Sperm penetration of the cumulus cell layer

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Chapter 2: Materials and Methods

Introduction

Materials

Chemicals

All water used was ultra pure pyrogen free water, and autoclaved where appropriate. All chemicals used were of analytical grade unless otherwise stated and were purchased from BDH (Pennsylvania,USA), BioLab (Clayton, Australia), Difco Laboratories (Detroit, USA), Invitrogen (Carlsbad, USA), Life Technologies (Gaithersburg, USA), Merck (Whitehouse Station, USA), Scharlau (Barcelona, Spain), Serva Electrophoresis GmbH (Heidelberg, Germany) and Sigma-Aldrich (St Louis, USA).

Enzymes

Taq DNA polymerases derived from the bacterium Thermus aquaticus were purchased from Qiagen (Hilden, Germany) (Qiagen Taq), which was used for general polymerase chain reaction (PCR) reactions. AccuPrime Pfx from Invitrogen (Carlsbad, USA) is a proofreading Taq and was used to amplify the SPRASA promoter regions. Advantage 2 Polymerase Mix, Clontech (Mountain View,USA) was used for the 5’ and 3’ RACE experiments. Amplification grade DNAse I, RNaseOUT, Oligo (dT)20 primer(s), Thermoscript reverse transcriptase kit, SYBR GreenER qPCR SuperMix were all purchased from Invitrogen. Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT),Epicentre (Madison, USA), was used to amplify the first-strand cDNA for 5’- and 3’-RACE PCR. Restriction endonucleases (Mlu I and Xho I) were purchased from New England Biolabs (NEB) (Ipswich, USA). TaqMan Gene Expression Mastermix, BigDye Terminator v3.1 Cycle Sequencing Kits were purchased from Applied Biosystems (ABI) (Foster City, USA).

Kits

Purifying columns for PCR products and plasmid isolation (small quantities) were purchased from Roche (Basel, Switzerland). Plasmid isolation (maxiprep) and columns for the extraction of total RNA from mammalian cells and tissues were purchased from Qiagen (Hilden, Germany). SMART RACE 5’and 3’ Amplification Kit was purchased from Clontech (California, USA). Maxwell 16 DNA Purification Kit was kindly supplied by Promega (Madison, USA). MasterPureTM Complete DNA and RNA Purification Kit were purchased from EPICENTRE Biotechnologies (Madison, USA).

Cell Culture Reagents

The cell culture media Roswell Park Memorial Institute (RPMI) 1640, Fetal Bovine Serum (FBS), trypsin and the cell culture antibiotics penicillin and streptomycin were all purchased from Invitrogen. The tissue culture flasks were purchased from Becton Dickinson (New Jersey, USA), and the Cryotubes were obtained from NUNC (Roskilde, Denmark).

Plasmids

The pGEM-T Easy is driven by the T7 and SP6 promoters and was used to increase the quantity of PCR products, to confirm that the orang-utan variants identified in exon two were on the same amplicon (Chaper 5.1.1 and Figure 5.3), as well as isolate and amplify the 5’ and 3’ RACE PCR products (Chaper 5.1.1). The pGL3-enhancer reporter plasmid, in which the expression of luciferase is modified from the firefly luciferase gene, contains an SV40 enhancer, and was used to express the SPRASA promoter region variants. The reporter plasmid pRL-TK, in which the expression of luciferase is derived from the sea pansy, Renilla, is driven by the thymidine kinase promoter and was used as a co-transfection control. The pShuttle-GFP plasmid which expresses the green fluorescent protein (GFP) driven by the CMV promoter was used to optimise the reporter assays and was kindly given to the laboratory by Melony Black (Liggins Institute and the School of Biological Sciences,University of Auckland). All other plasmids were obtained from Promega (Wisconsin, USA).

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Primers

All primers designed for this research were purchased from Invitrogen (Auckland, New Zealand) and were of standard purity. For PCR amplification, primers were resuspended in ultra pure pyrogen free water to a concentration of 20µM. For quantitative Reverse Transcription-PCR (qRT-PCR), primers were resuspended in 10mM Tris, 0.1mM EDTA, pH 8 to a stock concentration of 100µM and working concentrations of 3µM for Sybr GreenER master mixes and 0.9µM for TaqMan master mix. Quantitative RT-PCR probes were purchased from Applied Biosystems and were resuspended in10mM Tris, 0.1mM EDTA, pH 8 to a stock concentration of 5µM and a working concentration of 0.25µM. Quantitative Reverse Transcription-PCR (qRT-PCR) Mouse geNorm SYBRgreen detection primer kit (cytochrome c-1; CYC1, ubiquitin; UBC, ribosomal protein L13a; RPL13A, tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; YWHAZ, succinate dehydrogenase complex, subunit A; SDHA, and eukaryotic translation initiation factor 4A2; EIF4A2) was purchased from PrimerDesign (Southampton, United Kingdom).

