Initiation Of Embryo Development Research Articles

180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes

Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (> 70%), DNA integrity (<8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P<0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P<0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.

Regulation of the meiotic divisions of mammalian oocytes and eggs.

Initiated by luteinizing hormone and finalized by the fertilizing sperm, the mammalian oocyte completes its two meiotic divisions. The first division occurs in the mature Graafian follicle during the hours preceding ovulation and culminates in an extreme asymmetric cell division and the segregation of the two pairs of homologous chromosomes. The newly created mature egg rearrests at metaphase of the second meiotic division prior to ovulation and only completes meiosis following a Ca2+ signal initiated by the sperm at gamete fusion. Here, we review the cellular events that govern the passage of the oocyte through meiosis I with a focus on the role of the spindle assembly checkpoint in regulating its timing. In meiosis II, we examine how the egg achieves its arrest and how the fertilization Ca2+ signal allows the initiation of embryo development.

Phospholipase C zeta parameters in sperm from polymorphic teratozoospermic men

Teratozoospermia is a disorder associated with high abnormal sperm morphology which affects fertility in males. In recent years, it has been described that biomarker-based sperm quality evaluation can alleviate male infertility treatment. Phospholipase C zeta (PLCζ) is a sperm-specific factor which appears to be a predicting biomarker for fertilization potential of males. Following fertilization, PLCζ enters into oocyte cytoplasm and induces oocyte activation, a fundamental stage in initiation of embryo development. Currently, PLCζ parameters, including localization patterns, the proportion of PLCζ-expressing sperm and the expression level, are not defined in polymorphic teratozoospermic men. This study aimed to evaluate PLCζ parameters in polymorphic teratozoospermic men, and compare these parameters with fertile normozoospermic men. Semen samples from thirteen normozoospermic fertile men and twenty-three polymorphic teratozoospermic men were included in this study and evaluated using western blotting and immunofluorescence analyses. Our data indicated significantly lower expression of PLCζ in polymorphic teratozoospermic men, as compared with control men; however, there was no significant difference in localization patterns and the proportion of PLCζ-expressing sperm between polymorphic teratozoospermic patients and control men. Collectively, findings from the present study demonstrated that polymorphic teratozoospermic men did not show abnormal localization patterns or the absence of PLCζ, as compared to the control men; nonetheless, lower expression of PLCζ, considering its role in oocyte activation, might be one of the possible causes of infertility in these patients.

Investigation on Association of ESR2 polymorphism as a Candidate Gene for Duroc sperm motility and kinematic characteristics

For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Zygote arrest 1 (ZAR1) is oocyte-specific protein involved in the initiation of embryo development. but their functions have not been confirmed in pig until now. Therefore, this study was conducted to analyze their association with sperm motility and kinematic characteristics. DNA samples from 105 Duroc pigs with records of semen motility and kinematic characteristics〔Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)〕 were analyzed. A SNP in coding region of ZAR1 g.4095 C>A in intron 4 was associated with MOT in Duroc population. Therefore, we suggest that the porcine ZAR1 gene may be used as a molecular marker for Duroc boar semen quality, although its functional effects were not defined yet. These results might shed new light on the roles of ZAR1 in spermatogenesis as candidate gene for boar fertility, but still the lack of association across populations should be considered.

TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse.

The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development.

Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition

Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic change in the composition of the protein complex upon M-phase exit and the oocyte to embryo transition.

Transcriptome analysis during somatic embryogenesis of the tropical monocot Elaeis guineensis: evidence for conserved gene functions in early development

With the aim of understanding the molecular mechanisms underlying somatic embryogenesis (SE) in oil palm, we examined transcriptome changes that occur when embryogenic suspension cells are initiated to develop somatic embryos. Two reciprocal suppression subtractive hybridization (SSH) libraries were constructed from oil palm embryogenic cell suspensions: one in which embryo development was blocked by the presence of the synthetic auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-D: ) in the medium (proliferation library); and another in which cells were stimulated to form embryos by the removal of 2,4-D: from the medium (initiation library). A total of 1867 Expressed Sequence Tags (ESTs) consisting of 1567 potential unigenes were assembled from the two libraries. Functional annotation indicated that 928 of the ESTs correspond to proteins that have either no similarity to sequences in public databases or are of unknown function. Gene Ontology (GO) terms assigned to the two EST populations give clues to the underlying molecular functions, biological processes and cellular components involved in the initiation of embryo development. Macroarrays were used for transcript profiling the ESTs during SE. Hierarchical cluster analysis of differential transcript accumulation revealed 4 distinct profiles containing a total of 192 statistically significant developmentally regulated transcripts. Similarities and differences between the global results obtained with in vitro systems from dicots, monocots and gymnosperms will be discussed.

