Cat #: A9594) and vinculin (1:1000 dilution, CST, Cat #: 13901). The blots were finally captured using ECL Western Blotting Substrate (Tanon) after incubation with the secondary antibodies (1:5000 dilution, Abmart). To evaluate the protein degradation rates, HEK293T cells were treated with 50 µg/mL CHX (Sigma) after 30 hours of transfection. Cell lysates were collected after 0, 3, 6, 9 and 12 hours of exposure to CHX.
The c.546+5G>A mutation was located at the donor splice-site of intron 4. Due to the large size of intron 4, we failed to clone the full sequence of exon 4, exon 5 and intron 4 into the minigene vector. Thus, we integrate exon 4, exon 5 and the sequences 200–300 bp before and after the two exons by PCR and cloned the integrated fragment into a modified pcDNA3 plasmid. The control and c.546+5G>A plasmids were transfected into HEK293T cells. After incubation for 30 hours, total RNA was extracted using an RNeasy Mini Kit (Qiagen), and cDNA was obtained with the PrimeScript RT reagent kit (Takara, Osaka, Japan). The primers for constructing the minigene vectors and for detecting alternative splice sites are listed in online supplementary table S1.
In order to determine the effect of the C133G substitution resulting from the missense mutation in REC114 on the interaction of REC114 with other proteins, plasmids containing MEI4 and REC114 were cotransfected into HEK293T cells. We adjusted the amount of WT and C133G REC114 protein level to be equal. Cell proteins were extracted in NP-40 lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.5) with 1% protease inhibitor cocktail (Bimake). Total protein was incubated with Flag-IgG Sepharose beads (Bimake) at 4°C for 3 hours on a rotating wheel. The beads were washed and then boiled with SDS loading buffer for western blotting with anti-Flag and anti-HA antibodies.
Identification of mutations in REC114
We first focused on an individual diagnosed with primary infertility from a consanguineous family (Family 1, figure 1A). The proband (II-1) was 32 years old and had planned for pregnancy for at least 5 years. She had regular menstrual cycles and normal sex hormone levels, and no physical abnormalities were observed in her infertility examination.
Individual II-1 in Family 1 underwent 10 failures of assisted reproduction from 2013 to 2016, consisting of nine ICSI cycles and one IVF cycle. A total of 23 ovulated oocytes were obtained in the 10 cycles (table 1), and there were altogether 20 mature oocytes with a first polar body (PB1). Altogether, nine oocytes were fertilised abnormally, eight of which with MPN and one with no pronucleus. The frequency of MPN is around 40%. In normal condition, frequency of 3PN is 5%–7.1%16 and zygotes with more than 3PN have been reported on rare occasions. Therefore, we believe MPN in this patient is a unique phenotype with genetic basis. The rest zygotes with 2PN cleaved normally, but after cultivation 10 were arrested at the 2–6 cell stage at day 3. Only one morphologically normal 8 cell embryo was acquired and frozen, but it failed to establish pregnancy after implantation. Individual II-1 had two younger sisters, one of whom had given birth normally while the other suffered from two miscarriages (both at about 40 days) and did not try assisted reproductive technologies.
All members in family 1 underwent whole-exome sequencing as shown in the pedigree. A recessive model of inheritance for variant filtering was applied because of the consanguineous family history, and homozygosity analysis was performed to establish the homozygous regions (Family 1, figure 1B). Based on the criteria mentioned in the methods, we identified the homozygous missense mutation c.397T>G (p.C133G) in REC114 (Gene Bank: NM_001042367.2). The parents and the sister who had given birth were all heterozygous carriers, while the sister with miscarriages had the same homozygous variant as the proband. The functional impact of this variant was assessed to be damaging by SIFT and PolyPhen-2 (table 2). This homozygous mutation did not exist in the public databases or our in-house control database consisting of more than 1000 females with normal pregnancies.
