The purpose of this study was to observe in vitro matured equine oocytes with an objective computerized technique which involve the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n=922) were subjected to different in vitro maturation times (24, 36, or 45 h), but only 36-h matured oocytes were analyzed using PLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate quality and meiotic competence. Average maturation percentages per session in groups 1, 2, and 3 (24-, 36- and 45-h matured oocytes, respectively) were 29.31±13.85%, 47.01±9.90%, and 36.62±5.28%, whereas the average percentages of immature oocytes per session were 28.78±20.17%, 7.83±5.51%, and 22.36±8.39%, respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (p<0.001 and p<0.01, respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (p<0.01). These results show that the 36-h IVM protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes.
We present artificially motorized sperm cells-a novel type of hybrid micromotor, where customized microhelices serve as motors for transporting sperm cells with motion deficiencies to help them carry out their natural function. Our results indicate that metal-coated polymer microhelices are suitable for this task due to potent, controllable, and nonharmful 3D motion behavior. We manage to capture, transport, and release single immotile live sperm cells in fluidic channels that allow mimicking physiological conditions. Important steps toward fertilization are addressed by employing proper means of sperm selection and oocyte culturing. Despite the fact that there still remain some challenges on the way to achieve successful fertilization with artificially motorized sperms, we believe that the potential of this novel approach toward assisted reproduction can be already put into perspective with the present work.
Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis.
The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage.
Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle?
In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following klp-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.
The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs
- Animal : an international journal of animal bioscience
- Published over 8 years ago
Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 μM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 μM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP-DPAM, SNAP-OAG and SNAP-PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 μM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP-OAG 0, SNAP-DPAM 17.4 ± 2.5% and SNAP-PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca2+ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP-DPAM 20.7 ± 2.6%, SNAP-OAG 16.7 ± 2.9% or SNAP-PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.
The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30min, respectively. No differences (P>0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P<0.05) after exposure to 2mg/mL of MβCD compared to the group exposed to 0mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P<0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P<0.05) by MβCD. The blastocysts rate (P<0.05) on D7 was higher in the 2mg/mLMβCD group, but an identical finding was not observed on D8 (P>0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.
BACKGROUND: 15 % of oocytes collected from Assisted Reproductive Technology (ART) cycles are immature. These oocytes may be matured following in vitro maturation (IVM) program. It is possible to cryopreserve the immature oocytes for further use in ART after application of IVM. OBJECTIVE: The aim was to determine the maturation rate and viability of human oocytes that were matured in vitro after vitrification program. MATERIALS AND METHODS: 63 women (19-43 years old) who underwent controlled ovarian stimulation for ART were included in this study. 53 immature oocytes were used for fresh group (fIVM) and 50 immature oocytes for vitrification group (vIVM). The maturation medium was Ham’s F10 supplemented with 0.75 IU FSH, 0.75 IU LH and 40 % human follicular fluid (HFF). After 36 h, maturation and morphology of all oocytes were assessed. Also, the oocyte viability was assessed using PI/Hoechst immunostaining technique. RESULTS: The maturation rates were reduced in vIVM group (56.0 %) in comparison to fIVM group (88.7 %; P < 0.001). Oocyte viability rate were also reduced in vIVM group (56.0 %) in comparison to fIVM (86.8 %, P < 0.007). CONCLUSIONS: Cryopreservation via vitrification reduced both the maturation capacity and viability of human oocytes in IVM technology. It is, therefore, recommended to apply IVM on fresh immature oocytes, instead.
Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation?