Cables1 protects p63 from proteasomal degradation to ensure deletion of cells after genotoxic stress

The p63 gene product regulates epithelial morphogenesis and female germline integrity. In this study, we show that cyclin‐dependent kinase 5 and Abl enzyme substrate 1 (Cables1) interacts with the trans‐activating (TA) p63α isoform to protect it from proteasomal degradation. Using the female germline of Cables1‐null mice as an in vivo model, we demonstrate further that oocytes lacking Cables1 exhibit lower basal levels of TAp63α and reduced accumulation of phosphorylated TAp63α in response to genotoxic stress. This in turn enhances the survival of these cells after ionizing radiation exposure. Thus, Cables1 modulates p63 protein stability and function during genotoxic stress.     

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse

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Ultrastructure of follicular atresia in the rat

Observations were made on the sequence of morphologic changes in atresia of medium-sized preantral follicles in the rat. Ultrastructural studies indicated that in both control and hormonally treated animals granulosa cell changes, including nuclear condensation and alterations in cytoplasmic organelles, occurred prior to effects on the oocyte. In more advanced stages of atresia, extensive disruption of granulosa cell cytoplasm was associated with loss of microvilli and cytoplasmic vacuolization in oocytes. The findings are consistent with the view that follicular atresia begins with alterations in granulosa cells, effects on the oocyte occurring later in the atretic process.     

Selective protein transport: Characterization and solubilization of the phosvitin receptor from chicken oocytes

Phosvitin (PV), a subunit of a female-specific protein, vitellogenin, binds to oocyte membranes with a KD of 10^?6 M. Binding reaches equilibrium within 30 min after incubation at 25°C. Bound ¹²⁵I-PV dissociates from the membrane with a t1/2 of 13 h when incubated in buffer. However, when ¹²⁵I-PV-labeled membranes are incubated in buffer containing 10^?5 M unlabeled PV, 50% of the initially bound ¹²⁵I-PV dissociates from the membrane within 10 min. These results support the conclusion that PV binds to a membrane-associated receptor. Solubilization studies show that Triton X-100 solubilizes up to 45% of the total membrane-bound ¹²⁵I-PV. Gel-exculsion chromatography of the solubilized material yields a 500,000 dalton ¹²⁵I-PV-containing complex separated from free ¹²⁵I-PV. The 500,000 dalton complex completely dissociates to yield free ¹²⁵I-PV when incubated with excess unlabeled PV. However, when incubated with (1) no addition, (2) IgG, or (3) serum albumin, the extent of dissociation is significantly reduced and is consistent with that which would be predicted on the basis of the observed dissociation rate in the absence of unlabeled PV. These results suggest that bound ¹²⁵I-PV can only be displaced by unlabeled PV. These results also indicate that the 500,000 dalton species is a solubilized PV-receptor complex and that it is possible to solubilize the PV-receptor in an active form.     

Observation on the incorporation cone in the rat

At the time of in vivo sperm–egg fusion in the rat, a small region of the oolemma under the head of the fertilizing sperm is observed to be free of microvilli. The microvilli-free region increases in area, and by one hour after sperm–egg contact extends over an area 20–30 μ in circumference and bulges out to form an “incorporation cone” visible by light microscopy. The microvilli-free incorporation cone reaches its maximum size at about two hours after sperm–egg interaction. It soon becomes smaller and has disappeared three to four hours after sperm–oocyte fusion. The cone cytoplasm is characterized by a 0.1 μ zone of thin filaments below the plasma membrane. Cytochalasin-B, 2.5 μg/ml, prevents formation of the cone or destroys the intact cone. It is suggested that micro filaments may be involved in the formation of the incorporation cone.     

Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Embryonic poly(A)-binding protein is differently expressed and interacts with the messenger RNAs in the mouse oocytes and early embryos

