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Advanced concepts in reproduction > Week 2 > Flashcards

Week 2 Flashcards

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1
Q

What regulates sex determination in the Bipotential gonad?

A

In both XX and XY gonads at 11.25dpc Fgf9 transcripts are detected near the gonad surface whereas Wnt4 transcripts are detected near the gonad mesonephric boundary

A genetic or environmental switch initiates the male pathway by creating an imbalance between these signals

  • In mammals, this imbalance occurs through the up-regulation of Sox9
  • Sox9 upregulates Fgf9 and Fgf9 maintains SOX9, forming a positive feed-forward loop in XY gonads
  • The balance between Fgf9 and WNT4 signals is shifted in favour of Fgf9 and the dominance of the male pathway is established
  • In the absence of a feed-forward loop between SOX9 and Fgf9 (as in XX gonads), WNT4 blocks Fgf9 initiating the female pathway

Fate determination morphogenesis determines if a testis or an ovary is produced

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2
Q

What do Wnt4 and Rspo1 do?

A

Repress testicular fate and promote ovarian development

Beta-catenin is normally synthesised and degraded quickly

Wnt signalling causes B-catenin to stabilise, this levels increase in the cell

As levels increase, B-cat moves to nucleus to regulate gene expression

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3
Q

What does beta-catenin (B-cat) do?

A

Suppression of male gonadal development

Germ cell survival regulation of meiosis
Activation of ovary genes:
- FST, Foxl2, Bmp2

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4
Q

What lessons have been learned from different species regarding Foxl2?

A

MOUSE KO 1 (Schmidt et al 2004):

Insertion of LacZ gene into Foxl2 gene, removing amino acids 62-365
- Forms fusion of first 65aa of Foxl2 and LacZ
- No eyelid defect
- Follicles form, but fail to grow

MOUSE KO 2 (Uda et al 2004):

Deletion of entire Foxl2 gene
- Eyelid defects
- Ovarian failure due to failure to assemble enough follicles
- Sex reversal - Sox9 expression reactivated?

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5
Q

What is Foxl2 required for?

A

Granulosa cell differentiation

Pre-granulosa cells switch on Foxl2 as they migrate away from the Ovarian Surface Epithelium (OSE)

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6
Q

What determines the developmental fate of a germ cell?

A

A balance of signals

Fgf9
Cyp26B1
Nanos2

–> Male fate
(spermatogenesis, germ cell arrest)

Retinoic acid (RA)

–> Female fate
(Activation of Stra8 entry into meiosis)

In the ovary, absence of Cyp26b1 exposes germ cells to meiosis inducing RA

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7
Q

What does Fgf9 lead to in a germ cell?

A

Decrease in Stra8

Male fate
expression of
- Oct4
- Sox2
- Nanos2
- Dnmt3L
- P15

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8
Q

What does Retinoic acid (RA) lead to in a germ cell?

A

Increase in Stra8

Female fate

Meiosis

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9
Q

What do germ cells form in the developing ovary?

A

Interconnected cysts known as germ cell nests

A. Primordial germ cell, forming cysts
Days 11-13.5

B. Germline Cyst
Cysts
Days 13.5-~21

C. Primordial follicle
Primordial follicles
Days 21-25

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10
Q

What are KIF23 and TEX14?

A

Protein components of the cytoplasmic bridges between oocytes

Nests connected by intercellular bridges

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11
Q

Why are intercellular bridges between oocytes important?

A

Allow sharing of cytoplasm and organelles

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12
Q

What may germ cell cysts (nests) be?

A

A conserved feature of ovarian germ cell development

In the adult drosophila ovary , germ cell cysts also form - but only one cell becomes the oocyte.

The others, called nurse cells, sacrifice themselves, providing the oocyte with their cytoplasm and organelles

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13
Q

What does the nest breakdown do?

A

Releases oocytes to assist with somatic cells and form follicles

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14
Q

What are the steps to nest breakdown?

A
  1. Oogonial nests
    - Mitosis and meiotic prophase I
  2. Nest breakdown
    - Association of oocytes with somatic cells
  3. Primordial follicle formation
    - Meiotic arrest
    - Oocytes not assembled into follicles die
    - Follicles arrest until puberty
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15
Q

What factors regulate follicle formation?