Based on our findings in Family 1, we further identified another REC114 mutation in our whole-exome sequenced cohort of 579 patients with recurrent failure of IVF/ICSI characterised by abnormalities in fertilisation or early embryonic arrest and we identified one patient carrying a rare homozygous donor splicing mutation c.546+5G>A (table 2). Homozygosity analysis showed the consanguinity of family 2 (figure 1A and B). As shown in table 1, the individual was 35 years old and had undergone three failed IVF cycles. All 18 ovulated oocytes were mature with PB1, among which 12 were successfully fertilised and were 2PN, while 6 were abnormally fertilised, including 1 with no pronucleus and five with MPN. The frequency of MPN is 27.7%. Only four viable embryos were obtained, while the other inviable embryos failed to form blastocysts, but these morphologically normal embryos failed to establish pregnancy on implantation on three separate occasions.
Distribution and conservation of mutations in REC114
REC114 consists of a large REC114-like domain covering almost all amino acid residues (figure 2A). The missense variant c.397T>G (p.C133G) is located in exon 4, which encodes peptides within the REC114-like domain, while the splicing area of c.546+5G>A comes just after exon 4 (figure 2A). It is reported that there are seven conserved short signature sequence motifs (SSMs) in the REC114 protein,17 and the C133G substitution lies in the highly conserved SSM6 motif, implying the potential impairment of the protein (figure 2B).
The influence of the mutations on REC114 in vitro
To assess the impact of the missense mutation on the function of REC114, vectors with wild-type and C133G REC114 fused with a Flag-tag were constructed. These constructs were transfected into HEK293T cells to measure their relative expression. Western blot analysis revealed significant reductions in C133G REC114 compared with wild-type (figure 3A and B), suggesting the unstable or low expression nature of the C133G REC114.
The effect of the c.546+5G>A splicing variant was studied with a minigene assay. Agarose gel electrophoresis showed different-size bands for the c.546+5G>A REC114 compared with wild-type, indicating the abnormal and multiple alternative splicing isoforms with this mutation (figure 3C). Detecting all of the alternative splicing isoforms is impossible and cDNA sequence analysis of the smallest band in the c.546+5G>A lane is shown in figure 3D as an example. A 94 bp intronic sequence was inserted between exon 4 and exon 5, causing a frameshift and premature termination. This indicated that the c.546+5G>A mutation affected normal splicing of intron 4, resulting in a range of abnormal transcripts.
Effects of the C133G substitution on the stability of MEI4
It is known that REC114 can form a complex with MEI4 and CCDC36 during homologous chromosome pairing and recombination during meiosis.14 18 To assess the effect of the C133G substitution on binding to MEI4 or CCDC36, we performed immunoprecipitation and western blot analysis in transfected HEK293T cells. As shown in online supplementary figures S1 and S2A, the C133G substitution did not influence the interaction between REC114 and MEI4 or CCDC36. Interestingly, we observed increased MEI4 protein expression in response to the increased protein level of wild-type REC114, while the level of MEI4 did not change with the increased level of C133G REC114 (figure 4A and C). The protein level of CCDC36 showed no obvious difference between coexpression with wild-type REC114 and coexpression with C133G REC114 (online supplementary figure S2B). In order to verify whether the mutation affects gene expression or affects protein degradation, we performed CHX chase assays with transfected HEK293T cells. CHX treatment led to reduced MEI4 in a time-dependent manner (figure 4B and D). In contrast to coexpression with wild-type REC114, MEI4 exhibited rapid degradation when coexpressed with mutant REC114 as well as when expressed alone. We infer from this that wild-type REC114 plays an essential role in stabilising the state of MEI4, while C133G REC114 loses such function.
In this study, we identified two novel homozygous mutations in REC114 in two independent consanguineous families with female infertility. The patients from these two families shared similar infertile phenotypes, including MPN formation during fertilisation, early embryonic arrest and failed implantation of surviving embryos. The c.546+5G>A splicing variant following exon 4 was verified in vitro to affect the normal alternative splicing of intron 4, leading to aberrant REC114 transcripts (figure 3C and D), while the C133G REC114 protein lost the ability of maintaining the stability of its partner protein MEI4 (figure 4).
