Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.
Transcriptional activity and gene expression are critical for the development of mature, meiotically competent oocytes. Our study demonstrates that the absence of cyclin-dependent kinase 12 (CDK12) in oocytes leads to complete female sterility, as fully developed oocytes capable of completing meiosis I are absent from the ovaries. Mechanistically, CDK12 regulates RNA polymerase II activity in growing oocytes and ensures the maintenance of the physiological maternal transcriptome, which is essential for protein synthesis that drives further oocyte growth. Notably, CDK12-deficient growing oocytes exhibit a 71% reduction in transcriptional activity. Furthermore, impaired oocyte development disrupts folliculogenesis, leading to premature ovarian failure without terminal follicle maturation or ovulation. In conclusion, our findings identify CDK12 as a key master regulator of the oocyte transcriptional program and gene expression, indispensable for oocyte growth and female fertility. A schematic illustrating the effects of loss of CDK12 in mammalian oocytes on the regulation of transcription by polymerase II and the concomitant effects on translation. This disruption leads to an aberrant transcriptome and translatome, resulting in the absence of fully mature oocytes and ultimately female sterility.
- MeSH
- cyklin-dependentní kinasy * metabolismus genetika MeSH
- meióza genetika MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- RNA-polymerasa II metabolismus MeSH
- transkriptom genetika MeSH
- ženská infertilita * genetika patologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Smooth endoplasmic reticulum aggregates (SERa) are a type of dysmorphism in oocytes derived from controlled ovarian stimulation (COS). The effect of SERa on assisted reproductive techniques (ART) outcomes is debatable. Based on some evidence, SERa-positive (SERa+) oocytes cause complications including newborn demise, and compromise the outcome of the unaffected oocytes of the same cycle. While other reports demonstrated equal developmental competence between SERa + and SERa-negative (SERa-) oocytes/cycles. We conducted a prospective cross-sectional study on 315 women candidates for ART and compared the outcome among SERa+ (N = 73) and SERa- cycles (N = 217). Furthermore, for the first time, we investigated the prevalence of SERa + cycles in women with various infertility etiologies. Our results indicated that SERa + patients presented higher levels of Estradiol on the day of ovulation triggering (p = 0.02). Regarding the ART outcome, there were no differences in the number of retrieved oocytes, oocyte maturation and fertilization rates among the groups. However, the quality of the unaffected oocytes (p = 0.03), the rates of day-3 top-quality embryos (p = 0.01, and p = 0.03 for grades A and B, respectively), and clinical pregnancy (p = 0.05) in SERa + group were significantly reduced. Moreover, the prevalence of SERa + cycles gradually increased among endometriosis, POI/POR, PCOS, normal women, tubal factor, and idiopathic groups. Our study suggests that suboptimal situations such as elevated levels of Estradiol can increase the occurrence of SERa + oocytes. This suboptimal phenomenon can negatively influence the outcome of the cycle. Thus, optimization of COS, particularly in vulnerable groups such as women with idiopathic infertility may lower the SERa + cycle occurrence, improving the ART outcome.
