1

Stefan Baumgartner

Lund University Sept. 11, 2018

BIMA82: Developmental Biology and Genetics

Gametogenesis & Fertilization

What is wrong here?

What is Developmental Biology? ”Developmental Biology is at the core of all biology. It deals with the process by which genes in the fertilized egg control cell behaviour in the embryo and so determine its pattern, its form, and much of its behaviour.” Wolpert 2015

”For animals, fungi, and plants, the sole way of getting from egg to adult is by developing an embryo. Whereas most of biology studies adult structure and function, developmental biology finds the study of the transient stages leading up to the adult to be more interesting. Developmental biology studies the initiation and construction of organisms rather than their maintenance.” Gilbert and Barresi 2016

Phylogenetic tree showing the positions of some models organsims used in developmental biology

2

System DisadvantagesAdvantages

Yeast Very good genetic system. Easy to manipulate. Short generation time.

Unicellular organism. Limited cell-cell communication. Limited set of genes.

C. elegans Very good genetic system. Easy to manipulate. Short generation time.

Simple organism. 959 cells. Specialized genome.

DrosophilaExcellent genetic system. Excellent tools for manipulation. Short generation time.

No vertebra.

Xenopus Vertebrate. Eggs easily accessible. Transparent embryos. Ectopic studies.

No genetics. No mutants. Limited phenotypic studies.

Zebrafish Vertebrate genetic system. Embryos easily accessible. Transparent embryos.

New system. Genetically not well developed. Long generation time.

Chicken Vertebrate. Eggs easily accessible. Ectopic studies.

No genetics. No mutants. Limited phenotypic studies.

MouseMammalian genetic system. Good tools for experimental manipulation.

Complex organism. Long generation time. Embryos inaccessible. Expensive.

Developmental Biology Model Systems Comparative developmental milestones for the six model systems gastrulation segmentation

hatching/birth

last molt/ metamorphosis

Historical aspects I Early Scientific Period: William Harvey 1651 Book: “Generation of Animals”

Nikolas Hartsoeker 1695 “Homunculus” Marcello Malphigi 1673 chicken egg

1800-1880: Era of “Recapitulation” or “Embryological parallelism”

Karl von Baer Johann W. Goethe

Charles Darwin Ernst Haeckel

Zeus liberating living beings

weak magnifying glasses

primitive microscopes

future structure is already present at 1st stage of development

Historical aspects II Ernst Haeckel 1834-1919 “Entwicklungsmechanik”

1920 - 1950 Era of Biochemistry

1945 - 1985: Era of Cell Biology

1985 - : Era of Molecular Biology and Engineering

Thomas Hunt Morgan 1866-1945. “ Model systems”: Drosophila. Nobel price 1933

3

Concepts in Developmental Biology Axis

Germ Layers

Fate Map Developmental Commitment

not committed

committed

4

Cytoplasmic Determinant Induction

Lateral inhibition Stages of development presented in the course

Week 2: Fertilization, cleavage, C. elegans Stefan Baumgartner

Week 3: Early Development I: Early development in invertebrates: Drosophila Udo Häcker Week 4: Early Development II: Early development in vertebrates: amphibians & fish Udo Häcker + Labs

Week 6: Neurogenesis and eye development Per Ekström

Week 5: Early Development III: Early development in vertebrates: mammals and birds Udo Häcker + Labs

Week 7: Paraxial mesoderm / Limb development and regeneration Edgar Pera

Week 8: Organ development / Influence of the environment on development Edgar Pera, Stefan Baumgartner

2, 8 6 7, 8 3, 4, 5

5

Week 2: Gametogenesis, Fertilization and Cleavage

fertilization gametogenesis

cleavage

C. elegans

Gene targeting cloning iPS cells

Week 3: Early development in invertebrates: Drosophila

gastrulation

A-P axis formation in embryos Maternal genes Gap genes Pair-rule genes Segment polarity genes Hox genes

