Úspěch léčby metodami asistované reprodukce závisí zejména na kvalitě získaných oocytů. Luteinizační hormon významným způsobem ovlivňuje fyziologické procesy v ovariálním folikulu a samotném oocytu. Nedostatečný účinek luteinizačního hormonu v konečné fázi zrání folikulu významně snižuje podíl kvalitních oocytů získaných při použití metod asistované reprodukce. U žen se sníženou citlivostí k luteinizačnímu hormonu nebo s nedostatečnou produkcí tohoto hormonu je vhodné hormon substituovat podáním rekombinantního preparátu.
The success of the assisted reproduction treatment depends mostly on the quality of obtained oocytes. Luteinizing hormone significantly impacts physiological processes in the ovarian follicle and in the oocyte oneself. Insufficient effect of the luteinizing hormone in the final phase of follicle maturation significantly reduces ratio of high-quality oocytes acquired using assisted reproduction. Hormone substitution by the recombinant preparation is recommended by the women with luteinizing hormone lowered sensitivity or production.
There is increasing evidence that bisphenols BPS and BPF, which are analogues of BPA, have deleterious effects on reproduction even at extremely low doses. Indirect exposure via the maternal route (i.e. across the placenta and/or by breastfeeding) is underestimated, although it can be assumed to be a cause of idiopathic female infertility. Therefore, we hypothesised the deleterious effects of exposure to BPA analogues during breastfeeding on the ovarian and oocyte quality of offspring. A 15-day exposure period of pups was designed, whilst nursing dams (N ≥ 6 per experimental group) were treated via drinking water with a low (0.2 ng/g body weight/day) or moderate (20 ng/g body weight/day) dose of bisphenol, mimicking real exposure in humans. Thereafter, female pups were bred to 60 days and oocytes were collected. Immature oocytes were used in the in-vitro maturation assay; alternatively, in-vivo-matured oocytes were isolated and used for parthenogenetic activation. Both in-vitro- and in-vivo-matured oocytes were subjected to immunostaining of spindle microtubules (α-tubulin) and demethylation of histone H3 on the lysine K27 (H3K27me2) residue. Although very low doses of both BPS and BPF did not affect the quality of ovarian histology, spindle formation and epigenetic signs were affected. Notably, in-vitro-matured oocytes were significantly sensitive to both doses of BPS and BPF. Although no significant differences in spindle-chromatin quality were identified in ovulated and in-vivo-matured oocytes, developmental competence was significantly damaged. Taken together, our mouse model provides evidence that bisphenol analogues represent a risk to human reproduction, possibly leading to idiopathic infertility in women.
- MeSH
- aparát dělícího vřeténka účinky léků metabolismus patologie MeSH
- benzhydrylové sloučeniny metabolismus toxicita MeSH
- epigeneze genetická MeSH
- fenoly metabolismus toxicita MeSH
- fertilita účinky léků MeSH
- hodnocení rizik MeSH
- IVM techniky MeSH
- kojená zvířata MeSH
- laktace metabolismus MeSH
- matka - expozice noxám MeSH
- mléko metabolismus MeSH
- myši inbrední ICR MeSH
- oocyty účinky léků metabolismus patologie MeSH
- ovariální rezerva účinky léků MeSH
- ovarium účinky léků metabolismus patofyziologie MeSH
- sulfony metabolismus toxicita MeSH
- těhotenství MeSH
- vývojová regulace genové exprese MeSH
- ženská infertilita chemicky indukované metabolismus patologie patofyziologie MeSH
- zvířata MeSH
- Check Tag
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.
- MeSH
- cytoplazma účinky léků ultrastruktura MeSH
- dospělí MeSH
- endoplazmatické retikulum účinky léků ultrastruktura MeSH
- folikuly stimulující hormon farmakologie MeSH
- indukce ovulace MeSH
- lidé MeSH
- mitochondrie účinky léků ultrastruktura MeSH
- mladý dospělý MeSH
- oocyty účinky léků ultrastruktura MeSH
- oogeneze účinky léků fyziologie MeSH
- segregace chromozomů MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mladý dospělý MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bisphenol S (BPS) is widely used to replace the known endocrine disruptor BPA in various products. We evaluated the effect of acute in vivo BPS exposure on oocyte quality, simulating the oral route of exposure via oral gavage. Eight-week-old ICR female mice (N = 15 per experimental group) were exposed to vehicle or BPS1-BPS4 (0.001, 0.1, 10, and 100 ng BPS x g bw-1 day-1, respectively) for seven days. Oocytes were isolated and matured in vitro. We observed that BPS exposure increased aberrant spindle formation in mature oocytes and induced DNA damage. Moreover, BPS3 significantly increased the chromatin repressive marks 5-methyl cytosine (5meC) and H3K27me2 in immature oocytes. In the BPS2 group, the increase in 5meC occurred during oocyte maturation. Transcriptome analysis revealed differential expression of early embryonic development transcripts in BPS2-exposed oocytes. These findings indicate that the biological effect of BPS is non-monotonic, affecting oocyte quality even at concentrations that are orders of magnitude below those measured in humans.
