MOS mutation causes female infertility with large polar body oocytes

Abstract

The Moloney sarcoma oncogene (MOS) encodes a protein serine/threonine kinase and MOS is expressed at high levels in oocytes undergoing meiotic maturation. The MOS/MAPK pathway is normally required for the maintenance of microtubules and chromatin in a metaphasic state during the meiotic divisions. To determine the pathogenic genes in a female infertile patient due to large polar body oocytes, whole-exome sequencing was performed on the patient and available family members. We identified a novel homozygous missense mutation c.591T > G in MOS. Bioinformatics analysis showed that the mutation is harmful. These findings suggest that MOS mutation results in oocytes with a large polar body and poor embryonic development in patients. The MOS variant may regulate oocyte asymmetric division by MAPK/WAVE2/Arp2/3/actin signaling pathway. This will help to understand the comprehensive role of MOS in early human reproductive process and provide genetic markers for future genetic counseling for more individualized treatments.

摘要

莫洛尼肉瘤癌基因(MOS)编码蛋白丝氨酸/苏氨酸激酶, 在经历减数分裂成熟的卵母细胞中高水平地表达。MOS/MAPK途径通常需要在减数分裂过程中维持微管和染色质的变态状态需要MOS/MAPK途径。为了确定一位女性不孕患者因大极体卵母细胞而产生的致病基因。我们对该患者和现有的家庭成员进行了全外显子组测序。我们确定了一个新的同源错义突变c.591T > G的MOS。生物信息学分析表明, 该突变是有害的。这些发现表明, MOS突变导致患者的卵母细胞具有较大的极体, 胚胎发育不良。MOS变异可能通过以下方式调节卵母细胞的不对称性分裂MAPK/WAVE2/Arp2/3/actin信号通路。这将有助于了解MOS在人类早期生殖过程中的综合作用。这将有助于了解MOS在人类早期生殖过程中的全面作用, 并为未来的遗传咨询提供遗传标记, 以便进行更多的个性化治疗。更加个体化的治疗提供遗传标记。

Keywords:

• Infertility
• large polar body
• MOS
• meiosis

关键词:

• 不孕
• 大极体
• 莫洛尼肉瘤癌基因
• 减数分裂

Introduction

Proper oocyte maturation is essential for subsequent fertilization and early embryonic development, which is necessary for successful reproduction [1]. We can evaluate the morphology of human oocytes with the help of assisted reproductive technology [2]. Maturation of oocytes requires two meiosis. During meiosis, bipolar meiotic spindles form and homologous chromosomes are arranged through microtubule-organizing centers (MTOCs) [3–5]. After the spindle is assembled, with the extrusion of the first polar body, the oocyte extrudes half of its genetic material and then goes directly into the metaphase II (MII); the oocyte arrests at MII until fertilization [6, 7]. Unlike the symmetric cell division that occurs in mitosis, mammalian oocyte meiosis is characterized by asymmetric division, producing a highly polarized and large metaphase II-arrested oocyte and a small polar body [8]. The failure of asymmetric oocyte division results in the production of large polar bodies in oocytes, usually due to low oocyte quality or aging after ovulation [9]. In mice, some studies have identified abnormal gene expression leading to a meiotic spindle dysfunction phenotype; however, the genetic etiology of human oocytes remains largely unknown.

MOS protein is a serine/threonine kinase [10]. It was not detected in immature mouse oocytes, and after germinal vesicle breakdown, the translation of the maternal MOS mRNA was activated dependent on cytoplasmic polyadenylation [11]. Recently, mitogen-activated protein kinase (MAPK) has been highly activated throughout the oocyte maturation [12] and has been identified as one of the main downstream targets of MOS [13]. MOS and MAPK play a very important role in microtubule organization, and studies have found that MOS is partly located in the mitotic spindle [14]. Observations show that the MOS/MAPK pathway is usually required to maintain microtubules and chromatin in the metaphasic state during meiosis [15].

Mutations in TUBB8 and PATL2 have so far been identified to be responsible for the phenotype of large polar body in oocytes [16, 17]. In this study, we identified a novel homozygous missense mutation c.591T > G in MOS. We showed that mutation in the MOS gene led to the formation of large polar bodies in human oocytes. This will help to understand the overall role of MOS in the early human reproductive process and provide some candidate genetic markers for future individualized treatments. We also summarized the recently discovered factors that regulate the migration and anchoring of meiotic spindles, the formation of contractile ring, and the process of polar body extrusion, which may explore the formation mechanism of large polar bodies.

Description of the case

All samples from donors were obtained with informed consent. This study was approved by the Ethics Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University.