A

Oocyte-expressed factors

Factor in the germline alpha (Figla)

Figla gene expression peaks at birth - time of follicle formation in mice

By 2d after birth WT mouse ovaries are full of oocytes in primordial follicles

Figla KO oocytes never form follicles - all oocytes gone (dead?) by 2 days after birth

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16
Q

Does oestrogen determine the timing of the external regulation of follicle formation?

A

HYPOTHESIS:
Follicle formation occurs after birth because the newborn mouse is no longer exposed to high levels of oestrogen in utero, and this oestrogen is required for maintaining germ cell nests in tact

Newborn mouse ovaries cultured in vitro with oestrogens show delayed breakdown of cysts and follicle formation

BUT… mice lacking both oestrogen receptors (Era and Era) show largely normal cyst breakdown

AND… In humans, follicle formation happens whilst the foetus is still in the womb (18 weeks onwards) exposed to high levels of oestrogen

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17
Q

What happens when oocytes proceed through meiotic prophase I?

A

a. Prophase
First trimester
- Commitment to meiosis

b. Dictyate arrest
Second trimester
- Follicle formation
Birth
- Oocyte growth

c. Divisions
Adult
- Fertilisation

HUMAN OOCYTES CAN REMAIN ARRESTED FOR >50 years

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18
Q

What are the stages of meiotic prophase I?

A
  1. Leptotene
    First stage of meiotic prophase. Chromosomes begin to condense
  2. Zygotene
    Second stage of meiotic prophase. Homologous chromosomes pair.
  3. Pachytene
    Third stage of meiotic prophase. Homologous chromosomes are tightly held together by the synaptonemal complex and homologous recombination (‘crossing over’) begins.
  4. Diplotene
    The fourth stage of meiotic prophase. The synaptonemal complex breaks down, but homologous chromosomes are held together at sites of recombination
  5. Diakinesis
    Final stage of meiotic prophase. Chromosomes condense further, the nuclear envelope breaks down and the meiotic spindle begins to form. The bivalents are ready for metaphase

STOP HERE UNTIL PUBERTY

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19
Q

Where does massive loss of oocytes occur?

A

During foetal life

Successful meiotic recombination and arrest –> Survival

Failed meiotic recombination and arrest –> Death by defect

Germline cyst breakdown –> Death by self-sacrifice (altruistic death)

Growth factors present –> Survival

Growth factors limiting –> Death by neglect

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20
Q

What are Wnt4/RPSO1 and Foxl2 required for?

A

The primordial gonad to take the ovarian pathway

Wnt4 blocks testis development and regulates ovarian gene expression

Foxl2 further requires for granulosa cell differentiation

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21
Q

Basic stages of foetal ovarian development:

A

Germ cells in the ovary divide incompletely to form interconnected cysts, these then enter meiosis

Cysts break down to yield individual oocytes which are either assembled into follicles or die

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22
Q

Steps of follicle maturation in the ovary:

A

Prenatal

  1. Primordial germ cell

migration to ovary

  1. Oogonium

entry into meiosis I

Postnatal and post pubertal

  1. Primordial follicle
  2. Primary follicle
  3. Secondary follicle
  4. Tertiary (Graafian) follicle

Gonadotrophin surge at middle of menstrual cycle stimulates reentry of oocyte into meiosis as far as metaphase II

