Vitrification of Human Oocytes Before or After Rescue-IVM Does not Impair Maturation Kinetics but Induces Meiotic Spindle Alterations.

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16% and IVM post-VIT: 59.38%, p = 0.72) and timings (17.73 h in fresh IVM, 17.33 h in IVM post-VIT, p = 0.72) were observed whether IVM is performed freshly or after vitrification. Meiotic spindles were shorter in VIT + IVM (10.47 µm vs 11.23 µm in IVM and 11.40 µm in IVM + VIT, p = 0.012 and p = 0.043) and wider in IVM + VIT (9.37 µm vs 8.12 µm in IVM and 8.16 µm VIT + IVM, p = 0.027 and p = 0.026). The length-to-width ratio was lower in vitrified groups (IVM + VIT: 1.19 and VIT + IVM: 1.26) compared to IVM (1.38), p = 0.013 and p = 0.014. No differences in multipolar spindle and chromosome misalignment occurrence and cytoplasmic F-actin filament length and density were observed between groups. Our results suggest vitrification before or after rescue-IVM does not seem to impair maturation rates and kinetics parameters but induces meiotic spindle alterations.

An actin-dependent spindle position checkpoint ensures the asymmetric division in mouse oocytes.

Faithful chromosome segregation, during meiosis, is of critical importance to prevent aneuploidy in the resulting embryo. In mammalian oocytes, the segregation of homologous chromosomes takes place with the spindle located at the cell's periphery. The spindle is often assembled close to the centre of the cell, which necessitates the actin network for spindle transport to the cell cortex. In this study, we investigate how the segregation of chromosomes is coordinated with the positioning of the metaphase I spindle. We develop different assays to perturb the spindle's position and to delay its relocation to the cell periphery. We find that anaphase is delayed until the spindle is positioned in close proximity with the oocyte cortex. We further show that the metaphase arrest is dependent on a functional actin network, in addition to the spindle assembly checkpoint. Our work provides the first evidence for the existence of a functional spindle position checkpoint.

Lack of maternal Heat Shock Factor 1 results in multiple cellular and developmental defects, including mitochondrial damage and altered redox homeostasis, and leads to reduced survival of mammalian oocytes and embryos.

Heat Shock Factor 1 (HSF1) is a transcription factor whose loss of function results in the inability of Hsf1(-/-) females to produce viable embryos, as a consequence of early developmental arrest. We previously demonstrated that maternal HSF1 is required in oocytes to regulate expression of chaperones, in particular Hsp90alpha, and is essential for the progression of meiotic maturation. In the present work, we used comparative morphological and biochemical analytic approaches to better understand how Hsf1(-/-) oocytes undergo irreversible cell death. We found that the metaphase II arrest in mature oocytes, cortical granule exocytosis and formation of pronuclei in zygotes were all impaired in Hsf1(-/-) mutants. Although oogenesis generated fully grown oocytes in follicles, intra-ovarian Hsf1(-/-) oocytes displayed ultrastructural abnormalities and contained dysfunctional mitochondria as well as elevated oxidant load. Finally, the apoptotic effector, caspase-3, was activated in most mutant oocytes and embryos, reflecting their commitment to apoptosis. In conclusion, our study shows that early post-ovulation events are particularly sensitive to oxidant insult, which abrogates the developmental competence of HSF1-depleted oocytes. They also reveal that Hsf1 knock-out mice constitute a genetic model that can be used to evaluate the importance of redox homeostasis in oocytes.

Mammalian heat shock factor 1 is essential for oocyte meiosis and directly regulates Hsp90alpha expression.

Heat shock transcription factor 1 (HSF1) is the main regulator of the stress response that triggers the transcription of several genes encoding heat shock proteins (Hsps). Hsps act as molecular chaperones involved in protein folding, stability, and trafficking. HSF1 is highly expressed in oocytes and Hsf1 knock-out in mice revealed that in the absence of stress this factor plays an important role in female reproduction. We previously reported that Hsf1(-/-) females produce oocytes but no viable embryos. Consequently, we asked whether oocytes require HSF1 to regulate a particular set of Hsps necessary for them to develop. We find that Hsp90alpha (Hspaa1) is the major HSF1-dependent chaperone inasmuch as Hsf1 knock-out resulted in Hsp90-depleted oocytes. These oocytes exhibited delayed germinal vesicle breakdown (or G(2)/M transition), partial meiosis I block, and defective asymmetrical division. To probe the role of Hsp90alpha in this meiotic syndrome, we analyzed meiotic maturation in wild-type oocytes treated with a specific inhibitor of Hsp90, 17-allylamino-17-demethoxy-geldanamycin, and observed similar defects. At the molecular level we showed that, together with these developmental anomalies, CDK1 and MAPK, key meiotic kinases, were significantly disturbed. Thus, our data demonstrate that HSF1 is a maternal transcription factor essential for normal progression of meiosis.

Tshz1 is required for axial skeleton, soft palate and middle ear development in mice.

Members of the Tshz gene family encode putative zinc fingers transcription factors that are broadly expressed during mouse embryogenesis. Tshz1 is detected from E9.5 in the somites, the spinal cord, the limb buds and the branchial arches. In order to assess the function of Tshz1 during mouse development, we generated Tshz1-deficient mice. Tshz1 inactivation leads to neonatal lethality and causes multiple developmental defects. In the craniofacial region, loss of Tshz1 function leads to specific malformations of middle ear components, including the malleus and the tympanic ring. Tshz1(-/-) mice exhibited Hox-like vertebral malformations and homeotic transformations in the cervical and thoracic regions, suggesting that Tshz1 and Hox genes are involved in common pathways to control skeletal morphogenesis. Finally, we demonstrate that Tshz1 is required for the development of the soft palate.