Making Germ Cells Flashcards
where do germ cells come from
position dependent - specific location in embryo and depends on signalling (wnt, tgfbeta)
what determines whether they will become oocytes or sperm
gonadal environment - whether somatic cells of gonad follow ovarian or testicular pathway
what controls entry into meiosis
retinoic acid - produced by both embryos but degraded by male embryos (do not enter meiosis)
what happens by time female born
all recombination and crossing over of chromosomes has occurred in all oocytes
describe primordial germ cells
cells in early embryo
must choose cell fate = xx or xy (oogonia or spermatogonia)
how does pgc choose cell fate
Depends on environment = has no actual preference just responding to environment
are germ cells fundamentally different from somatic cells
yes and no
describe germ cells are fundamentally different from somatic cells
egg–> soma
egg–>germ
egg–>germ–>germ
germ cells = have infinite life sorta, egg becomes mebryo and keeps going on
somatic cells = finite, like house for germ cells to live and propagate indefinitely
describe germ cells are NOT fundamentally different from somatic cells
egg–>soma–>germ–>soma
mammals more like this
gives rise to many cell types - some germ and some soma
describe germ cells of nematode - ascaris
certain regions of chromatin degraded in somatic cells = limited subset of genes to work with, incomplete genome
all chromatin retained in germ cells = complete genome
describe germ cells of fly (drosophila)
poles segregated at posterior end of embryo (pole plasm) contain germ cell determinants
early spatial segregation
posterior pole cells - descendants form pcgs
describe germ cells of c elegans (worm)
aggregates of mitochondria, protein and rna in egg (p granules) become segregated into germ cells - germ cells set aside very early from somatic cells
intermingled germ and somatic then during cleavage the germ cells cluster into 2 cells –> descendants become germ cells
describe germ cells of mammals
Lessons from monozygotic siblings and embryo biopsies
identical twins/triplets = at early stage embryogenesis (20-50 cells) = fragments and each becomes embryo
serves as Argument against c elegans model also ivf (genetic testing done to check for mutations, can biopsy embryo and see if have mutations, must take one of cells and do genetic analysis, not a correct model of germ cells cause could end up taking cell with germ cell precursors)
describe differentiation of pcgs = mouse
gestation period 3 weeks
pcg determination –> pcg migration–> sex determination –> gametogenesis
cell fate assigned as pcgs = once start to develop must migrate to developing gonad
describe anatomy of embryo at pcg specification - mouse
all cells descended from fertilized egg
extraembryonic ectoderm = contributes to placenta
proximal posterior epiblast = posterior part tail
anterior epiblast = head part
epiblast = forms mouse itself
not linear but axis still present
describe anatomy of embryo at pcg specification - mouse vs human
much more linear
pgc’s originate from posterior side
tail and head regions
describe Anatomical position of newly specified PGCs
in mouse = one origin, primitive streak
in human = pcgs arise from posterior end (specific), could be 2 origins - hard to see, analogous region in hands
Why do the PGCs arise in the posterior epiblast?
can be explained by an experiment
cells from distal tip of epiblast which normally give rise to neuroectoderm , when transplanted to post epiblast = give rise to pcgs
recover 6.5 day embryo from mouse and dissect out cells from future trunk of embryo = transplant into new position and cells acquired fate of new position
not where they were originally from = positional
name 2 cell signalling pathways important in pcg specification
wnt
Transforming growth factor beta
describe wnt singling - absence of ligand
off
no wnt
beta catenin = made by cell
if no ligand it’s bound in complex and then degraded by ub
describe wnt singling - presence of ligand
wnt binds to frizzled (activates receptor on surface of cell)
starts intracellular signalling pathway
Complex disassembles and beta catenin is stabilized = not degraded so enters nucleus and activates transcription of target genes with help of partner protein
describe wnt
ligand that activates pathway encoded by wnt genes
widespread
describe tfgbeta signalling
ligand binds receptor on membrane = signalling pathway
smads stabilized/accumulated/translocated to nucleus and transcriptional activation of genes
involved with bmp = bone morphogenic protein
do wnt and tgfbeta activate same genes