Establishment of D-V-axis

A-P axis formation in the oocyte

Segmentation mutants

Week 4: Early development of vertebrates: amphibians and fish

Xenopus

zebrafish

Gastrulation in amphibia and zebrafish Convergent extension PCP pathway

Spemann experiment

Left-right symmetry

Formation of the Spemann Organizer and axis formation

Week 5: Early development of vertebrates: mammals and birds

6

Integrated in weeks 3-5 : signalling events during early development

Cyclopia due to defective Shh signaling

Wg (Wnt) signalling pathway

Shh and Bmp in neural patterning

RTK-signaling pathway Sevenless pathway Notch pathway Hippo pathway TGF β pathway

Hedgehog signaling pathway

Week 6: Neurogenesis and eye development

eye development, eye field specification Formation of lens, cornea, retina

Neural induction Neurulation

Birth and migration of neurons: Stratification

Neural crest cells

growth cones trophic factors

Week 7: Paraxial mesoderm / Limb development and regeneration

apical ectodermal ridge AER, zone of polarizing activity ZPA

salamander planaria

hydra

Epimorphosis: adult structures dedifferentiate, grow and respecify into new structures Morphallaxis: existing structures repatterns, little regrowth

Week 8: Organ development & Influence of the environment on development

Organs: • (endoderm) • pancreas• kidney

Contergan (Neurosedyn) temperature and sex determination

alcoholism

Bisphenol (BPA)

Zika virus

7

relative sizes of eggs Structure of the human egg

Structure of the sea urchin egg Stages of oogenesis

from first menstruation onwards

3-8 months of embryonic growth

~ 400-800 000 oocytes

!

8

Maturation of the follicle. oviduct = fallopian tubules

The hormonal cycle of women.

LH

FSH

progesterone estrogen

Pregnancy test: mab against hCG (human Chorionic Gonadotropin, 244 aa)

Spermatogenesis Stages of spermatogenesis

ca. 24d

ca. 35d

primary spermatocytes

secondary spermatocytes

�capacitation�

during embryogenesis

at birth

birth spermatogonia

at puberty:

mouse: ca. 35 d total ca. 60d

Meiosis

9

Maturation of sperm: ”capacitation” occurs during the travel to the oocyte.

important issues during capacitation: • removal of cholesterol • certain proteins and sugars lost • membrane potential more negative • protein phosphorylation takes place

adenylate kinase

Structure of the sperm

Location of sperm production: seminiferous tubules

Leydig cells secrete testosterone

Formation of syncytial clones of germ cells. mitotic divisions

meiotic phase

differentiation

10

Sex Determination Sex determination in mammals

Inititation of primary sex determination in mammals: the testis and ovary pathway.

testis: Sry, Sox9

ovary: Wnt4, Rspo1

XX mouse + Sry transgene: Which sex?

but males do not form functional sperm !

Differentiation of human gonads, shown in transverse section

11

Development of the gonads and their ducts in mammals. I Development of the gonads and their ducts in mammals. II

From: Tridimensional Visualization and Analysis of Early Human Development. Cell 169, 161-173 (2017)

3 Methods: -  whole mount immunostaining -  3DISCO clearing -  light-sheet imaging

development of urogenital system in male embryos comparative 3D analysis of male and females embryos development of Müllerian MD and Wolffian WD duct

A duplication in Sox9 leads to intersex in deer.

unbranched antlers

retro-posed penis testicles

small testicles

How can this observation be explained?

What are the primary sex determinants in the animal kingdom?

XX-XY type XX-XO type

ZW-ZZ type

ZO-ZZ type

mammal, insects: Drosophila

birds, fishes, reptiles, some insects

insects: bugs, cockroaches & grasshoppers

bees, moths, butterflies

mammals: Y determines male

Drosophila: ratio between sex chromosomes / autosomes

12

Fertilization The moment of fertilization

The acrosomal reaction

sperm: 1µm/min

ca. 7µm Prevention of polyspermy: fast block (only in sea urchin)

13

Film: sperm entry and cortical reaction => slow block (sea urchin)

cortical granule exocytosis

cortical granules serine proteases

Film: the cortical reaction and elevation of the vitelline envelope (fertilization membrane; sea urchin)

Sperm-egg binding models in mammals involving ZP3 (old) and ZP2 (new)

old model new model

mammals

The slow block system: cortical reaction (mammals)

Ovastacin=Astl cleaves ZP2

Localization of Ovastacin in cortical granules

14

Cleaved ZP2 is critical for inhibiting polyspermy (mammals)

From: Gahlay et al., 2010

ZP2Mut cannot be cleaved anymore

Humans have 4 ZP proteins (mice only 3), and only ZP2 binds sperms (mammals)

From: Baibakov et al., 2012

S p e r m a t o z o a - Z P interaction is largely species- species specific. Human spermatozoa bind only to ZP from humans and hominoid species (gibbon, gorrilla), but not to other sub-hominoid species (baboon, rhesus monkey, squirrel monkey).