The embryonic poly(A)-binding protein (EPAB) functions in the translational regulation of the maternal messenger RNAs (mRNAs) required during oocyte maturation, fertilization, and early embryo development. Since there is no antibody specific to mammalian EPAB protein, all studies related to the Epab gene could be performed at the mRNA levels except for the investigations in the Xenopus. In this study, we have produced an EPAB-specific antibody. When we examined its expressional distribution in the mouse gonadal and somatic tissues, the EPAB protein was found to be expressed only in the mouse ovary and testis tissues, but it is undetectable level in the somatic tissues including stomach, liver, heart, small intestine, and kidney. Additionally, the spatial and temporal expression patterns of the EPAB and poly(A)-binding protein cytoplasmic 1 (PABPC1) proteins were analyzed in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, one-cell, and two-cell embryos. While EPAB expression gradually decreased from GV oocytes to two-cell embryos, the PABPC1 protein level progressively increased from GV oocytes to one-cell embryos and remarkably declined in the two-cell embryos ( P < 0.05). We have also described herein that the EPAB protein interacted with Epab, Pabpc1, Ccnb1, Gdf9, and Bmp15 mRNAs dependent upon the developmental stages of the mouse oocytes and early embryos. As a result, we have first produced an EPAB-specific antibody and characterized its expression patterns and interacting mRNAs in the mouse oocytes and early embryos. The findings suggest that EPAB in cooperation with PABPC1 implicate in the translational control of maternal mRNAs during oogenesis and early embryo development.     

Effects of Bisphenol A and its Derivatives on the Response of GABAA Receptors Expressed in Xenopus Oocytes

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic γ-aminobutyric acid (GABA) receptors, GABA_A receptors were expressed in Xenopus oocytes by injecting both poly(A)⁺RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of α₁ and β₁ subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.     

Calcineurin role in porcine oocyte activation

Calcineurin is required for oocyte exit from meiotic block in metaphase II (MII) stage in invertebrates and also in lower vertebrates. However, the role of calcineurin in mammalian oocyte activation is still unclear. The aim of this study was to determine whether calcineurin is involved in the processes regulating porcine oocyte activation. Indirect immunofluorescence demonstrated localization of both calcineurin subunits, CnA and CnB, especially in the cortex area of MII oocytes, in vitro fertilized and also parthenogenetically activated oocytes. After activation, the fluorescence intensity of the protein in the cortex area of oocytes remains unchanged; the protein calcineurin in the cytoplasm was recorded mainly around the pronuclei. Treatment of matured oocytes with calcineurin inhibitors, cyclosporin A (CsA) and hymenistatin I (HS-I), followed by activation with calcium ionophore A23187, significantly decreased the rate of activated oocytes compared to oocytes that were treated only with calcium ionophore (Ca-Io), (CsA+Ca-Io 25.0% v. Ca-Io 83.3%; HS-I+Ca-Io 32.5% v. Ca-Io 85.0%). Compared to the control, CsA treatment of matured oocytes followed by activation with Ca-Io did not affect the activity level of metaphase-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in activated oocytes evaluated by kinase activity assay. Simultaneous staining of calcineurin and cortical granule content in matured oocytes showed that calcineurin distributed in the cortical area of the oocyte has not been colocalized with cortical granules content. On the other hand, the calcineurin inhibition before parthenogenetic activation leads to a reduction of the cortical reaction level compared to oocytes that were not treated with CsA (complete exocytosis: CsA+Ca-Io 2.6% v. Ca-Io 83.9%; sum of cortical granule brightness: CsA + Ca-Io 0.69 v. Ca-Io 0.15). Our results showed that calcineurin is involved in the process of pig oocyte activation and cortical granule exocytosis; however this regulation seems to be MPF and MAPK independent.     

Editorial: Twin peaks mission

The Y human chromosome has many ancient genes whose fidelity seems to have been preserved by tandem sequences and palindromic ‘hairpins’, compared/repaired by ‘gene conversion’. That a primary function of recombination machinery is DNA repair has been suggested, and rejected, several times; this new evidence is very persuasive. The process, better called gene conservation than gene conversion, could operate in all diploid organisms, accounting for the retention of long gene sequences without ‘informational meltdown’ ('concerted evolution'). It resembles rocket‐science computer‐redundancy error‐checking, comparison of three or four sequences, not just two. If recognition of errors in ‘converted’ sequences can be followed by either repair or rejection, the rejection option can account for the vast wastage of meiotic products. The repair option might be used in Drosophila oocytes and even zygotic nuclei, possibly other oocytes, ancient asexual lineages such as mycorrhizal fungi, perhaps the Y itself. Both evolutionary stasis (conservatism) and development and deployment of complex developmental modules can be understood in these terms so both the evolution of biodiversity and the practice of systematics may have these mechanisms as their bases. The main individual‐fitness and evolutionary advantages of diploidy were not primarily cloaking of recessive al‐leles, or allelic recombination and Mendelism, but conserving long DNA sequences.