  1. Ovulated oocyte

insemination

  1. Fertilised ovum (zygotę)
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23
Q

Describe a primordial follicle

A

Primary oocyte

Granulosa cell layer

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24
Q

Describe a primary follicle

A

Primary oocyte

Zona pellucida

Granulosa cell layer

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25
Describe a secondary follicle:
Nucleus ("germinal vesicle") of oocyte Primary oocyte Zona pellucida Granulosa cells grow like layers of an onion
26
Describe a tertiary (Graafian) follicle:
Follicle fluid in antrum Granulosa cell layers Oocyte Theca cell layers
27
Describe an ovulated oocyte:
Spindle First polar body Zona pellucida Cloud of granulosa ('cumulus') cells
28
Describe a fertilised ovum (zygote):
Male and female pronuclei First and second polar bodies
29
Describe the follicle as a niche for oocyte including interactions between germ cell and somatic cell:
Theca cells: Steroidogenesis --> Basal lamina, granulosa cells Inhibition of meiosis --> Granulosa cells Basal lamina: Steroidogenesis --> Follicular fluid Metabolic aid --> Follicular fluid Cytoplasmic maturation and capacitation --> Follicular fluid Serum: Metabolic aid --> Basal lamina Cytoplasmic maturation and capacitation --> Basal lamina LH surge: COC expansion --> Granulosa cells, Cumulus cells Resumption of meiosis --> Granulosa cells Granulosa cells: Steroidogenesis --> Follicular fluid, theca cells Resumption of meiosis --> Cumulus cells Cytoplasmic maturation and capacitation --> Follicular fluid Inhibition of meiosis --> Follicular fluid COC expansion --> Cumulus cells Follicular fluid: Metabolic aid --> Cumulus cells Steroidogenesis --> The oocyte, Cumulus cells Cytoplasmic maturation and capacitation --> Cumulus cells, the oocyte Inhibition of meiosis --> Cumulus cells Cumulus cells: Resumption of meiosis --> The oocyte Inhibition of meiosis --> The oocyte COC expansion --> The oocyte Steroidogenesis --> The oocyte Metabolic aid --> The oocyte Cytoplasmic maturation and capacitation --> The oocyte
30
Describe the integration of cumulus-oocyte functions:
Regulateion of maternal mRNA translation in the oocyte plays a critical role in mediating protein secretion and feedback regulations In the follicle microenvironment, there is bidirectional exchange of signals between the oocyte and the surrounding somatic cells The signals from the somatic cells stimulate the fully grown oocytes to increase translation of selected mRNAs In turn, oocyte secreted facotrs may control cumulus cell function
31
What is the role of antioxidant defence in the integration of cumulus-oocyte functions?
Metabolise and/or neutralise ROS and protect the oocyte from oxidative stress-induced apoptosis
32
What is the role of Gap junctions in the integration of cumulus-oocyte functions?
Intercellular channels responsible for small molecules (ions, pyruvate, cyclic nucleotides, metabolites, amino acids, and RNA transcripts) Transfer from CCs to the oocyte, which contribute to meiosis, ATP production, and pH balance into the oocyte
33
What is the role of KIT pathway in the integration of cumulus-oocyte functions?
This pathway induces molecular events that dictate oocyte growth and induces the production of oocyte factors which stimulate granulosa cell proliferation
34
What is the role of GDF9 and BMP15 in the integration of cumulus-oocyte functions?
Induce expansion, metabolism, differentiation, proliferation, apoptosis, and luteinisation of CCs
35
What influence does the follicular luid (FF) constituent ROS and oxidative stress markers have on oocyte quality?
ROS and oxidative stress affect microtubule organisation and chromosomal alignment of metaphase II (MII) meiotic spindles in mouse oocytes
36
What influence does the follicular luid (FF) constituent Progesterone (P4) have on oocyte quality?
Lower Progesterone levels in follicular fluid are associated with more germinal vesicle and less MI and MII oocytes than higher progesterone levels
37
What influence does the follicular luid (FF) constituent Growth differentiation factor 9 (GDF-9) have on oocyte quality?
Higher GDF-9 levels in the FF are correlated with oocyte nuclear maturation and high-embryo quality
38
What influence does the follicular luid (FF) constituent bone morphogenic protein 15 (BMP-15) have on oocyte quality?
Higher BMP-15 levels in FF are associated with oocyte fertilisation and cleavage and with the best embryo morphology
39
What influence does the follicular luid (FF) constituent Amphiregulin have on oocyte quality?
Amphiregulin levels in FF are positively correlated with the number of available embryos
40
What influence does the follicular luid (FF) constituent Matrix metalloproteinase 2 (MMP-2) have on oocyte quality?
Increased activity of MMP-2 in FF is related to 100% of matured oocytes
41