- MeSH
- asistovaná reprodukce škodlivé účinky MeSH
- dospělí MeSH
- hladké endoplazmatické retikulum * metabolismus MeSH
- indukce ovulace * škodlivé účinky MeSH
- lidé MeSH
- oocyty * MeSH
- prospektivní studie MeSH
- průřezové studie MeSH
- těhotenství MeSH
- úhrn těhotenství na počet žen v reprodukčním věku MeSH
- ženská infertilita * patologie etiologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
STUDY QUESTION: Can oocyte functionality be assessed by observing changes in their intracytoplasmic lipid droplets (LDs) profiles? SUMMARY ANSWER: Lipid profile changes can reliably be detected in human oocytes; lipid changes are linked with maternal age and impaired developmental competence in a mouse model. WHAT IS KNOWN ALREADY: In all cellular components, lipid damage is the earliest manifestation of oxidative stress (OS), which leads to a cascade of negative consequences for organelles and DNA. Lipid damage is marked by the accumulation of LDs. We hypothesized that impaired oocyte functionality resulting from aging and associated OS could be assessed by changes in LDs profile, hereafter called lipid fingerprint (LF). STUDY DESIGN, SIZE, DURATION: To investigate if it is possible to detect differences in oocyte LF, we subjected human GV-stage oocytes to spectroscopic examinations. For this, a total of 48 oocytes derived from 26 young healthy women (under 33 years of age) with no history of infertility, enrolled in an oocyte donation program, were analyzed. Furthermore, 30 GV human oocytes from 12 women were analyzed by transmission electron microscopy (TEM). To evaluate the effect of oocytes' lipid profile changes on embryo development, a total of 52 C57BL/6 wild-type mice and 125 Gnpat+/- mice were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human oocytes were assessed by label-free cell imaging via coherent anti-Stokes Raman spectroscopy (CARS). Further confirmation of LF changes was conducted using spontaneous Raman followed by Fourier transform infrared (FTIR) spectroscopies and TEM. Additionally, to evaluate whether LF changes are associated with developmental competence, mouse oocytes and blastocysts were evaluated using TEM and the lipid dyes BODIPY and Nile Red. Mouse embryonic exosomes were evaluated using flow cytometry, FTIR and FT-Raman spectroscopies. MAIN RESULTS AND THE ROLE OF CHANCE: Here we demonstrated progressive changes in the LF of oocytes associated with the woman's age consisting of increased LDs size, area, and number. LF variations in oocytes were detectable also within individual donors. This finding makes LF assessment a promising tool to grade oocytes of the same patient, based on their quality. We next demonstrated age-associated changes in oocytes reflected by lipid peroxidation and composition changes; the accumulation of carotenoids; and alterations of structural properties of lipid bilayers. Finally, using a mouse model, we showed that LF changes in oocytes are negatively associated with the secretion of embryonic exosomes prior to implantation. Deficient exosome secretion disrupts communication between the embryo and the uterus and thus may explain recurrent implantation failures in advanced-age patients. LIMITATIONS, REASONS FOR CAUTION: Due to differences in lipid content between different species' oocytes, the developmental impact of lipid oxidation and consequent LF changes may differ across mammalian oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Our findings open the possibility to develop an innovative tool for oocyte assessment and highlight likely functional connections between oocyte LDs and embryonic exosome secretion. By recognizing the role of oocyte LF in shaping the embryo's ability to implant, our original work points to future directions of research relevant to developmental biology and reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by National Science Centre of Poland, Grants: 2021/41/B/NZ3/03507 and 2019/35/B/NZ4/03547 (to G.E.P.); 2022/44/C/NZ4/00076 (to M.F.H.) and 2019/35/N/NZ3/03213 (to Ł.G.). M.F.H. is a National Agency for Academic Exchange (NAWA) fellow (GA ULM/2019/1/00097/U/00001). K.F. is a Diamond Grant fellow (Ministry of Education and Science GA 0175/DIA/2019/28). The open-access publication of this article was funded by the Priority Research Area BioS under the program "Excellence Initiative - Research University" at the Jagiellonian University in Krakow. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.
- MeSH
- dospělí MeSH
- embryonální vývoj fyziologie MeSH
- lidé MeSH
- lipidová tělíska metabolismus MeSH
- metabolismus lipidů MeSH
- myši inbrední C57BL * MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- oxidační stres MeSH
- Ramanova spektroskopie MeSH
- stárnutí metabolismus MeSH
- transmisní elektronová mikroskopie MeSH
- věk matky MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure.
- MeSH
- alfa-globuliny metabolismus MeSH
- C-reaktivní protein metabolismus MeSH
- extracelulární matrix * metabolismus MeSH
- kumulární buňky * metabolismus MeSH
- kyselina hyaluronová * metabolismus MeSH
- lidé MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- ovariální folikul * metabolismus MeSH
- sérový amyloidový protein metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
STUDY QUESTION: Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes? SUMMARY ANSWER: Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes. WHAT IS KNOWN ALREADY: Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis. STUDY DESIGN, SIZE, DURATION: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining. LARGE SCALE DATA: The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633). LIMITATIONS, REASONS FOR CAUTION: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.
