Making Germ Cells

Question 1

where do germ cells come from

Answer 1

position dependent - specific location in embryo and depends on signalling (wnt, tgfbeta)

Question 2

what determines whether they will become oocytes or sperm

Answer 2

gonadal environment - whether somatic cells of gonad follow ovarian or testicular pathway

Question 3

what controls entry into meiosis

Answer 3

retinoic acid - produced by both embryos but degraded by male embryos (do not enter meiosis)

Question 4

what happens by time female born

Answer 4

all recombination and crossing over of chromosomes has occurred in all oocytes

Question 5

describe primordial germ cells

Answer 5

cells in early embryo
must choose cell fate = xx or xy (oogonia or spermatogonia)

Question 6

how does pgc choose cell fate

Answer 6

Depends on environment = has no actual preference just responding to environment

Question 7

are germ cells fundamentally different from somatic cells

Answer 7

yes and no

Question 8

describe germ cells are fundamentally different from somatic cells

Answer 8

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

Question 9

describe germ cells are NOT fundamentally different from somatic cells

Answer 9

egg–>soma–>germ–>soma
mammals more like this
gives rise to many cell types - some germ and some soma

Question 10

describe germ cells of nematode - ascaris

Answer 10

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

Question 11

describe germ cells of fly (drosophila)

Answer 11

poles segregated at posterior end of embryo (pole plasm) contain germ cell determinants
early spatial segregation
posterior pole cells - descendants form pcgs

Question 12

describe germ cells of c elegans (worm)

Answer 12

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

Question 13

describe germ cells of mammals

Answer 13

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)

Question 14

describe differentiation of pcgs = mouse

Answer 14

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

Question 15

describe anatomy of embryo at pcg specification - mouse

Answer 15

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

Question 16

describe anatomy of embryo at pcg specification - mouse vs human

Answer 16

much more linear
pgc’s originate from posterior side
tail and head regions

Question 17

describe Anatomical position of newly specified PGCs

Answer 17

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

Question 18

Why do the PGCs arise in the posterior epiblast?

Answer 18

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

Question 19

name 2 cell signalling pathways important in pcg specification

Answer 19

wnt
Transforming growth factor beta

Question 20

describe wnt singling - absence of ligand

Answer 20

off
no wnt
beta catenin = made by cell
if no ligand it’s bound in complex and then degraded by ub

Question 21

describe wnt singling - presence of ligand

Answer 21

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

Question 22

describe wnt

Answer 22

ligand that activates pathway encoded by wnt genes
widespread

Question 23

describe tfgbeta signalling

Answer 23

ligand binds receptor on membrane = signalling pathway
smads stabilized/accumulated/translocated to nucleus and transcriptional activation of genes
involved with bmp = bone morphogenic protein

Question 24

do wnt and tgfbeta activate same genes

Answer 24

nah

Question 25

describe importance of wnts and bmps

Answer 25

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

Question 26

describe key genes of specification of primordial germ cells - gen

Answer 26

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

Question 27

describe key genes of specification of primordial germ cells -specific genes

Answer 27

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

Question 28

describe differentiation of pcgs

Answer 28

demethylation of dna

Question 29

describe methylation of dna - general process

Answer 29

carbon in position number 5 can be modified by methyl group change catalyzed by certain enzymes

Question 30

describe methylation of dna - sequence

Answer 30

specific sequence susceptible to methylation = 5'-->3' dir, CG (if G next to it = susceptible)

Question 31

describe methylation of dna - process specifics

Answer 31

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

Question 32

describe methylation of dna - why it doing that

Answer 32

easy for methylated cytosine to be propagated during cell division- easy for chromatin to be propagated

Question 33

can dna methylation be inherited

Answer 33

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

Question 34

describe dna methylation of gene promoters

Answer 34

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

Question 35

describe what happens to dna in pcgs

Answer 35

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

Question 36

how does dna demethylation help pcgs

Answer 36

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

Question 37

describe migration of pcgs

Answer 37

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

Question 38

describe biological sex determination in most mammals - ancient

Answer 38

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

Question 39

describe biological sex determination in most mammals - late 19th c 1959

Answer 39

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

Question 40

describe biological sex determination in most mammals - late 19th c 1990

Answer 40

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

Question 41

describe The genetic basis of (most) mammalian biological sex determination

Answer 41

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

Question 42

describe structure of sry

Answer 42

300 base pairs HMG domain = gives protein ability to bind dna, binds with preference for sequence specificity (ACAA and binds dna) glutamine rich domain

Question 43

how does sry work - males

Answer 43

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

Question 44

how does sry work - females

Answer 44

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

Question 45

how does sry work - sox9

Answer 45

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

Question 46

how does sry work - in genital ridge vs other tissues and cells

Answer 46

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

Question 47

describe crucial role of sry

Answer 47

sry expressed = male pathway sry not expressed = female pathway ONE OR OTHER NOT BOTH entirely depends on environment

Question 48

describe male differentiation pathway

Answer 48

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

Question 49

describe female differentiation pathway

Answer 49

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

Question 50

what is not required for sexual differentiation of gonad

Answer 50

GERM cells are not required for sexual differentiation of gonad - and of individual Determined by somatic cells - expression of sry/sox9

Question 51

describe downstream of sox9

Answer 51

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

Question 52

describe biological sex determination of females - wnt

Answer 52

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)

Question 53

describe biological sex determination of females - mutations

Answer 53

wnt 4 mutations (inactivating) and r spondin mutations (inactivating) = partial female to male sex reversal

Question 54

describe morphological aspects of sexual differentiation of males

Answer 54

clusters sertoli cells, blood vessels = becomes somniferous tubules eventually forms seminiferous tubules and germ cells all clustered together

Question 55

describe morphological aspects of sexual differentiation of females

Answer 55

cells remain individual eventually covered in granulosa cells Beginning of formation of follicles - each houses one oocyte

Question 56

describe development of germ cells within gonad

Answer 56

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)

Question 57

what is molecular basis of decision to enter meiosis

Answer 57

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

Question 58

What turns on expression of Stra8?

