Errores Meioticos y Espermatogenesis.dra.Martin

8/2/2019 Errores Meioticos y Espermatogenesis.dra.Martin

1/44

Meiotic Errors and

SpermatogenesisMexico City, 2007

Rene Martin, Ph.D., FCCMG

Professor, Dept. of Medical GeneticsUniversity of Calgary

Canada Research Chair in Genetics


2/44

Chromosome Abnormalities

very common in humans

.6% newborns

6% stillborns

60% spontaneous abortions

estimates at conception: 20 - 50%


3/44

Animal Models

laboratory rodents 0 - 5%

domestic animals 1 - 8% primates 12%

Frequency of chromosome abnormalitiesin gametes and zygotes:


4/44

Cause of ChromosomalAbnormalities

very little information

produced in eggs and sperm, but most dieas embryos - information lost

need to study chromosome abnormalitiesin human eggs and sperm


5/44

Methods to Study Chromosomes in

Human Spermatozoa

sperm karyotypes hamster oocytes fused with human sperm

fluorescence in situ hybridization (FISH) chromosome-specific DNA probes

polymerase chain reaction (PCR) single sperm analysis for research

analysis of single genes cytoimmunofluorescence of meiotic

chromosomes analysis of recombination


6/44


7/44


8/44


9/44

Distribution of Aneuploidy

Among Chromosome Groups

clues about etiology of aneuploidy

all chromosomes equal frequency?

certain chromosomes predisposed


10/44

Aneuploidy in Humans

newborns: trisomy 13

18

21

sex chromosomes

susceptible to nondisjunctionor

compatible with survival


11/44

11,615 Sperm Karyotypes

hyperhaploidy in all chromosome groups

all chromosomes susceptible to

nondisjunction

significant increase for chromosome 21and sex chromosomes (p=.0001)


12/44

Sperm Karyotyping

plus

precise sperm karyotypes

numerical and structural abnormalities

information on all chromosomes

minus

technically difficult, slow, expensive

available in only a few labs worldwide

requires sperm with fertilization capability


13/44

FISH Studies in Human Sperm

study interphase sperm

no fertilization

rapid, inexpensive

increase sample size from 11,000 to >10,000,000


14/44


15/44

Mean Disomy Frequency in Sperm

Chromosome % Disomy1 .092 .084 .11

9 .1412 .1613 .1915 .1116 .1118 .1120 .1221 .29* p


16/44

Nondisjunction in IndividualChromosomes

FISH results corroborate results from

sperm karyotypes

increased frequency of aneuploidy for Ggroup chromosomes (21 & 22) and sexchromosomes in human sperm


17/44

Other FISH Studies

increased frequency of disomy for sexchromosomes

Williams et al., 1993

Spriggs et al., 1995

Scarpato et al., 1998

Kunathikom et al., 2002

increased frequency of disomy 21 Spriggs et al., 1996

Blanco et al., 1996

Oliver-Bonetet al.

, 2001


18/44

Increased Susceptibility toNondisjunction

G-group (21 and 22) and X-Y bivalent

have only one crossover

if recombination absent or reduced, mayincrease the chances of nondisjunction


19/44


20/44

Most Sex Chromosomal AneuploidiesResult from Paternal Nondisjunction

paternal:

47,XYY 100%

45,X 80% (Hassold et al., 1988)

47,XXY 50% (Jacobs et al., 1988) 47,XXX 7% (May et al., 1990)


21/44

Meiotic Pairing of SexChromosomes

recombination required in

pseudoautosomal region necessary for proper segregation of sex

chromosomes

decreased recombination in 47,XXY ofpaternal origin (Hassold et al., 1991)


22/44

Single Sperm Typing

to determine if there is a relationship between

recombination in the pseudoautosomal regionand nondisjunction

compared frequency of recombination betweenSTS/STS pseudogene (sex specific locus) andDXYS15 (pseudoautosomal locus) in unisomicvsdisomic sperm


