Nlrp5 and Tle6 expression patterns in buffalo oocytes and pre-implantation embryos.

Maternal-effect genes (MEGs) accumulate in oocytes during oogenesis and mediate the pre-implantation embryo developmental programme until activation of the zygote genome. Nlrp5 and Tle6 are required for normal pre-implantation and embryonic development. However, the precise function of these MEGs in buffalo (Bubalus bubalis) remains to be elucidated. The aim of this study was to characterize Nlrp5 and Tle6 sequences and analyse their mRNA and protein expression patterns in somatic tissues, oocytes and pre-implantation embryos of buffalo. The coding sequences of each gene were successfully cloned and characterized. Real-time quantitative reverse transcription PCR results revealed an absence of Nlrp5 or Tle6 transcripts in somatic tissues, with the exception of ovary. Expression levels of Nlrp5 and Tle6 in oocytes increased from the germinal vesicle stage to metaphase II stage and then gradually decreased during morula and blastocyst stages. Protein expression patterns were confirmed by immunofluorescence analysis. This study lays a foundation for further validation of the function of MEGs in buffalo.

A novel TLE6 mutation, c.541+1G>A, identified using whole-exome sequencing in a Chinese family with female infertility.

BACKGROUND: Oocytes have a lot of maternal RNAs and proteins, which are used by the early embryo before zygotic genome activation. Transducin-like enhancer of split 6 (TLE6) is a component of a subcortical maternal complex which plays a critical role in early embryonic development. METHODS: The patient had been diagnosed with primary infertility for 6 years and had undergone multiple failed in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycles. Genomic DNA samples were extracted from her parents' peripheral blood as well as hers. Whole-exome sequencing and Sanger validation were performed to identify candidate variants. RESULTS: We identified a novel transducin-like enhancer of split 6 (TLE6) gene mutations in the female patient with recurrent IVF/ICSI failure. The patient carried a homozygous mutation (NM_001143986.1(TLE6): c.541+1G>A) and had viable but low-quality embryos. Her parents both had heterozygous mutations at this locus. CONCLUSION: Our study expands the mutational and phenotypic spectrum of TLE6 and suggests the important role of TLE6 during embryonic development. Our findings have implications for the genetic diagnosis of female infertility with recurrent IVF/ICSI failure.

Identification of Novel Biallelic TLE6 Variants in Female Infertility With Preimplantation Embryonic Lethality.

Preimplantation embryonic lethality is a rare cause of primary female infertility. It has been reported that variants in the transducin-like enhancer of split 6 (TLE6) gene can lead to preimplantation embryonic lethality. However, the incidence of TLE6 variants in patients with preimplantation embryonic lethality is not fully understood. In this study, we identified four patients carrying novel biallelic TLE6 variants in a cohort of 28 patients with preimplantation embryonic lethality by whole-exome sequencing and bioinformatics analysis, accounting for 14.29% (4/28) of the cohort. Immunofluorescence showed that the TLE6 levels in oocytes from patients were much lower than in normal control oocytes, suggesting that the variants result in the lower expression of the TLE6 protein in oocytes. In addition, a retrospective analysis showed that the four patients underwent a total of nine failures of in vitro fertilization and intracytoplasmic sperm injection attempts, and one of them became pregnant on the first attempt using donated oocytes. Our study extends the genetic spectrum of female infertility caused by variants in TLE6 and further confirms previously reported findings that TLE6 plays an essential role in early embryonic development. In such case, oocyte donation may be the preferred treatment.

Knockout of the Transducin-Like Enhancer of Split 6 Gene Affects the Proliferation and Cell Cycle Process of Mouse Spermatogonia.

Tle6 (Transducin-like enhancer of split 6) is a member of the Tle co-repressor superfamily, which is expressed in various tissues of invertebrates and vertebrates and participates in the developmental process. However, the current research has only found that the TLE6 mutation is related to infertility, and the key regulatory mechanism of TLE6 remains to be explored. In this study, we combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 and the Tet-on system to construct mouse spermatogonia cell lines that induced TLE6 protein knockout (KO), and studied the effect of Tle6 on mouse spermatogonia proliferation and the cell cycle. The results showed that, after drug induction, the Tle6 gene in mouse spermatogonia was successfully knocked out at the genome and protein levels, and the Tle6 gene knockout efficiency was confirmed to be 87.5% with gene-cloning technology. At the same time, we also found that the mouse spermatogonia proliferated slowly after the Tle6 knockout. Using flow cytometry, we found that the cells did not undergo significant apoptosis, and the number of cells in the S phase decreased. After real-time quantity PCR (qRT-PCR) analysis, we found that the expression of cell-proliferation-related genes, CCAAT enhancer-binding protein α(C/ebp α), granulocyte-colony stimulating factor(G-csf), cyclin-dependent kinases 4(Cdk 4), Cyclin E, proliferating cell nuclear antigen(Pcna), and S-phase kinase-associated protein 2 (Skp2) was significantly reduced, which further affected cell growth. In summary, Tle6 can regulate spermatogonia cell proliferation and the cell cycle and provide a scientific basis for studying the role of TLE6 on spermatogenesis.

