![Anatomy of the mature (antral) ovarian follicle. Source: Erickson, 1983 [1]](https://www.researchgate.net/profile/Marie-Christou-Kent/publication/349074151/figure/fig2/AS:988116743630848@1612596798227/Anatomy-of-the-mature-antral-ovarian-follicle-Source-Erickson-1983-1_Q320.jpg)
![La dérégulation du transcriptome des ovocytes VG de souris PATL2 -/-et ses conséquences morphologiques et développementales. Comparaison des ovocytes de souris PATL2 -/-et sauvages : (1) diminution de l'abondance des ARN messagers (ARNm) mesurée par analyse transcriptomique (micropuce), (2) différence de morphologie analysée par immunofluorescence (marquage de la β-tubuline et de l'ADN, en rouge et bleu respectivement) et (3) détermination des taux de développement obtenus par fécondation in vitro. (VG : vésicule germinale, MII : métaphase II). Ovocyte : en beige ; zone pellucide : en gris ; vésicule germinale/globules polaires : en marron ; fuseau méiotique/microtubules : en rouge ; et chromosomes : en bleu (figure adaptée de [10]).](https://www.researchgate.net/profile/Marie-Christou-Kent/publication/349074151/figure/fig47/AS:988116752019479@1612596800831/La-deregulation-du-transcriptome-des-ovocytes-VG-de-souris-PATL2--et-ses-consequences_Q320.jpg)
Content uploaded by Marie Christou-Kent
Author content
All content in this area was uploaded by Marie Christou-Kent on Feb 15, 2021
Content may be subject to copyright.
A preview of the PDF is not available
Characterising human gametogenesis arrest from gene to protein
Abstract and Figures
Infertility is considered a global public health issue since it affects more than 50 million couples worldwide. Current assisted reproductive technologies (ARTs) have minimal requirements for gametes that are competent for fertilisation and subsequent embryo development. In cases where genetic abnormalities lead to arrested gametogenesis and the production of immature, defective or degraded gametes, treatment is not usually possible. Identifying the molecular causes of these types of infertility is crucial for developing new strategies to treat affected couples. Moreover, these patients represent a unique opportunity to discover new actors of oogenesis and spermatogenesis and to decipher the molecular pathways involved in the production of competent gametes.Genetic analysis of cohorts of infertile patients with shared ancestry can allow the identification of inherited genetic variants as possible causal factors. Using whole exome sequencing, we identified a homozygous pathogenic variant of the gene PATL2 in a cohort of patients with a phenotype of arrested oogenesis due to oocyte meiotic deficiency (OMD). OMD is a rare pathology characterised by the recurrent ovulation of immature oocytes. PATL2 encodes an oocyte ribonucleoprotein whose amphibian orthologue had been shown to be involved in oocyte translational control and whose function in mammals was poorly characterised. We also identified a pathogenic variant of the gene SPINK2 in a familial case of azoospermia. SPINK2 encodes a serine protease inhibitor essential for the neutralisation of acrosin activity during sperm acrosome formation.We showed, through generation of Patl2 and Spink2 knockout (KO) mice and Patl2 tagged mice (the latter using CRISPR-Cas9), that both corresponding proteins play essential respective roles in gametogenesis. We demonstrated that Patl2 is strongly expressed in growing mouse oocytes and that its absence leads to the dysregulation of numerous transcripts necessary for oocyte growth, meiotic maturation and preimplantation embryo development. This was accompanied by a phenotype of subfertility in KO females in natural mating, a large proportion of ovulated oocytes lacking a polar body (immature) and/or displaying spindle assembly defects in immunostaining, and high rate of oocytes with an aberrant response to fertilisation in IVF experiments. In Spink2 KO mice, we demonstrated that absence of Spink2 protein, which is located in the acrosome of maturing and mature spermatozoa, leads to arrested spermiogenesis and azoospermia due to autophagy at the round-spermatid stage. This is plausibly due to aberrant acrosin activity in the absence of its inhibitor, corroborated by fragmentation of the Golgi and absence of the acrosome in immunostaining.We have thus characterised two genetic subtypes of human infertility associated with mutation of these two genes. In doing so, we have furthered our understanding of the respective roles of these crucial actors of mammalian gametogenesis, potentially paving the way for improvement of current ARTs and development of new, personalised therapies.
Figures - uploaded by Marie Christou-Kent
Author content
All figure content in this area was uploaded by Marie Christou-Kent
Content may be subject to copyright.