Human sperm devoid of PLC, zeta 1 fail to induce Ca2+ release and are unable to initiate the first step of embryo development

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca(2+)](i) oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca(2+)](i) oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca(2+)](i) oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

PLCζ, a sperm-specific PLC and its potential role in fertilization

A dramatic rise in intracellular calcium plays a vital role at the moment of fertilization, eliciting the resumption of meiosis and the initiation of embryo development. In mammals, the rise takes the form of oscillations in calcium concentration within the egg, driven by an elevation in inositol trisphosphate. The causative agent of these oscillations is proposed to be a recently described phosphoinositide-specific phospholipase C, PLCzeta, a soluble sperm protein that is delivered into the egg following membrane fusion. In the present review, we examine some of the distinctive structural and functional characteristics of this crucial enzyme that sets it apart from the other known forms of mammalian PLC.

PLCζ, a sperm-specific PLC and its potential role in fertilization

A dramatic rise in intracellular calcium plays a vital role at the moment of fertilization, eliciting the resumption of meiosis and the initiation of embryo development. In mammals, the rise takes the form of oscillations in calcium concentration within the egg, driven by an elevation in inositol trisphosphate. The causative agent of these oscillations is proposed to be a recently described phosphoinositide-specific phospholipase C, PLCζ, a soluble sperm protein that is delivered into the egg following membrane fusion. In the present review, we examine some of the distinctive structural and functional characteristics of this crucial enzyme that sets it apart from the other known forms of mammalian PLC.

A specific alpha-tubulin is associated with the initiation of parthenogenesis in 'Salmon' wheat lines.

The 'Salmon system' consists of isogenic but alloplasmic wheat lines with either sexual or autonomous embryo development. Using two-dimensional gel electrophoresis these lines have been screened for proteins potentially involved in the initiation of parthenogenesis. A temporally altered expression of the polypeptide 'P 115.1' in the sexual and parthenogenetic 'Salmon' lines seems to be related with the autonomous embryo formation. Around anthesis when most of the egg cells begin the parthenogenetic development, the polypeptide 'P 115.1' was present in ovaries of the parthenogenetic lines but not in ovaries of the sexual line. Moreover, this polypeptide is only expressed in the ovaries of amphidiploid parthenogenetic plants containing differentiated embryo sacs. It is absent from ovaries of the analogous polyhaploid plants, which lack any embryo sac structure within their ovules. Furthermore, the polypeptide was neither detectable in meristematic tissue of root tips nor in leaves. N-terminal amino acid sequencing identified 'P 115.1' as an alpha-tubulin. Thus, 'P 115.1' apparently represents an embryo sac-specific isoform of alpha-tubulin involved in the initiation of embryo development.

Nicotinamide alters the calcium release pattern and the degradation of MPF activity after fertilisation in ascidian oocytes.

Fertilisation in ascidian oocytes triggers a plasma membrane current, the release of intracellular calcium and the degradation of Maturation Promoting Factor (MPF) activity leading to the completion of meiosis and the initiation of embryo development. We have previously shown that the fertilisation current in ascidians is produced through the metabolism of nicotinamide nucleotide (NN) metabolites to ADP ribose. In this study we have used nicotinamide to test whether NN metabolism plays additional roles in fertilisation in ascidians. Nicotinamide treatment blocked calcium-induced calcium release (CICR) and arrested the cell cycle prior to the completion of meiosis I. Nicotinamide further prevented the abolition of MPF activity after fertilisation. Interestingly, nicotinamide treatment caused ascidian oocytes to form interphase-like pronuclei after fertilisation, despite the high MPF activity. The data demonstrate that NN metabolism is involved in calcium signalling through CICR and further suggest that a NN metabolite acts as a messenger connecting MPF activity to the formation of the meiotic apparatus.

Embryology and fruit development in four species of Pistacia L. (Anacardiaceae)

Pistacia atlantica, P. palaestina, P. lentiscus and P. saportae, were found to have great similarity in their embryology and fruit development. The anatropous, pendulous and crassinucellate ovule was initially unitegmic; later, the integument split close to the micropyle, forming a partial second integument. After anthesis there was a development of a hypostase and an obturator. The development of the Polygonum-type embryo sac followed division of a megaspore mother cell, giving a tetrad or triad of megaspores. The functional megaspore was the chalazal one. The ovary developed into a mature pericarp after anthesis, even when pollination was prevented, and before the zygote divided. Therefore, the fruit can be parthenocarpic. The ovule started to grow after initiation of embryo development until it filled the cavity within the pericarp. The zygotes were dormant for 4–18 weeks after pollination. In P. saportae reproduction became arrested during the development of the embryo sac; only very few abnormal embryos were found. No fixed pattern of embryo development could be discerned. The endosperm was initially nuclear, becoming cellular when the embryo started to develop. The seed coat was derived from the integument and the remnants of the nucellus.