nah
describe importance of wnts and bmps
genes located near where germ cells arise
wnt3 = more locally secreted, influences cells around/same cells to become pgcs
Bmp4 (tgfbeta fam member) = bone morphogenic protein, extra embryonic ectoderm secretes bmp to act on epiblast cells (activate expression of certain genes)
BOTH PATHWAYS important for cells to become pcgs - both work together with other factors = interaction of multiple signalling pathways
describe key genes of specification of primordial germ cells - gen
what happens inside cells to give them this property of being pcgs
expression of these genes needed in pcgs
genes identified as being important for germ cells to acquire expression identity
expressing identity - right place and time
describe key genes of specification of primordial germ cells -specific genes
lfitm3/fragilis = transmembrane protein/vrial entry
dppa3/stella = uncertain
prdm1/blimp1= dna binding transcriptional regulator
prdm14= dna binding transcriptional regulator
tcfap2c (AP2y)= dna binding transcriptional regulator
like transcription factors
do no know if activating or repressing
describe differentiation of pcgs
demethylation of dna
describe methylation of dna - general process
carbon in position number 5 can be modified by methyl group
change catalyzed by certain enzymes
describe methylation of dna - sequence
specific sequence susceptible to methylation = 5’–>3’ dir, CG (if G next to it = susceptible)
describe methylation of dna - process specifics
daughter cell has same methylation as parent because of selective nature of methyl transferase = dna methylation by DNMT1
very rapid and selective, loves to add methyl to cytosine opposite a methylated cytosine
other cytosines (not opposite methylated cytosine) = remains unmethylated
describe methylation of dna - why it doing that
easy for methylated cytosine to be propagated during cell division- easy for chromatin to be propagated
can dna methylation be inherited
can be transmitted after dna rep and therefore is heritable during cell proliferation
some methylations start de novo = from no methylation
if see hemi methylated dna = rapidly methylates other side
describe dna methylation of gene promoters
linked to repression of gene expression
highly methylated transposable element = shut down expression
in promoter region of highly expressed genes = high density of CG residues, unmethylated in promoter region = expression
describe what happens to dna in pcgs
once germ cells specified = lots of demethylation of dna
function = returns chromatin to ground state, all cells being made are in undifferentiated state - can activate methylation characteristics of different cells
so they can be methylated for specific function = can become anything
how does dna demethylation help pcgs
may help give germ cells totipotency
demethylation occurs in pcgs shortly after they have been specified
descendants of totipotent germ cells have ability to acquire any cell type pattern - adopt future fate
describe migration of pcgs
originate in one spot then move to different spot
Experiment = put labelled marker that can be seen (gene) into pgcs = GFP (labels germ cells)
can see cells move to left and right sides and cluster into 2 groups = colonize left and right gonads
describe biological sex determination in most mammals - ancient
old wives tails =
position in uterus
sperm from left = girl or right = boy testis
higher = boy and lower = girl temp of uterine environment
none are true just theories
describe biological sex determination in most mammals - late 19th c 1959
discovery of chromosomes
turner syndrome = xo, do not need 2 x chrom to be female
klinefelter syndrome = xxy male, only need one y chrom to be male
describe biological sex determination in most mammals - late 19th c 1990
human sry discovered - part of y present in xx males
mouse sry (homologs) discovered also on y chrom
xx mice carrying sry develop as males
conclusion = if sry active in right place and time will look phenotypically like male, enough to be males
y chrom = mostly genes involved in regulating spermatogenesis and has sex determining region on short arm
describe The genetic basis of (most) mammalian biological sex determination
Experiment= males not making any sperm so came into clinic to be tested
2 x chroms and small portion of y translocated onto x
portion of y chrom translated onto x chrom in different phenotypic males having xx genotype
all discovered there was on area with overlap in the 6 different cases = male determining gene must be in this region - sry gene
describe structure of sry