REC114 is evolutionarily conserved and is known to form a complex with CCDC36 and MEI4 in mice (Mer2 and MEI4 in Saccharomyces cerevisiae). This complex plays important roles in DSB formation during homologous chromosome pairing and recombination.14 17–21 Null mutations and overexpression of REC114 both prevent DSB formation in S. cerevisiae, resulting in inviable spores,15 and Rec114-null mutant mice are defective in both spermatogenesis and oogenesis.14 Recently, Nguyen et al reported that a homozygous splicing mutation in REC114, which is located directly before exon 4 in the splicing region (purple arrowhead, figure 2A), might cause complete hydatidiform moles (CHMs) in humans, and the patient with the mutation in their paper was reported to have had one miscarriage, two spontaneous CHMs and one CHM after intrauterine sperm injection.22 She had not tried IVF or ICSI technologies, thus the state of fertilisation and embryo development were unknown. In our results, the two probands underwent several failed IVF/ICSI attempts characterised by MPN formation during fertilisation and early embryonic arrest. The younger sister in Family I (II-3, figure 1A) did not experience CHMs, and the miscarriages that occurred in early pregnancy might have been due to poor-quality embryos. These results demonstrated that patients with mutations in REC114 have phenotypic variability, including MPN formation during fertilisation, early embryo arrest, miscarriage and CHM formation. The phenotypic variability might depend on different effects of mutations similar to our observations in our previous studies.10 12 23
Homologous chromosome recombination is critical for the viability of gametes and is initiated by the formation of DSBs catalysed by SPO11.24 25 In S. cerevisiae, the REC114-MEI4-MER2 subcomplex promotes DNA cleavage by SPO11.20 In mice, MEI4, REC114 and CCDC36 form one of the mammalian DSB machineries that localises to unsynapsed chromosome axes.17 18 26 In vitro experiments indicated that the splicing variant c.546+5G>A leads to aberrant transcripts (figure 3C and D), and thus the function of REC114 is assumed to have been lost in Family 2. The C133G REC114 showed reduced protein levels of REC114 (figure 3A and B) and its partner MEI4 (figure 4). The part or complete dysfunction of REC114 may cause abnormal DSBs, resulting in abnormal chromosomal paring and recombination and further resulting in misaligned chromosome. It has been known that oocytes/embryos with incomplete chromosome segregation or diffused chromosome tend to exhibit abnormal fertilisation and early embryonic development.4 27 28 Therefore, we postulate that the C133G REC114 lost the ability to maintain the stability of MEI4, leading to loss of functional REC114-MEI4-CCDC36 complexes and abnormal DSB formation during chromosome synapsis, eventually leading to dysfunctional oocytes characterised by MPN formation during fertilisation and early embryonic arrest. In addition, it is reported that REC114 is involved in facilitating the initial chromatin binding of SPO11.25 Normal chromosome localisation of MEI4 depends on REC114 and MER2,19 and thus the decrease in REC114 might cause the abortive loading of SPO11 (or its homologs in humans) to DSB hotspot sequences or might lead to the reduced association between MEI4 and chromosomes, which might further result in abnormal oocyte meiosis and cytoplasmic maturation and subsequent phenotypes of MPN and early embryonic arrest.
In conclusion, we have identified homozygous mutations in REC114, an important gene related to DSB formation that cause MPN formation and early embryonic arrest. Our findings reveal the important roles of REC114 in human oocyte meiosis and female fertility.
We thank the patients, their families and healthy volunteers for participating in this study.
WW, JD and BC are joint first authors.
Contributors WW, JDo and BC contributed equally to this work. WW, JDo, Z Zhang, QS and LWa conceived and designed this study. WW, JDo, BC, JDu, YK, XS, JF, BL, LWu, XM and ZY collected the samples. WW, BC, XM and JF organised the medical records. WW and JDo performed the experiments. WW, JDo, BC, JDu, JM, Z Zhang, Z Zhou, ZL, QL, LH, QS and LWa analysed the data. WW and LWa wrote the manuscript. All authors reviewed and approved the manuscript.
Funding This work was supported by the National Key Research and Development Program of China (2018YFC1003800; 2017YFC1001500; 2016YFC1000600), the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81571501), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Shanghai Rising Star Program (17QA1400200), the Natural Science Foundation of Shanghai (17ZR1401900) and the Foundation of Shanghai Health and Family Planning Commission (20154Y0162).
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request. Main data relevant to the study are included in the article or uploaded as supplementary information.
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