ResearchGate has not been able to resolve any citations for this publication.
During oocyte maturation, changes in gene expression depend exclusively on translation and degradation of maternal mRNAs rather than transcription. Execution of this translation program is essential for assembling the molecular machinery required for meiotic progression, fertilization, and embryo development. With the present study, we used a RiboTag/RNA-Seq approach to explore the timing of maternal mRNA translation in quiescent oocytes as well as in oocytes progressing through the first meiotic division. This genome-wide analysis reveals a global switch in maternal mRNA translation coinciding with oocyte re-entry into the meiotic cell cycle. Messenger RNAs whose translation is highly active in quiescent oocytes invariably become repressed during meiotic re-entry, whereas transcripts repressed in quiescent oocytes become activated. Experimentally, we have defined the exact timing of the switch and the repressive function of CPE elements, and identified a novel role for CPEB1 in maintaining constitutive translation of a large group of maternal mRNAs during maturation.
Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.
Acute myeloid leukemia (AML) has a high mortality rate and its clinical management remains challenging. The aim of the present study was to identify the hub genes involved in AML. In order to do so, the gene expression data of the GSE9476 database, including 26 AML and 10 normal samples, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were then identified via bioinformatics analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed on DEGs. Furthermore, the most upregulated genes were selected for further investigation in the Oncomine, gene expression profiling interactive analysis and UALCAN datasets. In total, 1,744 upregulated and 1,956 downregulated genes were detected. The GO and KEGG results revealed that upregulated genes were enriched in metabolic processes, while downregulated genes were associated with the immune response. Serine protease inhibitor Kazal-type 2 (SPINK2) ranked first among all the upregulated genes and was regarded as a hub gene in the development of AML. The overexpression of SPINK2 was validated in 12 patients with AML from the Linyi Central Hospital and in data from the Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) databases. Furthermore, the UALCAN and GEPIA datasets demonstrated that patients with high SPINK2 levels had shorter survival times. In conclusion, the results from the present study revealed that the SPINK2 gene was upregulated in patients with AML and that elevated SPINK2 expression was associated with poor outcomes in these patients.
The Musashi family of mRNA translational regulators control both physiological and pathological stem cell self-renewal primarily by repressing target mRNAs that promote differentiation. In response to differentiation cues, Musashi can switch from a repressor to an activator of target mRNA translation. However, the molecular events that distinguish Musashi-mediated translational activation from repression are not understood. We have previously reported that Musashi function is required for the maturation of Xenopus oocytes, and specifically for translational activation of specific dormant maternal mRNAs. Here, we employed mass spectrometry to identify cellular factors necessary for Musashi-dependent mRNA translational activation. We report that Musashi1 needs to associate with the embryonic poly(A) binding protein (ePABP) or the canonical somatic cell poly(A) binding protein PABPC1 for activation of Musashi target mRNA translation. Co-immunoprecipitation studies demonstrated an increased Musashi1 interaction with ePABP during oocyte maturation. Attenuation of endogenous ePABP activity severely compromised Musashi function, preventing downstream signaling and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP attenuated oocytes. Consistent with these Xenopus findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Since association of Musashi1 with poly(A) binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly[A] binding protein family members in response to extracellular cues that control cell fate.
Background
Male infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) is a genetically heterogeneous disorder. Previous studies revealed several MMAF-associated genes, which account for approximately 60% of human MMAF cases. The pathogenic mechanisms of MMAF remain to be illuminated.
Methods and results
We conducted genetic analyses using whole-exome sequencing in 50 Han Chinese probands with MMAF. Two homozygous stop-gain variants (c.910C>T (p.Arg304*) and c.3400delA (p.Ile1134Serfs*13)) of the SPEF2 ( sperm flagellar 2 ) gene were identified in two unrelated consanguineous families. Consistently, an Iranian subject from another cohort also carried a homozygous SPEF2 stop-gain variant (c.3240delT (p.Phe1080Leufs*2)). All these variants affected the long SPEF2 transcripts that are expressed in the testis and encode the IFT20 (intraflagellar transport 20) binding domain, important for sperm tail development. Notably, previous animal studies reported spontaneous mutations of SPEF2 causing sperm tail defects in bulls and pigs. Our further functional studies using immunofluorescence assays showed the absence or a remarkably reduced staining of SPEF2 and of the MMAF-associated CFAP69 protein in the spermatozoa from SPEF2 -affected subjects.
