BIOL30001 reproductive physiology Molecular techniques for reproductive research Hongshi Yu Research...

BIOL30001 reproductive physiology

Molecular techniques for reproductive research

Hongshi YuResearch AssociateDepartment of ZoologyUniversity of Melbourne

Reading and References

• Green M.R. and Sambrook J. Molecular Cloning: A Laboratory Manual (4th Edition) 2012 Cold Spring Harbor Laboratory Press

• Nicholl D.S.T. An Introduction to Genetic Engineering. 2013 (3rd 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 1921XX, female XY, male

• Y chromosome determines sex was recognized in 1960sXO [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

Homogenization Separation

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 toamplify

5’3’ 5’

3’

Polymerase Chain Reaction (PCR)5’3’ 5’

3’

5’ 3’5’

3’ 5’

5’

Denature to separateAnneal to add primers

Add Taq polymerase

5’ 3’3’ 5’

+ 3’ 5’5’ 3’

Denature to separateAnneal to add primers

5’ 3’

3’ 5’

5’5’

3’ 5’

5’5’ 3’

5’

Add Taq polymerase

5’3’ 5’

3’ 5’5’ 3’

3’ 5’5’

5’ 3’5’

Amplifications of target

Repeat cycles

Cycles Copies1 22 4

4 1610 1,024

15 32,76820 1,048,576

25 33,554,43230 1,073,741,824

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’-CapmRNA

(dT)12~18 primer anneal

5’-Cap

AAA(A)n

3’ 5’

Reverse transcriptasedNTP

5’-Cap

AAA(A)n

5’

cDNA:mRNA hybrid

Koopman et al (1990) Nature 348(6300):450-452.

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.2010

0

1

2

3

4

-4 -2 0 2 4 6 8

SR

Y m

RN

A e

xpre

ssio

n re

lativ

e to

β-A

ctin birth

days from birth

Testis differentiates

mRNA in situ hybridization

Target RNA

Hybridize withLabeled probe (complementary sequence)

Detect probeReveal cellCell interest

Which cells / tissues express the gene of interest?

Swain et al (1998) Nature 391(6669):761-767.

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

Sem

inife

rous

tubu

les

Unpublished data, Yu et al

gonadtestis

SRY expressed in Sertoli cells, starting just before onset of differentiation

Tammar SRY in situ

Day 8 PY testis

Day 17 PY testis

Seminiferous tubuleSeminiferous 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 137:3921-3930

SRY immunofluorescence

Western blot

Is the antibody detectingjust 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

MicroscopyScale 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 0.00 001 μm(large molecules)

Synchrotron beam: 0.000 001 μm(small molecules)

Atomic force microscopy atomic scale

Types of microscopyLight microscopy

thin sections – paraffin; frozen – 4-10 μmgood for tissue/cellular levelrelatively low costmany ways to stain to highlight things of interest

Confocal microscopylight microscopy with special opticscan get higher resolutioncan build up 3-D structure

Transmission Electron microscopyextremely thin sections (0.05 μm)good for cellular and subcellular levelexpensive machinerymore limited staining options (heavy metals)

Scanning EMshows surface structures

Interpreting Microscope Slides

• 2-D vs

oblique

TS

longitudinal,sagittal

central tangential

oblique

TS

longitudinal,sagittal

central tangential

transverse sectionstransverse sections

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

testisepididymis

epididymis

fat

seminiferous tubules

germ cells

lumenblood

ovaryantral 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