Abstract 
Acknowledgements
List of figures 
List of tables 
Abbreviations
Chapter 1: Introduction 
1 Foreword 
1.1 Female reproductive system 
1.2 Male reproductive system
1.3 Mammalian fertilisation
1.3.1 Epididymal maturation
1.3.2 Capacitation
1.3.3 Vaginal environment
1.3.4 Cervical mucus
1.3.5 Uterine environment
1.3.6 The oviductal environment
1.3.7 Sperm penetration of the cumulus cell layer
1.3.8 Sperm-oocyte binding
1.3.9 Acrosomal reaction
1.3.10 Sperm-oocyte adhesion and fusion
1.3.11 Sperm-oocyte adhesion proteins
1.3.12 Sperm-oocyte fusion proteins
1.4 Infertility
1.4.1 Female causes of infertility
1.4.2 Ovulation infertility
1.4.3 Blocked tubes in the female reproductive system
1.4.4 Uterine infertility
1.4.5 Cervical infertility
1.4.6 Male causes of infertility
1.4.7 Sperm infertility
1.4.8 Blocked tubes in the male reproductive system
1.4.9 Immunological infertility
1.4.10 Genetic abnormalities
1.4.11 Combined infertility
1.4.12 Unexplained infertility
1.5 Lysozyme family 
1.5.1 c-type Lysozyme
1.5.2 Alpha-lactalbumin
1.5.3 SPRASA
1.6 Conclusions from the literature 
1.7 Thesis hypotheses
Chapter 2: Materials and Methods
2 Introduction
2.1 Materials 
2.1.1 Chemicals
2.1.2 Enzymes
2.1.3 Kits
2.1.4 Cell Culture Reagents
2.1.5 Plasmids
2.1.6 Primers
2.2 Methods
2.2.1 Ethics Statement and Patient Recruitment
2.2.2 DNA Extraction
2.2.3 Polymerase Chain Reaction (PCR)
2.2.4 Sequencing Reactions
2.2.5 Plasmids: Maintaining, Propagation, Isolating and Purification
2.2.6 Cell culture
2.2.7 Luciferase Assays
2.2.8 Total RNA
2.2.9 Quantitative RT-PCR (qRT-PCT)
2.2.10 Reverse Transcription
2.2.11 Relative Quantification
2.2.12 Absolute quantification RT-PCR
2.2.13 Rapid amplification of cDNA Ends
2.2.14 In silico analyses
2.2.15 3D model determination
2.2.16 Phylogenetic analysis
2.2.17 Statistical analysis
Chapter 3: SPRASA expression
3 Introduction
3.1 Results 
3.1.1 In silico analysis of SPRASA expression profile
3.1.2 Quantification real-time PCR
3.1.3 Spatial and temporal expression analysis of SPRASA
3.1.4 Expression analysis of SPRASA in human tissues and cell lines
3.1.5 Absolute qRT-PCR expression analysis of human SPRASA
3.1.6 In silico analysis of SPRASA promoter regions
3.1.7 Transcription factor binding site analysis
3.1.8 Functional analysis of SPRASA promoters
3.2 Discussion 
3.2.1 In silico analysis of the SPRASA protein
3.2.2 Spatial and temporal expression of SPRASA
3.2.3 Expression pattern of SPRASA mRNA in the male mouse reproductive system
3.2.4 Human SPRASA expression
3.2.5 Absolute expression of SPRASA in human samples
3.2.6 SPRASA promoter analysis
3.2.7 Analysis of the promoter regions of SPRASA
3.3 Summary
Chapter 4: Mutational analysis of the SPRASA gene
4 Introduction
4.1 Results 
4.1.1 Optimisation of PCR amplification
4.1.2 Calculation of allele, homozygote and heterozygote frequencies
4.1.3 Nucleotide analysis of exon one
4.1.4 Functional analysis of the SPRASA promoter variant g.-22TGC(4_5)
4.1.5 Nucleotide analysis of exon two
4.1.6 In silico analysis of c.314G>A (p.C80Y) variant
4.1.7 Nucleotide analysis of exon three and exon four
4.1.8 Nucleotide analysis of exon five
4.1.9 Variant analysis within couples
4.2 Discussion
4.2.1 Tri-nucleotide insertion at the quadruple tri-nucleotide repeat region
4.2.2 Luciferase assay characterisation of the SPRASA g-22TGC(4_5) variant
4.2.3 The SPRASA c.314G>A possible association with infertility
4.2.4 Genetic analyses of exon three and exon four of the SPRASA gene
4.2.5 Novel nucleotide change in the 3’ UTR of the SPRASA gene
4.2.6 Variation analysis within infertile and fertile couples
4.3 Summary 
Chapter 5: Evolution of the SPRASA gene 
5 Introduction
5.1 Results
5.1.1 SPRASA orthologues
5.1.2 Gene organisation and structural homology
5.1.3 SPRASA motif
5.1.4 Phylogenetic analysis of amino acid sequence data
5.1.5 Tertiary structure modelling
5.2 Discussion 
5.2.1 SPRASA orthologue analysis
5.2.2 Analysis of the orang-utan SPRASA gene
5.2.3 SPRASA gene organisation and structural homology
5.2.4 Catalytic residues
5.2.5 Analysis of SPRASA invariant residues
5.2.6 Analysis of the SPRASA motif
5.2.7 Phylogenetic analysis
5.2.8 Analysis of the tertiary structure modelling
5.3 Summary
Chapter 6: Concluding remarks 
6 Discussion 
6.1 The determination of the spatial and temporal expression of SPRASA 
6.2 To determine whether genetic variants in the SPRASA gene sequence are associated with infertility in humans 
6.3 To examine the evolutionary conservation of SPRASA 
6.4 Future directions
6.5 Summary 
Appendix I
7 SPRASA promoter and gene sequence
Appendix II
8 Buffer and Solutions
8.1 Stock solutions for DNA isolation 
8.2 Buffers and solutions for gel electrophoresis 
8.3 RNA visualisation on agarose gel 
8.4 Cell transformation 
8.5 General solutions
8.6 Sequencing cleanup 
8.7 Cell Culture 
8.8 Competent Cells Solutions
Appendix III
References 

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