BIOL30001 reproductive physiology Molecular techniques for reproductive research Hongshi Yu Research Associate Department of Zoology University of Melbourne

Reading and References Green M.R. and Sambrook J. Molecular Cloning: A Laboratory Manual (4 th Edition) 2012 Cold Spring Harbor Laboratory Press Nicholl D.S.T. An Introduction to Genetic Engineering (3 rd Edition). Cambridge University Press Current protocols | Book series |Wiley Methods in Cell Biology | Book series | Elsevier Methods in Molecular Biology | Book series |Springer

The discovery of the SRY gene sex chromosome was discovered in 1921 XX, female XY, male Y chromosome determines sex was recognized in 1960s XO [Turner] XXY [Klinefelter] Mutation, Sequencing and DNA analysis to discover SRY in 1990 “Three men” with XX; “”a woman” with XY

Materials Genomic DNA RNA Protein cell functions transcription translation

Isolation of DNA, RNA and protein HomogenizationSeparation Aqueous phase Interphase Organic phase RNA DNA Protein Precipitation TRI Reagent® protocol

PCR Polymerase Chain Reaction – Amplifies small amounts of DNA so we have enough to use in tests. – Can assess if a gene present eg SRY in males or females eg is gene present in different species?

Sequence to amplify 5’ 3’ 5’ 3’ Polymerase Chain Reaction (PCR) 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Denature to separate Anneal to add primers Add Taq polymerase 5’ 3’ 5’ + 3’ 5’ 3’ Denature to separate Anneal to add primers 5’ 3’ 5’ 3’ 5’ 3’ 5’ Add Taq polymerase 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Amplifications of target Repeat cycles

CyclesCopies , , ,048, ,554, ,073,741,824 CyclesCopies , , ,048, ,554, ,073,741,824 Real-time PCR

Assessing gene transcription mRNA present? How much? mRNA breaks down quickly mRNA  cDNA (stable)  PCR

mRNA to cDNA: Reverse transcriptase AAA(A) n 5’-Cap mRNA (dT) 12~18 primer anneal 5’-Cap AAA(A) n 3’3’5’5’ Reverse transcriptase dNTP 5’-Cap AAA(A) n 5’5’ cDNA:mRNA hybrid

Koopman et al (1990) Nature 348(6300): Mouse Sry gene expression pattern RT-PCR Northern blot When is it expressed? Where is it expressed?

SRY expression in tammar Pask, Calatayud, Shaw and Renfree BMC Biol SRY mRNA expression relative to β-Actin birth days from birth Testis differentiates

mRNA in situ hybridization Target RNA Hybridize with Labeled probe (complementary sequence) Detect probe Reveal cell Cell interest Which cells / tissues express the gene of interest?

Swain et al (1998) Nature 391(6669): Whole mount in situ hybridization of Sry gene male female SRY expressed in testis, starting at posterior, and not it ovary

Day 25 fetal testis Tammar SRY in situ hybridisation Day 2 PY testis 50µm Meso 50µm Meso Setoli Cell Germ Cell Seminiferous tubules Unpublished data, Yu et al gonad testis SRY expressed in Sertoli cells, starting just before onset of differentiation

Tammar SRY in situ Day 8 PY testis Day 17 PY testis Seminiferous tubule Seminiferous tubules Sertoli cells Germ cells Sertoli cells Germ cells Unpublished data, Yu et al

Immunohistochemistry Hopefully antibody is specific to particular protein or part of protein

Kashimada and Koopman (2010) Development 137: SRY immunofluorescence Western blot Is the antibody detecting just one band, and of the appropriate size? quantitation.

Molecular techniques PCR to determine if gene is absent or present Real-time PCR to quantify how much mRNA present mRNA in situ hybridization to show mRNA spatio-temporal expression Northern blot to check RNA sizes, abundance, and expression Immunohistochemistry to localize protein distribution Western blot to confirm identity (correct protein size), and assess abundance

Microscopical analysis: Geoff Shaw Structure   Function Gross anatomy (macroscopic) tells us what is happening on a gross scale Microscopical anatomy tells us what is happening on a cellular scale BIOL30002 Experimental Reproductive Physiology

Microscopy Scale limited by detection method visible light: wavelength ~ 0.38 – 0.74 μm  cannot reveal structures less than about 1 μm (organelles) Electron beam: at 10kV  μm (large molecules) Synchrotron beam:  μm (small molecules) Atomic force microscopy  atomic scale

Types of microscopy Light microscopy thin sections – paraffin; frozen – 4-10 μm good for tissue/cellular level relatively low cost many ways to stain to highlight things of interest Confocal microscopy light microscopy with special optics can get higher resolution can build up 3-D structure Transmission Electron microscopy extremely thin sections (0.05 μm) good for cellular and subcellular level expensive machinery more limited staining options (heavy metals) Scanning EM shows surface structures

Interpreting Microscope Slides 2-D vs

histology light microscopy vs TEM vs SEM vs confocal stains –haematoxylin & eosin - good general purpose –trichrome - good connective tissue discrimination –PAS - sugar containing components –lipophilic dyes - fats and oils –nucleic acid stains –enzyme histochemistry –immunohistochemistry Resources: –Kerr (2000) Atlas of Functional Histology –Wheater et al, (1987) Functional Histology

Basic tissue types blood connective tissue, bone fat (adipose tissue) muscle nerves epithelium (epithelia)

epithelia columnar cuboidal squamous … uterine luminal epithelium lumen Junctions between cells  barrier

pituitary posterior (nerve ends) anterior stalk (with blood vessels)

testis epididymis fat

seminiferous tubules germ cells lumen blood

ovary antral follicle oocyte

Drawings Use a SHARP pencil Tissue outlines – don’t attempt elaborate shading unless you are very skilled Use appropriate size scales Don’t spend forever replicating fine detail – just do a bit to show Label and annotate with observations and info from reference material