300 base pairs
HMG domain = gives protein ability to bind dna, binds with preference for sequence specificity (ACAA and binds dna)
glutamine rich domain
how does sry work - males
sry expressed in fetal testis for only brief period of time
in somatic cells of xy gonad
for 1-2 days = binds to promoter of sox9 and turns on expression and sox9 can feedback on itself and keep it on
drives all somatic cells of gonad = makes males
1-2 days of sry expression = enough to trigger development of males
how does sry work - females
sox9 not turned on in xx gonad
sry not present at time of sex determination
not active in somatic cells of gonad during time of sex determination bc sry not present
how does sry work - sox9
autosomal - human chrom 17
dna binding protein (transcription factor, expression of bunch of genes)
mutations cause severe skeletal abnormalities, sex reversal = campomelic dysplasia
sox 9 not only involved in sex determination - famles have sox9 expressed elsewhere, does many things in cell types
how does sry work - in genital ridge vs other tissues and cells
in genital ridge = somatic cells of gonad sox9 expression requires SRY
other cells and tissues = sox9 expression does not require sry - other factors turn it on
describe crucial role of sry
sry expressed = male pathway
sry not expressed = female pathway
ONE OR OTHER NOT BOTH
entirely depends on environment
describe male differentiation pathway
sry expressed - somatic cells of genital ridge - not germ cells
sry turns on sox9
Differentiation of sertoli cells - start expressing genes, growth factors or hormones
–>differentiation of leydig cells (in testis, produce testosterone)
–> production of testosterone = male secondary sexual differentiation
describe female differentiation pathway
sry not expressed in somatic cells of genital ridge
differentiation of pre-granulosa cells - cells that surround and nourish oocyte in ovary during oocyte growth and development
–> differentiation of pre theca cells
–> female secondary sexual differentiation
what is not required for sexual differentiation of gonad
GERM cells are not required for sexual differentiation of gonad - and of individual
Determined by somatic cells - expression of sry/sox9
describe downstream of sox9
genital ridge formation = mesonephros (gives rise to kidney) + gonad
if +sry = male, fibroblast growth factor 9, amh, expression of genes needed for male development - phenotypic differentiation –> testicular development to male
if -sry = wnt4 and rspo1(enhances wnt signalling) - different set of genes activated in somatic cell –> ovarian development to female
describe biological sex determination of females - wnt
wnt –(r spondins)–> nuclear (active) b catenin –> transcription of target genes
wnt 4 = initially expressed in xx and xy, later then becomes restricted to xx (ovaries develop in females)
describe biological sex determination of females - mutations
wnt 4 mutations (inactivating) and r spondin mutations (inactivating) = partial female to male sex reversal
describe morphological aspects of sexual differentiation of males
clusters
sertoli cells, blood vessels = becomes somniferous tubules eventually
forms seminiferous tubules and germ cells all clustered together
describe morphological aspects of sexual differentiation of females
cells remain individual
eventually covered in granulosa cells
Beginning of formation of follicles - each houses one oocyte
describe development of germ cells within gonad
pcgs arrive in genital ridge –> 2-3rounds of cell proliferation –> proliferative arrest
if male = mitotic arrest maintained until puberty
if female = germ cells enter meiosis (early events, undergo synaptonemal complex formation and recomb between homologus chroms, happens before birth - doesnt go through divisions tho)
what is molecular basis of decision to enter meiosis
depends on expression of stra8 - correlates with entry into meiosis
Regulated by retinoic acid
Turned on in females not males
Experiment = in situ hybridization
stra8 expressed in female gonads but not males
not just marker but need for meiosis
required for germ cells to enter meiosis
What turns on expression of Stra8?
retinoic acid
retinol binds to receptor (stra8) then metabolized to retinoic acid
can activate expression of certain genes = retinoic acid receptor
explain stra8 mutations - experiment
in vitro
RAR antagonist = blocks receptor of retinoic acid in both males and females
if flood with high amounts retinoic acid = on both male and females turns on stra8 - if RAR-alpha or beta agonist
Why don’t XY germ cells respond to environmental retinoic acid?