What influence does the follicular luid (FF) constituent Tumour necrosis facotr alpha (TNF-a) have on oocyte quality?
FFs with higher TNF-a concentration originate poor quality oocytes
42
What influence does the follicular luid (FF) constituent interleukins (Il-6, IL-1, IL-15) have on oocyte quality?
IL-6 level in FF associated with decreased chance of clinical pregnancy, higher levels of IL-1 are related to higher chance of embryo implantation after IVF, lower IL-15 levels are related to clinical pregnancy after IVF
43
Describe the breakdown of the oocyte niche:
A. Large bilateral cysts attached to the uterine horns of a 1-yr-old Bmp15-/-Gdf9-/-mouse B. Ovary from a five month old mouse showing primary follicles around the periphery and follicular nests (arrows) in the centre C. Ovary from a 4 month old mouse showing increased magenta-coloured ZP remnants (arrows) throughout the centre, a sign of increased oocyte turnover D. Ovary from a 9 month old showing few oocytes and a further accumulation of ZP remnants (arrows)
44
What do we know about soluble TGFß ligands in the oocyte niche?
Oocyte: - GDF-9 - BMP-15 - BMP-6 Granulosa: - Inhibin - Activin - AMH - BMP-2,-5,-6 Theca: - BMP-4 - BMP-7 - TGF-ß
45
What is the fate of the gonad?
One gonad, two fates: Bipotential gonad Sex determination Testis or ovary
46
What is the cellular makeup of the testis?
Spermatozoa Germ cells Sertoli cells Basement membrane Peritubular myoid cells Leydig cells Blood Vessels
47
What do we mean by sex? Genetic? Gonadal? Phenotypic?
Genetic (Or chromosomal sex): - Determined at fertilisation by inheritance of X and/or Y chromosomes Gonadal sex: - Presence of testis or ovaries Phenotypic sex: - External appearance of the individual - external genitalia and secondary sexual characteristics Possible to be a mixture of the above - e.g. Gonadally male (with internal undescended testis) and phenotypically female
48
Where do gonadal ridges arise from? What do they express?
Arise from thickenings of the coelomic epithelium around e10.0 Express Wt1, Lhx9, Emx2 and Sf-1 genes
49
What is Gata4 essential for?
Gata4 is essential for the formation of the genital (gonadal) ridge In the absence of Gata4 (conditional KO) the coelomic epithelium does not thicken to form the genital ridge Sf-1 and Lhx9 expression is not activated in the genital ridge in the absence of Gata4
50
What is male gonadal sex determined by?
The inheritance of SRY - Sex-determining region Y Transcription factor on Y chromosome Starts to be expressed in the somatic cells of the genital ridge e10.5, decreases after E12.5 (in mice) Mutations in SRY in humans cause XY>XX sex reversal Expression of SRY in XX mice results in testis formation and male phenotype Switched on by Wt-1 and Sf-1 proteins
51
Where does Sry expression begin?
In the centre of the developing testis and spreads out along it Expression starts in the central zone of the gonad 11.0dpc Expression spreads outwards along the long axis of the gonads in waves 11.5dpc Timing is also critical. If Sry is not switched on in a 6 hour window between e11 and e11.25 the gonad will develop as an ovary
52
What is the spreading of Sry expression along the gonad important for?
Masculinisation of the gonad Some mouse strains undergo partial XY>XX sex reversal, resulting in ovotestis formation Ovotestis structure frequently has male testis tissue at centre, and female ovarian tissue at poles of the gonad - due to a failure of Sry to spread along the gonadal axis
53
What does Sry do to Sox9?
Activates Sox9 to trigger sertoli cell formation Bipotential gonad cells +Sry --> Sox9 positive Sertoli cell precursor specialised --> Reinforcement of male fate recruitment of more Sertoli cells
54
Is Sox9 more important than Sry?
Only around 10% of XY>XX sex reversal cases have mutations in SRY 75% of XY patients with Campomelic Dysplasia (CD) show complete XY>XX sex reversal CD caused by heterozygous mutations in SOX9 Expression of Sox9 in XX mice results in testis formation and male phenotype - can bypass SRY entirely Sox9 is found in birds, fish, reptiles and frogs, which lack SRY
55
How is Sox9 the master regulator of testis development?
Sry causes an increase in Sox9 An increase in Sox9 causes an increase in - AMH - Pdgf - Ahh (regulation of other cell lineages) an increase in - FGF9 - Ptgds (Stops germ cells adopting female fate) Inc in FGF9 and Ptgds causes an increase in Sox9 Positive feedback a decrease in - Foxl2 Blocks formation of granulosa cells
56
What is Leydig cell development dependent on?
Paracrine signals from sertoli cells Activin B - Promotes the proliferation of Sertoli cells leading to expansion and elongation of testis cords Sertoli cells promote the differentiation of Leydig cells Leydig cells express LH receptor and Steroidogenesis enzymes e.g. 3betaHSD
57
What do Leydig cells express?
Leydig cells express LH receptor and Steroidogenesis enzymes e.g. 3betaHSD
58
How do the cords know to form?