Clinical characterization

The patient (II-1) was diagnosed with primary infertility at age 25 after 5 years of attempts without contraception and was not pregnant. She was slender (167 cm; 45 kg), normal physically and mentally. The anatomical structure of the reproductive tract was normal, and she had no potential diseases such as infectious diseases and immune dysfunction. She was not from a consanguineous family.

She had regular menstrual cycles and normal concentrations of sex hormone. Blood samples of the patient revealed endocrine normality, with normal levels of follicle-stimulating hormone (FSH) (6.87 mIU/mL), serum luteinizing hormone (LH) (7.47mIU/mL), progesterone (P) (0.655 ng/mL), estradiol (E2) (35.79 pg/mL), testosterone (T) (0.380 ng/mL), and antimullerian hormone (AMH) (2.053 ng/mL). Additional blood testing included prolactin (PRL) (20.91 ng/mL), thyroid stimulating hormone (TSH) (2.49mIU/L), free thyroxine (FT4) (13.96 pmol/L), and negative antinuclear antibody (ANA) (1:40>), all within normal ranges.

She underwent three in vitro fertilization attempts

In the first cycle, the patient used the long protocol and down-regulated with GnRH agonist triptorelin (Jinlei, Changchun, China), then administered with follicle-stimulating hormone (Jinsaiheng, Changchun, China) daily on the basis of patient’s age and ovarian reserve. Ovarian response was monitored with transvaginal ultrasound (TV-USG) and serum estradiol level. To induce the oocyte maturation, recombinant human chorionic gonadotrophin (Merck Serono, Geneva, Switzerland) or hCG (Lizhu Group Co., Ltd, Zhuhai, China) was injected when ≥3 follicles reached the diameter of 16–18 mm. After hCG injection, oocytes were collected at 36–38 h and cultured at incubators until insemination. In the second cycle, the patient still used a long protocol to promote ovulation and the patient used a mini-stimulation protocol during the third cycle.

Twelve oocytes were retrieved by TV-USG guided aspiration. After 4 h, granule cells were removed and only three oocytes were with two normal polar bodies (Figure 1A). There were five oocytes with large polar bodies (Figure 1C,E) and one was at the metaphase-II stage and the remaining three oocytes were immature. The following day, three oocytes showed normal fertilization with two pronuclei (2PN, Figure 1B). All five large polar bodies oocytes showed abnormal fertilization, three of which were fertilized with one pronuclei (1PN, Figure 1D), two of which had already undergone cleavage (Figure 1F). All embryos were with unequal-sized blastomeres and many fragments on day 3. Five embryos were cultured until day 6, with no blastocysts formed. In the second cycle, eight oocytes were retrieved and the patient had a similar phenotype that six oocytes with large polar bodies apart from one 2pb oocyte and one immature oocyte. In the third cycle, three of the five oocytes had large polar bodies. Both cycles of embryonic development were similar to the first cycle, ultimately without blastocyst formation. The oocytes and embryos outcomes in all the three in vitro fertilization (IVF)/ICSI attempts for the patient were shown in Table 1.

Figure 1. Clinical characteristics of oocytes retrieved from the infertile patient. The day of oocyte retrieval was defined as day 0. Scale bar = 20 µm. (A) The morphology of a normal mature PB2 oocyte. (B) The morphology of a normal fertilized oocyte with two pronuclei (2PN). (C) The morphology of an abnormal mature oocyte with multiple large-polar bodies. (D) The morphology of an abnormal fertilized oocyte with one pronuclei (1PN). (E) The morphology of an abnormal mature oocyte with a large-polar body. (F) The morphology of an oocyte that has undergone cleavage 18 h after fertilization

Table 1. The oocytes and embryos outcomes in all the three IVF/ICSI attempts for the patient.

Attempt	IVF/ICSI	Retrieved oocytes	MII oocytes	Abnormal morphology oocytes	Fertilized outcomes (2PN + 1PN + MPN + 0PN)	Cleaved/Fragmented embryo	Blastocysts
1	IVF	12	10	5	4 + 3 + 0 + 3	9/9	0
2	IVF + ICSI	8	7(4 discarded)	6	0 + 1 + 0 + 2	3/3	0
3	IVF	6	5	3	1 + 1 + 0 + 3	5/4	0

MII: Metaphase II; MPN: multi-pronucleus; PN: pronucleus.