23/44

Heterozygous Male


24/44

Methods

sperm sonicated to remove tails

flow cytometer separates individual sperm

single sperm into 96-well plates(+ve and -ve controls)

disomic sperm (24,XY) identified by FISH,

scraped off slide & micromanipulated intoindividual tubes

PCR: DXYS15 and STS/STS pseudogene


25/44

Results of Single Sperm Typing

329 unisomic sperm - 38% recombination

150 disomic (24,XY) - 25% recombination

significant decrease in recombination in XYsperm (p=.001)

lack of recombination directly linked tonondisjunction

Ch Ab li i i


26/44

Chromosome Abnormalities inMale Infertility

somatic chromosomal abnormalities

consequences in sperm

infertile men with normal karyotypes

chromosomal abnormalities in sperm


27/44

Sperm Chromosomal Abnormalitiesin Infertile Men

oligo-, aestheno- or teratozoospermia

46,XY somatic karyotype

518 sperm karyotypes

significant increase in aneuploidy (4X)

FISH on 103,130 sperm chromosome 1,12, X, Y

significant increase in disomy 1 and XY (3X)


28/44

Infertile Men

FISH analysis for chromosomes 13 & 21

90,809 sperm from 9 infertile men significant increase in the frequency of disomy

for chromosomes 13 (p


29/44

Infertile Men: Other 3-ColourFISH Studies

>30 multicolour FISH studies

most show an increased frequency of

disomy for autosomes and sex

chromosomes

mainly severe infertility (OAT) or mixed

infertility

I d F f


30/44

Increased Frequency ofChromosome Disorders After ICSI

Van Opstal et al., 1997 6/71 prenatal diagnosis: 8% abnormal sex chrom. abnormalities: all paternal origin

Aboulghar et al., 2001 15/430 consecutive babies abnormal: 3.5% sex chromosomes and autosomes

Devroey & van Steirteghem, 2004 2622 fetal karyotypes: 3% abnormal 1.6% de novo: sex chromosomes and autosomes 1.4% inherited

I fl M th d


31/44

Immunofluorescence Methodsto Study Meiosis

allows study of recombination in allchromosomes

analysis of chromosome pairing byvisualization of synaptonemal complex

antibodies to:

synaptonemal complex (SCP1/SCP3)

recombination foci (MLH1)

centromeres (CREST)

Lepto diplotene


32/44

Lepto-diplotenemeiosis diagram


33/44


34/44

8-1-20

Analysis of Synaptonemal


35/44

Analysis of SynaptonemalComplexes 27 Normal men

testicular samples from vasectomyreversals (15) and cancer patients (12)

recombination foci - mean 48.5/cell 90% cells in pachytene

5% cells have at least 1 bivalent with no

recombination foci

no significant difference in vasectomy

reversals vs cancer patients


36/44

Infertile Men with Pachytene Cells

6/6 men with obstructive azoospermia(OA) had pachytene cells

mainly congenital absence of the vasdeferens (CF)

14/29 men with nonobstructive

azoospermia (NOA) had pachytene cells 14 men with no meiotic cells

1 man with a block at zygotene


37/44

SC 12-3-2

Mean Frequency of


38/44

Mean Frequency ofRecombination

49

47

43

Control OA* NOA*

*p


39/44

% Cells with at Least 1 Bivalent

with no Recombination

5

9

29

Control OA NOA*

*p=0.0005, Z-test


40/44

noa sc h7-3-5


41/44

cenM-FISH / Karyotyped SC


42/44


43/44

Summary

in both NOA and OA, abnormalities in:

chromosome pairing

decreased frequency of recombination

increased frequency of bivalents with no

recombination foci

could lead to meiotic arrest or increased frequency

of aneuploid sperm


44/44

Acknowledgements

Qinghua Shi

Maria Oliver-Bonet

Fei Sun Nafisa Moosani

Monica Mikhaail-Philips

Jena Smith

Brenda McInnes

Evelyn Ko

Paul Turek

Cal Greene Peter Moens

Marv Fritzler

Terry Ashley

Fred Rademaker

Canadian Institutes of Health Research

Errores Meioticos y Espermatogenesis.dra.Martin

Documents

Transcript of Errores Meioticos y Espermatogenesis.dra.Martin