Cords form in testis lacking germ cells - so these can't be the nucleation points Invasion of cells from mesonephros forms blood vessels and triggers cord formation Blocking mesonephric cell invasion results in cords failing to organise Invasion of cells from mesonephros forms blood vessels and triggers cord formation Blocking mesonephric cell invasion results in cords failing to organise
59
What does the somatic cell environment determine?
The germ cell sex, not the genetic sex of germ cells
60
What is germ cell sex determined by?
Gonadal sex (to a point): XY germ cells from e11.5 embryos can adopt a female oocyte if aggregated with female gonadal somatic cells But, by e12.5, XY germ cells are locked into male fate E11.5 and e12.5 XX germ cells can adopt male germ cell phenotype if aggregated with male gonadal somatic cells By e13.5, XX germ cells are locked into female fate XY germ cells become locked into male fate a day earlier than XX germ cells become restricted to female fate
61
What determines the fate of a germ cell?
A balance of signals MALE FATE Testis Fgf9 Cyp26B1 Nanos2 Spermatogenesis, germ cell arrest Cyp26b1: retinoic acid degrading enzyme expressed by foetal testis cells FEMALE FATE Ovary Retinoic Acid Activation of Stra8, entry into meiosis
62
What happens in the absence of meiosis-inducing signals?
The germ cells in the testis enter cell cycle arrest Remain arrested or 'quiescent' until after birth Normal regulatory processes happening here as well Reduction of cell division at this time
63
What is germ cell arrest accompanied by?
Changes in expression of cell cycle regulators Loss of phosphorylation of pRB - blocks progression through cell cycle Increase in level of cell cycle inhibitor p16INK4a - blocks phosphorylation of pRB
64
Summarise foetal testis development:
The mammalian gonad initially biopotential, irrespective of genetic sex Activation of SRY in a narrow window is necessary for testis formation SRY activates SOX9 which triggers Sertoli cell formation signals from the Sertoli cells pattern the other cell types in the foetal testis PGCs are initially biopotential too PGCs in the foetal testis progressively enter cell cycle arrest
65
What is the spermatogonial stem cell (SSC) niche?
Stem cell - ready to go into something but quietly sits until something comes and pokes it The thing that 'pokes' it is called the niche Niche - prompts stem cell division Spermatogonial stem cell niche is generally the Sertoli cell STRA8 allows for the progression of the Sertoli cell Testis biggest example of stem cell renewal - always new sperm
66
Describe the pathways involved in the Spermatogonial Stem Cell (SSC) Niche:
The daily production of millions of spermatozoa in mammals is ensured by the presence within the male germ line of stem cells able to maintain their own stock and to differentiate and continuously initiate new waves of spermatogenesis. The balance between the maintenance of spermatogonial stem cells, their proliferation and their differentiation into mature spermatogonia is triggered by intrinsic factors, such as PLZF, and by signals from the spermatogonial stem cell niche, including Sertolian signals, such as GDNF, ERM or Kit ligand. GDNF: glial cell line-derived neurotrophic factor ERM: Ets-related molecule PLZF: Promyelocytic leukaemia zinc finger
67
What happens to the SSC niche during ageing?
As mice age, the weight of the testis decreases The number of stem cells in the testis also decreases Ageing compromises the SSC niche NOT SSC renewal capacity
68
What is SSC niche exhaustion?
Decline in the number and renewal capacity of spermatogonial stem cells (SSCs) within the testis
69
What do we know about early loss of SSC in ETS related Molecule4 (ERM) KO mice?
Division of spermatogonial stem cells1 produces daughter cells that either maintain their stem cell identity or undergo differentiation to form mature sperm. The Sertoli cell, the only somatic cell within seminiferous tubules, provides the stem cell niche through physical support and expression of surface proteins and soluble factors 2,3. The Ets related molecule 4 (ERM) is expressed exclusively within Sertoli cells in the testis and is required for spermatogonial stem cell self-renewal. Mice with targeted disruption of ERM have a loss of maintenance of spermatogonial stem cell self-renewal without a block in normal spermatogenic differentiation and thus have progressive germ-cell depletion and a Sertoli-cell-only syndrome Microarray analysis of primary Sertoli cells from ERM-deficient mice showed alterations in secreted factors known to regulate the haematopoetic stem cell niche. These results identify a new function for the Ets family transcription factors in spermatogenesis and provide an example of transcriptional control of a vertebrate stem cell niche

Advanced concepts in reproduction (9 decks)

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