Identification of mutation in MOS

To investigate the genetic cause of the infertility, we performed whole-exome sequence (WES) on peripheral blood samples from the affected individuals. We selected candidate variants with the following criteria: (1) had a under 1% frequency in public databases (such as the genome Aggregation Database (gnomAD) and the Exome Aggregation Consortium (ExAC) Browser), (2) variants located in exon or splice site, (3) mRNAs/proteins that were highly expressed or specifically expressed in oocytes. The variant score and functional prediction were assessed by Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (Polyphen2). The three-dimensional structure of wild-type MOS (NM_005372.1) was predicted using the Swiss Model web server. We identified mutation in MOS that may be responsible for the phenotypes of the infertile female. Whole-exome sequence analysis of the family members (Figure 2A I-1, I-2, II-1) implicated a homozygous missense mutation (c.591T > G: p.I197M) in the coding region of MOS. Sanger sequencing further confirmed that the patient (Figure 2B II-1) carried a homozygous missense mutation (c.591T > G: p.I197M) in the coding region of MOS in exon 1. This mutation was inherited from her parents, who showed a heterozygous mutation in MOS. The analysis of the reading frame of the gene suggested that the missense mutation in MOS caused an amino acid substitution of the encoded protein, in which the 197th isoleucine was replaced by methionine, resulting in an abnormal protein structure (Figure 2C). According to the analysis of the conserved domains of the NCBI database, the 197th isoleucine of the protein is highly conserved in nine species (Figure 2D).The family followed a recessive inheritance pattern. The variant p.I197M have not been reported in the gnomAD exome database and 1000 Genome database (Table 2). Based on the three-dimensional (3D) structures of the MOS protein used to assess effect of the missense variant, the variant had no obvious effect on the protein structure but the hydrogen bond of 197 amino acids had changed (Figure 2E), which may affect the function of the mutant protein. In addition, the variant was predicted pathogenic by SFIT or Polyphen with probably damaging (Table 2). Collectively, these results indicated that the MOS variant is likely to be pathogenic.

Figure 2. Identification of MOS mutation in the family of the infertile patient. (A) Pedigree of the family. Squares indicate male family members, circles indicate female family members, solid symbol indicates affected members, open symbols indicate unaffected family members, and equal sign denotes no offspring. (B) Sanger sequence analysis in the coding region of MOS in family members is implicated with a homozygous missense mutation. (C) The missense mutation in MOS causes an amino acid substitution of the encoded protein. (D) Conservation analysis of altered amino acids among nine species. (E) MOS variant encoding amino acid disrupted the ion pairs formed by wild-type MOS protein.

Table 2. The hazard analysis of the MOS variant observed in the family.

Genomic position On chr8(bp)	cDNA change	Protein change	Mutation type	Genotype	gnomAD allele frequency	1000 genome	Polyphen score	Polyphen	SIFT score	SIFT
56113392	c.591T > G	p.I197M	missense	homozygous	N/A	N/A	0.999	D	0	D

D: Deleterious; N/A: not available.

Discussion

In this study, we found a novel homozygous missense mutation (c.591T > G: p.I197M) in MOS leading to meiosis defects in human oocyte. This mutation affects oocyte maturation, resulting in symmetrical division and abnormal spindle and eventually leading to morphological defects of large polar body.

MOS plays an extremely important role in oocyte meiotic maturation, which activates MAP kinase and is involved in microtubule organization in oocytes. The MOS^-/-oocytes undergo the first meiosis, usually with symmetrical division or an abnormally large polar body [18, 19]. The phenotype of mutation p.I197M in MOS is similar to that of MOS ^-/-oocytes, resulting in a decreased number of MOS, because of the decline in MOS mRNA. Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation [20, 21]. During asymmetric division, the spindle is first located at the center of the oocytes and then migrated to the cortex to produce a small polar body with a diameter of 15–20 μm and the remaining larger capacity secondary ocytes. In phenotypic, the first meiosis of MOS^-/-oocytes is usually similar to mitotic division or producing an abnormally large polar body. In these oocytes, the shape of the spindle changes, and the spindle is not translocated to the cortex, leading to a change in the cleavage plane [18]. The reduction in zinc can reduce the expression of MOS, leading to similar phenotypes, including the formation of large polar bodies [22]. ADP-ribosylation factor 1(Arf1) which is a small GTPase seems to be necessary for spindle morphology, possibly by regulating MAPK, because the spindle is prolonged and MAPK expression is reduced in Arf1 mutation oocytes [23]. Inhibition of MEK1/2 through U0126 interferes with the asymmetric division of the oocytes, leading to the formation of large polar bodies [24].