Subcortical maternal complex (SCMC) expression during folliculogenesis is affected by oocyte donor age in sheep.

PURPOSE: The age-associated decline in female fertility is largely ascribable to the decrease in oocyte quality. The subcortical maternal complex (SCMC) is a multiprotein complex essential for early embryogenesis and female fertility and functionally conserved across mammals. The present work evaluated expression dynamics of its components during folliculogenesis in relation to maternal age in sheep. METHODS: The expression of the SCMC components (KHDC3/FILIA, NLRP2, NLRP5/MATER, OOEP/FLOPED, PADI6, TLE6 and ZBED3) was analyzed by real-time PCR in pools of growing oocytes (GO) of different diameters (70-90 µm (S), 90-110 µm (M), or 110-130 µm (L)) derived from non-hormonally treated adult (Ad; age < 4 years), prepubertal (Pr; age 40 days), or aged ewes (age > 6 years). RESULTS: Specific expression patterns associated with donor age were observed during folliculogenesis for all genes, except ZBED3. In oocytes of adult donors, the synthesis of NLRP2, NLRP5, PADI6, and ZBED3 mRNAs was complete in S GO, while FILIA, TLE6, and OOEP were actively transcribed at this stage. Conversely, Pr GO showed active transcription of all mRNAs, except for ZBED3, during the entire window of oocyte growth. Notably, aged GO showed a completely inverse pattern, with a decrease of NLRP2, TLE6, FILIA, and PADI6 mRNA abundance during the latest stage of oocyte growth (L GO). Interestingly, MATER showed high expression variability, suggesting large inter-oocyte differences. CONCLUSION: Our study describes the SCMC expression dynamics during sheep oogenesis and reports age-specific patterns that are likely involved in the age-related decline of oocyte quality.

Expanding the genetic and phenotypic spectrum of female infertility caused by TLE6 mutations.

PURPOSE: The present study was intended to identify genetic causes of infertile patients with recurrent failure of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) attempts. METHODS: Infertile patients with recurrent IVF/ICSI failure from Shanghai Ji Ai Genetics & IVF Institute and the Ninth Hospital affiliated with Shanghai Jiao Tong University were recruited. Genomic DNA samples were extracted from their peripheral blood. Whole-exome sequencing and Sanger validation were performed to identify candidate variants. RESULTS: We identified novel transducin-like enhancer of split 6 (TLE6) gene mutations in three patients with recurrent IVF/ICSI failure. One patient carried a homozygous missense mutation (c.1226G>A; p.Arg409Gln) with subsequent fertilization failure, while the other two patients carried either a homozygous missense mutation (c.1621G>A; p.Glu541Lys) or a compound heterozygous missense mutation (c.388G>A/c.1507G>A; p.Asp130Asn/p.Val503Ile) and had viable but low-quality embryos. CONCLUSIONS: Our study expands the mutational and phenotypic spectrum of TLE6 and suggests the important role of TLE6 during embryonic development. Our findings have implications for the genetic diagnosis of female infertility with recurrent IVF/ICSI failure.

Novel mutations in genes encoding subcortical maternal complex proteins may cause human embryonic developmental arrest.

Successful human reproduction initiates from normal gamete formation, fertilization and early embryonic development. Abnormalities in any of these steps will lead to infertility. Many infertile patients undergo several failures of IVF and intracytoplasmic sperm injection (ICSI) cycles, and embryonic developmental arrest is a common phenotype in cases of recurrent failure of IVF/ICSI attempts. However, the genetic basis for this phenotype is poorly understood. The subcortical maternal complex (SCMC) genes play important roles during embryonic development, and using whole-exome sequencing novel biallelic mutations in the SCMC genes TLE6, PADI6 and KHDC3L were identified in four patients with embryonic developmental arrest. A mutation in TLE6 was found in a patient with cleaved embryos that arrested on day 3 and failed to form blastocysts. Two patients with embryos that arrested at the cleavage stage had mutations in PADI6, and a mutation in KHDC3L was found in a patient with embryos arrested at the morula stage. No mutations were identified in these genes in an additional 80 patients. These findings provide further evidence for the important roles of TLE6, PADI6 and KHDC3L in embryonic development. This work lays the foundation for the genetic diagnosis of patients with recurrent IVF/ICSI failure.

The subcortical maternal complex: multiple functions for one biological structure?

The subcortical maternal complex (SCMC) is a multiprotein complex uniquely expressed in mammalian oocytes and early embryos, essential for zygote progression beyond the first embryonic cell divisions. Similiar to other factors encoded by maternal effect genes, the physiological role of SCMC remains unclear, although recent evidence has provided important molecular insights into different possible functions. Its potential involvement in human fertility is attracting increasing attention; however, the complete story is far from being told. The present mini review provides an overview of recent findings related to the SCMC and discusses its potential physiological role/s with the aim of inspiring new directions for future research.