Answer 58

retinoic acid retinol binds to receptor (stra8) then metabolized to retinoic acid can activate expression of certain genes = retinoic acid receptor

Question 59

explain stra8 mutations - experiment

Answer 59

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

Question 60

Why don’t XY germ cells respond to environmental retinoic acid?

Answer 60

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

Question 61

Molecular control of entry into meiosis: summary FEMALES

Answer 61

retinol= from mesonephros or elsewhere and gives retinol (retinoic acid) stimulates germ cells to enter meiosis

Question 62

Molecular control of entry into meiosis: summary MALES

Answer 62

retinol but cyp26b1 from sertoli cells inhibits retinol = germs cells in mitotic arrest * cyp26b1 produced by sertoli cells

Question 63

describe gonochorism

Answer 63

Individuals reproduce as one sex throughout lifetime = male or female

Question 64

describe protogyny

Answer 64

female first sequential hermaphroditism individuals first reproduce as females, change sex once with increasing size/age and then reproduce as males

Question 65

describe protandry

Answer 65

male first sequential hermaphroditism individuals first reproduce as males, change sex once with increasing size/age and then reproduce as females

Question 66

describe bidirectional sex

Answer 66

Individuals can change sex more than once in either direction throughout lifespan - usually starts from protogyny

Question 67

describe simultaneous sex

Answer 67

Individuals produce gametes of both sexes at same time or in a short period of time

Question 68

describe development of gonads - gen

Answer 68

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

Question 69

describe development of gonads - males

Answer 69

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

Question 70

describe development of gonads - females

Answer 70

no male hormones so wolffian duct degenerates and mullerian gives rise to oviduct, uterus, cervix and vagina

Question 71

describe rare patterns of sexual differentiation = GENE = SRY CHROM. SEX = XY describe effect of genetic modification, phenotype and characteristics

Answer 71

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

Question 72

describe rare patterns of sexual differentiation = GENE = SRY CHROM. SEX = XXsry (translocation) describe effect of genetic modification, phenotype and characteristics

Answer 72

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

Question 73

describe rare patterns of sexual differentiation = GENE = sox9 CHROM. SEX = xx describe effect of genetic modification, phenotype and characteristics

Answer 73

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

Question 74

describe rare patterns of sexual differentiation = GENE = sox9 CHROM. SEX = xy describe effect of genetic modification, phenotype and characteristics

Answer 74

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

Question 75

describe rare patterns of sexual differentiation = GENE = wnt4 CHROM. SEX = xx describe effect of genetic modification, phenotype and characteristics

Answer 75

effect of genetic modification = lethal - partial or full ovary to testis reversal - bc needed in MANY TISSUES

Question 76

describe rare patterns of sexual differentiation = GENE = androgen receptor (AR x chrom) CHROM. SEX = xx describe effect of genetic modification, phenotype and characteristics

Answer 76

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

Question 77

describe rare patterns of sexual differentiation = GENE = androgen receptor (AR x chrom) CHROM. SEX = xy describe effect of genetic modification, phenotype and characteristics

Answer 77

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

Question 78

describe rare patterns of sexual differentiation = GENE = anti mullerian hormone or its receptor CHROM. SEX = xx describe effect of genetic modification, phenotype and characteristics

Answer 78

effect of genetic modification = nothing obvious (human) bc do nto want amh anyways phenotype = female Characteristics = fertile

Question 79

describe rare patterns of sexual differentiation = GENE = anti mullerian hormone or its receptor CHROM. SEX = xy describe effect of genetic modification, phenotype and characteristics

Answer 79

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

Question 80

describe rare patterns of sexual differentiation = GENE = sex chrom aneuploidy CHROM. SEX = xo describe effect of genetic modification, phenotype and characteristics

Answer 80

effect of genetic modification = turner syndrome phenotype = female Characteristics = 99% die before birth, no functional oocytes

Question 81

describe rare patterns of sexual differentiation = GENE = sex chrom aneuploidy CHROM. SEX = xxx describe effect of genetic modification, phenotype and characteristics

Answer 81

effect of genetic modification = triple x syndrome - 2 xs become inactivated phenotype = female Characteristics =fertile

Question 82

describe rare patterns of sexual differentiation = GENE = sex chrom aneuploidy CHROM. SEX = xxy describe effect of genetic modification, phenotype and characteristics

Answer 82

effect of genetic modification = klinefelter syndrome phenotype = male, tend to be taller than avg Characteristics = few/no functional sperm

Question 83

describe rare patterns of sexual differentiation = GENE = 5-alpha reductase (SRD5A2) CHROM. SEX = xy describe effect of genetic modification, phenotype and characteristics

Answer 83

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