The MOS/MAPK signaling pathway may regulate the asymmetric division of the oocytes to influence normal polar body formation. The destruction of the MOS/MAPK pathway in mouse oocytes usually causes elongation of the spindle, which may inhibit the spindle movement toward the cortex, as the poles of the elongated spindle are already in contact with the cortex [25]. This suggests that the contact between the spindle poles and the cortex may be a signal of the termination of the spindle migration. While the elongation of the spindle seems sufficient to affect asymmetric division, actin may also be involved in the process. Actin regulates the migration of chromosomes and spindles and the formation of contraction rings during polar body extrusion, so actin may be a key molecule in asymmetrical division of oocytes [26]. The actin-associated protein (ARP)2/3 complex is a major actin nucleation factor consisting of two core components, ARP2 and ARP3, which may be involved in the asymmetric division of oocytes. The ARP2/3 complex binds to one side of the existing actin filament and initiates the new filament assembly [27]. In many species, inhibition of ARP2/3 complex through RNA interference or inhibitory antibodies destroys the establishment of cell migration, adhesion and cell polarity during mitosis [27]. The involvement of the ARP2/3 complex in oocyte maturation may begin to promote spindle movement shortly after the meiotic spindle forms in the center of the oocyte, as live cell imaging showed that when the Arp2/3 complex was interfered by specific inhibitor CK666 or RNAi, the spindle cannot migrate to the oocyte cortex and arrested in the oocyte center [28, 29]. The ARP2/3 complex involved in the formation of new branched actin filaments relies on interactions with nucleation promotion factors (NPFs) such as WAVE1-3, WASP, N-WASP, which are necessary for the activation of the ARP2/3 complex [30]. MOS-MAPK activates the actin nucleation regulator WAVE2 during actin-mediated lamellipodia protrusion [31]. In this way, the MAPK pathway is associated with the actin nucleation factors. Therefore, MOS may modulate oocyte asymmetry through the WAVE2/ARP2/3/actin signaling pathway and influence the size of the polar body.

MOS variant may also cause maternal mRNA decay disorder inducing the meiotic defect. In vertebrates, fully grown oocytes are arrested at the diplotene stage of meiosis I, and contain a large amount of maternal mRNAs that are translationally dormant [32]. Upon meiotic maturation, many maternal mRNAs are translationally activated followed by degradation during maternal to zygotic transition (MZT) [33]. The abnormality of maternal mRNA degradation is associated with human early embryonic arrest and embryonic genome activation failure [34]. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a key oocyte factor that regulates translation of maternal mRNA encoding B-cell translocation gene 4 (Btg4), a MZT licensing factor [35]. ERK triggers the phosphorylation and degradation of CPEB1 at an early stage of oocyte meiotic resumption [36, 37]. Furthermore, ERK1/2 increases maternal mRNA translation by phosphorylating the poly(A) polymerase at three sites [38]. As MOS is a maternal-effect gene that is transiently translated during oocyte maturation and has been reported to regulate the translation of some mRNAs [39]. We suspected that MOS/ERK signal cascade may participate in mRNA decay during human oocytes maturation.

PAT1 homolog 2(PATL2) encodes an RNA-binding protein that is an inhibitor participated in the translational regulation of maternal mRNAs during oocyte maturation. The missense mutation c.649 T > A p.Tyr217Asn in PATL2 disrupted oocyte maturation and morphological defects of large polar body were observed. The mutation p.Tyr217Asn in PATL2 inhibits the MOS translation, thereby damaging MAPK signaling pathway and oocyte meiosis [17]. Biallelic mutations in PATL2 leads to female infertility, which is characterized by oocyte maturation arrest and Pb1 oocytes with large polar body [40]. Tubulin class 8VIII (TUBB8) is a specific β-tubulin subtype that is expressed mainly in primate oocytes and early embryos and has been identified as a pathogenic gene for human oocyte maturation arrest. TUBB8 variation also causes oocytes with a large polar body [16, 41].

Conclusions

We identified a novel homozygous missense mutation c.591T > G in MOS, resulting in patient’s oocytes with a large polar body and poor embryonic development. The MOS variants may regulate oocyte asymmetric division by MAPK/WAVE2/Arp2/3/actin signaling pathway to disrupt spindle morphology and inhibit spindle movement to the cortex. Due to the scarcity of human oocytes available for experimental purposes, the underlying mechanism needs to be further clarified in more oocytes from patients or studied in transgenic mice with the corresponding MOS variants. Future studies are needed to explore the formation mechanism of large polar bodies by constructing MOS p.I197M transgenic mice.

Author’s contribution

Each author has met the requirements for authorship. X.Y.L. and G.Z.J. designed and wrote the main manuscript text. Acquisition of data was made by H.Y.L. and L.L.C. Analysis and interpretation of data were made by J.H.X. Bioinformatics analysis of the mutation was made by Z.L.D. All authors have provided final approval for the submission of this manuscript.

Ethics approval and consent to participate

The present study was approved by the institutional ethics committee review board of the Affiliated Yantai Yuhuangding Hospital of Qingdao University, Shandong, China. The patient gave written informed consent prior to the start of the study.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The author(s) reported there is no funding associated with the work featured in this article.