- MeSH
- choriogonadotropin farmakologie MeSH
- embryonální vývoj * fyziologie MeSH
- IVM techniky * MeSH
- kumulární buňky * metabolismus MeSH
- myši inbrední ICR * MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- proteosyntéza MeSH
- transkriptom MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- zygota metabolismus MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Cellular proteins and the mRNAs that encode them are key factors in oocyte and sperm development, and the mechanisms that regulate their translation and degradation play an important role during early embryogenesis. There is abundant evidence that expression of microRNAs (miRNAs) is crucial for embryo development and are highly involved in regulating translation during oocyte and early embryo development. MiRNAs are a group of short (18-24 nucleotides) non-coding RNA molecules that regulate post-transcriptional gene silencing. The miRNAs are secreted outside the cell by embryos during preimplantation embryo development. Understanding regulatory mechanisms involving miRNAs during gametogenesis and embryogenesis will provide insights into molecular pathways active during gamete formation and early embryo development. This review summarizes recent findings regarding multiple roles of miRNAs in molecular signaling, plus their transport during gametogenesis and embryo preimplantation.
- MeSH
- asistovaná reprodukce * MeSH
- embryonální vývoj * genetika MeSH
- gametogeneze genetika MeSH
- lidé MeSH
- mikro RNA * genetika metabolismus MeSH
- oocyty metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Mammalian oocyte development depends on the temporally controlled translation of maternal transcripts, particularly in the coordination of meiotic and early embryonic development when transcription has ceased. The translation of mRNA is regulated by various RNA-binding proteins. We show that the absence of cytoplasmic polyadenylation element-binding protein 3 (CPEB3) negatively affects female reproductive fitness. CPEB3-depleted oocytes undergo meiosis normally but experience early embryonic arrest due to a disrupted transcriptome, leading to aberrant protein expression and the subsequent failure of embryonic transcription initiation. We found that CPEB3 stabilizes a subset of mRNAs with a significantly longer 3'UTR that is enriched in its distal region with cytoplasmic polyadenylation elements. Overall, our results suggest that CPEB3 is an important maternal factor that regulates the stability and translation of a subclass of mRNAs that are essential for the initiation of embryonic transcription and thus for embryonic development.
- MeSH
- 3' nepřekládaná oblast genetika MeSH
- embryonální vývoj genetika MeSH
- meióza genetika MeSH
- messenger RNA genetika metabolismus MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- polyadenylace MeSH
- proteiny vázající RNA * metabolismus genetika MeSH
- stabilita RNA genetika MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.
- MeSH
- blastocysta účinky léků metabolismus MeSH
- fertilizace in vitro MeSH
- fibroblastový růstový faktor 2 * farmakologie MeSH
- insulinu podobný růstový faktor I * farmakologie MeSH
- kultivační média * chemie farmakologie MeSH
- leukemický inhibiční faktor * farmakologie MeSH
- oocyty MeSH
- prasata embryologie genetika MeSH
- proteomika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The fertilizing spermatozoa induce a Ca2+ oscillatory pattern, the universal hallmark of oocyte activation, in all sexually reproducing animals. Assisted reproductive technologies (ARTs) like intracytoplasmic sperm injection (ICSI) bypass the physiological pathway; however, while a normal Ca2+ release pattern occurs in some species, particularly humans, artificial activation is compulsory for ICSI-fertilized oocytes to develop in most farm animals. Unlike the normal oscillatory pattern, most artificial activation protocols induce a single Ca2+ spike, undermining proper ICSI-derived embryo development in these species. Curiously, diploid parthenogenetic embryos activated by the same treatments develop normally at high frequencies and implant upon transfer in the uterus. We hypothesized that, at least in ruminant embryos, the oscillatory calcium waves late in the first cell cycle target preferentially the paternal pronucleus and are fundamentally important for paternal nuclear remodeling. We believe that Ca2+ signaling is central to full totipotency deployment of the paternal genome. Research in this area could highlight the asymmetry between the parental genome reprogramming timing/mechanisms in early development and impact ARTs like ICSI and cloning.
- MeSH
- cytoplazma metabolismus MeSH
- fertilizace MeSH
- lidé MeSH
- oocyty metabolismus MeSH
- sperma * metabolismus MeSH
- spermie metabolismus MeSH
- vápník * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