- MeSH
- embryonální vývoj účinky léků MeSH
- fenoly toxicita MeSH
- metylace DNA účinky léků MeSH
- myši inbrední ICR MeSH
- oocyty účinky léků metabolismus MeSH
- poškození DNA MeSH
- stanovení celkové genové exprese MeSH
- sulfony toxicita MeSH
- těhotenství MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Culture media used in assisted reproduction are commonly supplemented with gonadotropin hormones to support the nuclear and cytoplasmic maturation of in vitro matured oocytes. However, the effect of gonadotropins on protein synthesis in oocytes is yet to be fully understood. As published data have previously documented a positive in vitro effect of follicle-stimulating hormone (FSH) on cytoplasmic maturation, we exposed mouse denuded oocytes to FSH in order to evaluate the changes in global protein synthesis. We found that dose-dependent administration of FSH resulted in a decrease of methionine incorporation into de novo synthesized proteins in denuded mouse oocytes and oocytes cultured in cumulus-oocyte complexes. Similarly, FSH influenced methionine incorporation in additional mammalian species including human. Furthermore, we showed the expression of FSH-receptor protein in oocytes. We found that major translational regulators were not affected by FSH treatment; however, the amino acid uptake became impaired. We propose that the effect of FSH treatment on amino acid uptake is influenced by FSH receptor with the effect on oocyte metabolism and physiology.
- MeSH
- aminokyseliny metabolismus MeSH
- folikuly stimulující hormon farmakologie MeSH
- IVM techniky metody MeSH
- kultivační média chemie farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- myši MeSH
- oocyty účinky léků metabolismus MeSH
- prasata MeSH
- savci MeSH
- skot MeSH
- stadium rýhování vajíčka účinky léků metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.
- MeSH
- anafázi podporující komplex metabolismus MeSH
- embryo savčí účinky léků metabolismus MeSH
- embryonální vývoj účinky léků MeSH
- kontrolní body M fáze buněčného cyklu * MeSH
- myši MeSH
- oocyty účinky léků růst a vývoj metabolismus MeSH
- prekurzory léčiv MeSH
- skot MeSH
- tosylargininmethylester aplikace a dávkování farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The oocyte GV/GVs (germinal vesicle/germinal vesicles) and zygot PN/PNs (pronucleus/pronuclei) of some mammals contain clearly visible nucleoli which exhibit an atypical morphological structure. These nucleoli (NCLs) can be relatively easily manipulated, i.e. removed from GVs/PNs or eventually transferred into another oocyte/zygote. Thus, with the help of micromanipulation techniques it was possible to uncover the real function(s) they play in processes of oocyte maturation and early embryonic development. The purpose of our review is to describe briefly the micromanipulation techniques that can be used for oocyte/zygote nucleoli manipulation. Moreover, we present some examples of results that were obtained in nucleolus manipulation experiments.
- MeSH
- buněčné jadérko metabolismus transplantace MeSH
- mikromanipulace metody MeSH
- myši MeSH
- oocyty cytologie účinky léků MeSH
- partenogeneze MeSH
- prasata MeSH
- zvířata MeSH
- zygota cytologie MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The serine proteases, tissue- and urokinase-type plasminogen activators (PLAT and PLAU) and their inhibitors SERPINE1/2 are regulators of plasminogen to plasmin conversion. They are widely expressed in ovarian tissues, including granulosa and cumulus cells, and their expression is regulated by gonadotropins. The aim of this work was to assess the effect of serine protease inhibitors (aprotinin and AEBSF) and SERPINE1/2 on FSH-induced cumulus cell expansion, the production of prostaglandin E2 (PGE2) and retention of hyaluronic acid (HA) in expanding cumulus. The serine protease activity proved to be essential for the production of PGE2 and also for the retention of HA; the inhibition of plasminogen activators by SERPINE1/2 had the same effect. Collectively, these data indicate that plasmin is required for proper function of expanding cumulus cells in vitro and presumably also in vivo in the pre-ovulatory follicles.
- MeSH
- aprotinin farmakologie MeSH
- dinoproston metabolismus MeSH
- folikuly stimulující hormon farmakologie MeSH
- inhibitor aktivátoru plazminogenu 1 farmakologie MeSH
- inhibitory serinových proteinas farmakologie MeSH
- kumulární buňky cytologie účinky léků metabolismus MeSH
- kyselina hyaluronová metabolismus MeSH
- oocyty cytologie účinky léků metabolismus MeSH
- prasata MeSH
- serpin E2 farmakologie MeSH
- sulfony farmakologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.
- MeSH
- blastocysta cytologie účinky léků fyziologie MeSH
- časové faktory MeSH
- cyklopentany farmakologie MeSH
- embryo savčí MeSH
- embryonální vývoj účinky léků MeSH
- IVM techniky metody veterinární MeSH
- kultivované buňky MeSH
- multiproteinové komplexy antagonisté a inhibitory metabolismus MeSH
- oocyty cytologie účinky léků fyziologie MeSH
- oogeneze účinky léků MeSH
- proteinligasy komplexu SCF antagonisté a inhibitory metabolismus fyziologie MeSH
- pyrimidiny farmakologie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide-amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1-the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.
- MeSH
- aktivace transkripce MeSH
- amfiregulin metabolismus MeSH
- erbB receptory metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy genetika MeSH
- folikuly stimulující hormon farmakologie MeSH
- IVM techniky MeSH
- kultivované buňky MeSH
- kumulární buňky cytologie účinky léků metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 1 genetika MeSH
- mitogenem aktivovaná proteinkinasa 3 genetika MeSH
- oocyty cytologie účinky léků metabolismus MeSH
- prasata MeSH
- signální transdukce účinky léků MeSH
- skupina kinas odvozených od src-genu metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