Assay: mouse eggs transgenic for each of the 4 human ZP proteins. Human sperm added ⇒  only ZP2 binds sperms.

Acrosome-reaction can already occur in the cumulus layer and acrosome-reacted sperm can bind the ZP (mammals)

From: Jin et al., 2011

Sperm with white arrow wins the race!

GFP = acrosomal reaction not yet completed (yellow arrows)

Juno – Izumo binding is essential for fertilization and may contribute to the membrane - block to polyspermy (mammals).

from: Cell Cycle Vol. 13, issue 13 (2014)

Juno- females are infertile

Rapid shedding of Juno = possible block to polyspermy, but probably not a fast block.

Note: reaction takes place at plasma membrane

GPI- anchored

15

Juno is the egg Izumo receptor and is GPI anchored.

from: Bianchi et al., Nature (2014)

recombinant Izumo1 protein

recombinant Izumo1 protein + juno cDNA

antibody staining using Juno ab’s

preincubation with Juno ab’s + recombinant Izumo1 protein

PIP lipase C treatment

Table of events during sea urchin fertilization.

Possible mechanisms of egg activation in sea urchin. Possible mechanisms of egg activation in mammals.

from: Nat Rev Mol Cell Biol. 2013 Sep;14(9):549-62.

Look at sperm! What is wrong?

16

Fusion of sperm and egg pronuclei (mammals). Film: parental chromosomes and how they fuse (mammals)

Brandnew: Dual spindle formation in mammals (mammals)

Reichmann et al., Science 361 (July 2018)

Non-disjunction of chromosomes.

17

Trisomy of the Autosomes: I (hyperdiploidy). Trisomy of the Autosomes: II

Trisomy 21

Trisomy 13

Trisomy 18 caused by: ~ 80% in females / ~ 20% in males

Robertsonian translocation as a cause for familial DS

�︎ DS �︎ �︎ wt︎ wt︎

Hypodiploidy

Turner Syndrome 45, X = female

18

Trisomy of the sex chromosomes.

Klinefelter Syndrome 47, XXY = male with female look

Other unusual chromosomal anomalies. Mosaicism: a person who has at least two cell lines with two or more different genotypes, either in autosomes or sex chromosomes. Occurs due to nondisjunction during early cleavage. Triploidy: 69 chromosomes. Triploid fetuses have severe intrauterine growth retardation with small trunk + other anomalies. Tripoloidy occurs in about 2% of all embryos, but most of them abort spontaneously. What could be causes for triploidy ?? Tetraploidy: 92 chromosomes. Doubling of the diploid chromosomal number to 92 probably occurs during the first cleavage division. Abort very early. Tetrasomy and Pentasomy: females: 48, XXXX and 49, XXXXX males: 48, XXXY; 48, XXYY; 49, XXXYY and 49, XXXXY Symptoms can be relatively minor or more severe. They can include learning challenges, social difficulties, congenital heart defects, and various physical attributes common to many chromosome disorders.

The case of Caster Semenya.

Gold medal in 800 m at Rio Olympics in 2016

Sex test results were never published officially, but resulting in claims that Semenya had an intersex trait. In 2015, the IAAF suspended the policy on hyperandrogenism (= high levels of testosterone) in women.

Literature

Gilbert, 11th ed., pp 9- 22, pp 181- 250 Wolpert and Tickle, 4th ed., pp 329-350 Alberts et al., 5th ed., chapter 21, pp 1287-1301 Slack: Chapter 4 (added to course library) Good review: Restarting life: fertilization and the transition from meiosis to mitosis. Clift, D. and Schuh, M. Nat Rev Mol Cell Biol. 2013 Sep;14(9):549-62. The cell biology of mammalian fertilization. Okabe, M. (2013), Development 140, 4471-4479.