Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation

PAWP, a candidate sperm-borne oocyte activating factor, induces oocyte activation and acts upstream of the calcium signalling pathway, however, PAWP’s downstream signalling pathway in oocyte cytoplasm remains to be uncovered. Data from our lab suggested that the interacting partner of PAWP, at least in the frog (Xenopus laevis) model may be YAP, a highly expressed protein in amphibian and mammalian oocytes. Therefore, the objectives of this study were to confirm that PAWP’s predominant binding partner in Xenopus laevis oocyte is YAP; to determine if mammalian oocyte activation is also dependent on PAWP-YAP interaction; and to verify that the PAWP-YAP interaction during oocyte activation is dependent on the WWI domain module. By immunohistochemistry, YAP was localized predominantly in the cytosol of metaphase II-arrested Xenopus laevis oocytes, where presumably the PAWP-YAP interaction occurs. Utilizing Far Western blotting, YAP was identified as the predominant binding partner of PAWP, in metaphase II-arrested frog (Xenopus laevis), swine (Sus scrofa) and mouse (mus musculus) oocytes. The specificity of this interaction was then tested on Far Western blotting of mouse ovarian and oocyte cytosolic extracts, by competition with both wild-type and point-mutated recombinant WWI domains derived from YAP. The removal of GST from the wild-type WWI-GST fusion protein was a requirement for effective blockage of WWI module interaction between PAWP and YAP. As expected, the mutated WWI domain was ineffective in inhibiting the PAWP-YAP interaction. To conclude, this study identified YAP as the predominant binding partner of PAWP in both amphibian and mammalian oocytes, and showed this interaction is dependent on the WWI modular interaction. The results allow us to test the functional relevance of this WWI modular interaction during oocyte activation in vivo, in the future.

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice

Background: The use of mechanical and enzymatic techniques to isolate preantral follicles before in-vitro culture has been previously described. The aim of this study was to assess the effect of the isolation procedure of mouse preantral follicles on their subsequent development in vitro. Methods: Follicles were isolated either mechanically or enzymatically and cultured using an individual non-spherical culture system. Follicular development and steroidogenesis, oocyte in-vitro maturation and embryo development were assessed for both groups. Results: After 12 days of culture, follicles isolated mechanically had a higher survival rate but a lower antral-like cavity formation rate than follicles isolated enzymatically. Enzymatic follicle isolation was associated with a higher production of testosterone and estradiol compared with mechanical isolation. A stronger phosphatase alkaline reaction was observed after enzymatic isolation, suggesting that follicles isolated enzymatically had more theca cells than those isolated mechanically. However, both isolation techniques resulted in similar oocyte maturation and embryo development rates. Conclusions: Enzymatic follicular isolation did not affect theca cell development. Follicular steroidogenesis was enhanced after enzymatic isolation but the developmental capacity of oocytes was comparable to that obtained after mechanical isolation.

A FUNCTIONAL, COMPARATIVE AND CLINICAL ANALYSIS OF SPERM-BORNE OOCYTE ACTIVATING FACTOR, PAWP

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte.

Expression of α6 integrin subunit in bovine oocyte and its potential role during fertilization

Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α6 integrin subunit antibody. The α6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α6 integrin subunit antibody at concentration 5 and 20 μg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.

Histone-specific RNA 3' processing in nuclear extracts from mammalian cells.

The mature 3' ends of histone mRNAs are formed by endonucleolytic cleavage of longer precursor transcripts. This process occurs in the nucleus and can be regarded as the equivalent of the polyadenylation reaction involved in 3′-end-generation of all other mRNAs. A sea urchin H3 gene that failed to be properly processed in the Xenopus oocyte system proved particularly useful, because it allowed the identification of a processing component from sea urchins by a complementation assay. Nuclear extracts prepared from cells under various growth conditions have helped to reveal proliferation-dependent changes in the efficiency of histone RNA 3′ processing. RNA substrates for in vitro processing are best prepared by runoff transcription of specific DNA templates with bacterial or phage RNA polymerases. For this purpose, a restriction fragment containing the 3′-terminal region of a histone gene and including the conserved palindrome and spacer motifs is cloned into a polylinker sequence downstream of a strong promoter.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

Renal bioenergetics during early gram-negative mammalian sepsis and angiotensin II infusion

Purpose: To measure renal adenosine triphosphate (ATP) (bioenergetics) during hypotensive sepsis with or without angiotensin II (Ang II) infusion. Methods: In anaesthetised sheep implanted with a renal artery flow probe and a magnetic resonance coil around one kidney, we induced hypotensive sepsis with intravenous Escherichia coli injection. We measured mean arterial pressure (MAP), heart rate, renal blood flow RBF and renal ATP levels using magnetic resonance spectroscopy. After 2 h of sepsis, we randomly assigned sheep to receive an infusion of Ang II or vehicle intravenously and studied the effect of treatment on the same variables. Results: After E. coli administration, the experimental animals developed hypotensive sepsis (MAP from 92 ± 9 at baseline to 58 ± 4 mmHg at 4 h). Initially, RBF increased, then, after 4 h, it decreased below control levels (from 175 ± 28 at baseline to 138 ± 27 mL/min). Despite decreased RBF and hypotension, renal ATP was unchanged (total ATP to inorganic phosphate ratio from 0.69 ± 0.02 to 0.70 ± 0.02). Ang II infusion restored MAP but caused significant renal vasoconstriction. However, it induced no changes in renal ATP (total ATP to inorganic phosphate ratio from 0.79 ± 0.03 to 0.80 ± 0.02). Conclusions:During early hypotensive experimental Gram-negative sepsis, there was no evidence of renal bioenergetic failure despite decreased RBF. In this setting, the addition of a powerful renal vasoconstrictor does not lead to deterioration in renal bioenergetics.