xy express cyp26b1 = degrades retinoic acid
expressed in embryonic male gonads
if mutate cyp26b1 - male mice lacking it, the germ cells enter meiosis during embryogenesis - stra8 and meiotic genes turned on
Molecular control of entry into meiosis: summary FEMALES
retinol= from mesonephros or elsewhere and gives retinol (retinoic acid)
stimulates germ cells to enter meiosis
Molecular control of entry into meiosis: summary MALES
retinol but cyp26b1 from sertoli cells inhibits retinol = germs cells in mitotic arrest
* cyp26b1 produced by sertoli cells
describe gonochorism
Individuals reproduce as one sex throughout lifetime = male or female
describe protogyny
female first sequential hermaphroditism
individuals first reproduce as females, change sex once with increasing size/age and then reproduce as males
describe protandry
male first sequential hermaphroditism
individuals first reproduce as males, change sex once with increasing size/age and then reproduce as females
describe bidirectional sex
Individuals can change sex more than once in either direction throughout lifespan - usually starts from protogyny
describe simultaneous sex
Individuals produce gametes of both sexes at same time or in a short period of time
describe development of gonads - gen
when embryo = develop 2 kinds of ducts = wolffian and mullerian
has 2 structures but one retained and other disappears depends on if male or female
describe development of gonads - males
male hormones =
AMH (anti mullerian hormone) = mullerian ducts Disappear
Testosterone = wolffian duct derivatives form (differentiates into epididymis, vas deferens, helps seminal vesicle form)
amh also produced by females but not at this place or time
describe development of gonads - females
no male hormones so wolffian duct degenerates and mullerian gives rise to oviduct, uterus, cervix and vagina
describe rare patterns of sexual differentiation =
GENE = SRY
CHROM. SEX = XY
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = do not initiate testis dev - wont turn on sox9
phenotype = female
Characteristics = streak ovaries (do not develop well), no functional germ cells, mullerian duct derivatives bc no amh, no wolffian duct derivatives bc no testosterone
describe rare patterns of sexual differentiation =
GENE = SRY
CHROM. SEX = XXsry (translocation)
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = initiate testis dev as embryos
phenotype = male
Characteristics = small testis, no sperm, no mullerian duct derivatives bc make amh, wolffian duct derivatives bc make testosterone
describe rare patterns of sexual differentiation =
GENE = sox9
CHROM. SEX = xx
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = no effect on ovarian differentiation bc sox9 downstream sry
phenotype = female
Characteristics = campomelic dysplasia - bc sox9 needed in other cells of body
describe rare patterns of sexual differentiation =
GENE = sox9
CHROM. SEX = xy
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = do not initiate testis dev as embryos
phenotype = female
Characteristics = no functional germ cells, mullerian duc derivatives bc no amh, no wolffian duct derivatives bc no testosterone
describe rare patterns of sexual differentiation =
GENE = wnt4
CHROM. SEX = xx
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = lethal - partial or full ovary to testis reversal - bc needed in MANY TISSUES
describe rare patterns of sexual differentiation =
GENE = androgen receptor (AR x chrom)
CHROM. SEX = xx
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = very rare, females hard to find bc homozygous mutant for receptor is uncommon
phenotype = female
Characteristics =no functional germ cells, no wolffian duct derivatives bc no testosterone, no mullerian duct derivatives - start xy males develop so amh
describe rare patterns of sexual differentiation =
GENE = androgen receptor (AR x chrom)
CHROM. SEX = xy
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = androgen insensitivity - only one x mutant, start developing as males but no receptors for testosterone = do not know they are making it, leydig cells make but cannot do anything with the testosterone
phenotype = female
Characteristics =no functional germ cells, no wolffian duct derivatives bc no testosterone, no mullerian duct derivatives - start xy males develop so amh
describe rare patterns of sexual differentiation =
GENE = anti mullerian hormone or its receptor
CHROM. SEX = xx
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = nothing obvious (human) bc do nto want amh anyways
phenotype = female
Characteristics = fertile
describe rare patterns of sexual differentiation =
GENE = anti mullerian hormone or its receptor
CHROM. SEX = xy
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = if have amh = uterine development - undescended testes
phenotype = male
Characteristics = usually infertile but in vitro fertilization possible - not very many sperms being made
describe rare patterns of sexual differentiation =
GENE = sex chrom aneuploidy
CHROM. SEX = xo
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = turner syndrome
phenotype = female
Characteristics = 99% die before birth, no functional oocytes
describe rare patterns of sexual differentiation =
GENE = sex chrom aneuploidy
CHROM. SEX = xxx
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = triple x syndrome - 2 xs become inactivated
phenotype = female
Characteristics =fertile
describe rare patterns of sexual differentiation =
GENE = sex chrom aneuploidy
CHROM. SEX = xxy
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = klinefelter syndrome
phenotype = male, tend to be taller than avg
Characteristics = few/no functional sperm
describe rare patterns of sexual differentiation =
GENE = 5-alpha reductase (SRD5A2)
CHROM. SEX = xy
describe effect of genetic modification, phenotype and characteristics
effect of genetic modification = cannot convert testosterone to di-hydro testosterone = more active form of it, makes testosterone but just not super active form
looks like female at birth- internally lacking uterus (bc make amh)
male external genitalia form at puberty (due to increased production of testosterone or maybe become more sensitive to testosterone so do not need super active form to make genitalia)
some individuals produce functional sperm- some remain as intersex
wolffian duct precursors of males = do not degenerate, remain in quioescent phase then more testosterone or increased sensitivity = differentiation begins