Conclusions
We identified SPEF2 as a novel gene for human MMAF across the populations. Functional analyses suggested that the deficiency of SPEF2 in the mutated subjects could alter the localisation of other axonemal proteins.
Endometriosis is an enigmatic disease that could start at birth. Its pathogenesis is supported by different theories. Accumulating facts relate it to a multigenic disorder. In this review of recent publications, the principal symptoms of the disease, pain and infertility, as well as its pathogenesis, diagnosis, and classification will be addressed. Endometriosis presents three main variants: superficial peritoneal disease, deep infiltrating endometriosis, and ovarian endometriomas. The management of the disease, surgery, and medical and alternative therapies will be discussed. Special reference will be made to the quality of surgery and how to understand patients with endometriosis and endometriosis.
Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression, and mRNA decay. A critical step in all 5′-3′ mRNA decay pathways is removal of the 5′ cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5′-3′ decay, the Pat1/Lsm1-7 complex engages mRNA at the 3′ end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5′ end through an unknown mechanism. We reconstitute Pat1 with 5′ and 3′ decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Second, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping messenger ribonucleoprotein (mRNP) that promotes 5′-3′ mRNA degradation.
Post‐transcriptional regulation of gene expression is largely achieved at the level of splicing in the nucleus, and translation and mRNA decay in the cytosol. While the regulation may be global, through the direct inhibition of central factors, such as the spliceosome, translation initiation factors and mRNA decay enzymes, in many instances transcripts bearing specific sequences or particular features are regulated by RNA‐binding factors which mobilize or impede recruitment of these machineries. This review focuses on the Pat1 family of RNA‐binding proteins, conserved from yeast to man, that enhance the removal of the 5′ cap by the decapping enzyme Dcp1/2, leading to mRNA decay and also have roles in translational repression. Like Dcp1/2, other decapping coactivators, including DDX6 and Edc3, and translational repressor proteins, Pat1 proteins are enriched in cytoplasmic P‐bodies, which have a principal role in mRNA storage. They also concentrate in nuclear Cajal‐bodies and splicing speckles and in man, impact splice site choice in some pre‐mRNAs. Pivotal to these functions is the association of Pat1 proteins with distinct heptameric Lsm complexes: the cytosolic Pat1/Lsm1‐7 complex mediates mRNA decay and the nuclear Pat1/Lsm2‐8 complex alternative splicing. This dual role of human Pat1b illustrates the power of paralogous complexes to impact distinct processes in separate compartments. The review highlights our recent findings that Pat1b mediates the decay of AU‐rich mRNAs, which are particularly enriched in P‐bodies, unlike the decapping activator DDX6, which acts on GC‐rich mRNAs, that tend to be excluded from P‐bodies, and discuss the implications for mRNA decay pathways.
This article is categorized under:
• RNA Turnover and Surveillance > Regulation of RNA Stability
• RNRNA Processing > Splicing Regulation/Alternative Splicing
• Translation > Translation Regulation
Technological and other advances over the past decades have led to the discovery of thousands of gene–disease associations for autosomal and X-linked recessive Mendelian disorders. Combined with recent improvements in assessing individual variants in each human genome, these developments offer the possibility of testing populations for all known severe recessive genetic disorders. Past experience has provided the framework for expanded carrier screening, but many challenges remain regarding which disorders to include, how to interpret variants and how to incorporate newly discovered gene–disease links into existing screening programmes. This Review discusses the current status of expanded carrier screening, including existing recommendations and limitations. The author reviews the framework that is needed for successful comprehensive carrier screening programmes for all autosomal recessive disorders in various populations.
Oocyte maturation arrest results in primary female infertility, but the genetic etiology of this phenotype remains largely unknown. Previously, we and other groups have reported that biallelic mutations in PATL2 are mainly responsible for human oocyte germinal vesicle-stage arrest and that the specific phenotype varies for different mutations. Here, we identified four novel missense mutations (p.V260M, p.Q300*, p.T425P, and p.D293Y), a novel frameshift mutation (p.N239Tfs*9), and a reported splicing mutation (p.R75Vfs*21) in PATL2 in seven affected individuals from five unrelated families, showing a multiplicity of phenotypes in oocyte maturation arrest, fertilization failure, or embryonic developmental arrest, which further expands the mutational and phenotypic spectrum in patients with PALTL2 mutations. This work further indicates the critical role of PATL2 in oocyte maturation and early embryo development and will provide a basis for pursuing the determination of genetic variation in PALT2 as an additional criterion for evaluating the quality of oocytes and embryos for assisted reproduction techniques.