Molecular Biology of Reproductive Processes

8/7/2019 Molecular Biology of Reproductive Processes

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THE MolecularBiology ofreproductiveprocesses

Dr. Bhaskar Ganguly


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ZSexual reproduction is favored byEVOLUTION.

Random recombination of genetic

information increases the chancesof producing at least some

offspring with better adaptability

and survival.


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Z Reproductive cycle: An alternation ofDiploid & Haploid states

Diploid cells:

Haploid cells {fuse to form the zygote which develops into a diploid organism}

Somatic cells {exist to help the cells of germ line}

Germ cells {produce gametes}


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The Big Decision:

MITOSIS or MEIOSIS!

Germ cells in the gonads have to decide

between the two.

The decision is controlled by the

DISTAL TIP CELL.


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DTC: A single, non-dividing cell at the end ofeach gonad.

Cells around the DTC divide mitotically, migrateaway & begin dividing meiotically.

DTC extends long filaments, containing Lag2, tothe Primordial Germ Cells (PGCs) that haveGLP1, a receptor forLag2.

Lag2maintains the cells in mitosis & inhibitsmeiotic differentiation; glp1 mutations inhibit

mitosis and all PGCs begin dividing meiotically.


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SPERM or EGG ?In Caenorhabditis elegans, a hermaphrodite, theproximal germ cells of the ovotestis form sperm

while the distal germ cells i.e. those near the tip

become eggs.

Germ cells entering meiosis early form sperms!


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1)Initiation is regulated by synthesis of BMP8b by spermatogonia.

BMP8b reaches critical concentration

Germ cells begin differentiation

Differentiating cells produce more BMP8b

Further stimulation of differentiation

2)Spermatogenic germ cells are bound to Sertoli cells by:

i. N-cadherin on both surfaces

ii. Galactosyltransferase molecule of germ cell to carbohydrate receptor on Sertoli cells

Division of PGCs & differentiation occurs in the recesses between

Sertoli cells.

SPERMATOGENESIS (Vertebrates)


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PGCs A1 spermatogonia A3 A4

Intermediate Spermatogonia

B Spermatogonia

Primary Spermatocytes

SPERMATOGENESIS (contd)

Mediated by Glial cell line Derived Neurotropic Factor {GDNF} secreted by Sertoli

cells.

Low level : Spermatogonia spermatocytes

High level : Spermatogonia spermatogonia {i.e. self-renewal}

GDNF is upregulated by FSH.


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In spermatogonial division,cytokinesis is incomplete &

cytoplasmic bridges persist throughwhich ions, nutrients, and signals areexchanged so that the cohorts mature

synchronously.


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Spermiogenesis (differentiation of the sperm)

Also known as spermateliosis i.e.

preparation for functions of motility &

interaction.

S i i


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STEPS:

1. Construction of acrosomal vesicle from Golgi apparatus {covers sperm nucleus}.

2. Nucleus rotates so that acrosomal cap faces the basement membrane of the seminiferoustubule; tail forming from centriole extends into the lumen.

3.Nucleus flattens & condenses, remaining cytoplasm (residual body/cytoplasmic droplet) isdiscarded; mitochondia form a ring around the base of the tail.

Spermiogenesis(differentiation of the sperm)

In the nucleus HISTONES are replaced by PROTAMINES {small proteins with >60% Arginine}.

The nucleosome dissociates, transcription shuts down & nucleus assumes crystalline

structure. Resulting sperm are released into the lumen. Unused sperm are resorbed or

passed out in urine.Sperm mitochondria are highly modified to fit the streamlined cells. Mitochondria fuse; in

flies the fusion is controlled by fuzzy oniongenes.


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OOGENESIS: Oogenic MeiosisSpermatogenic meiosis

(in mammals)

4 Gametes/meiosis

(absence of meiotic arrest)

Oogenic meiosis

1 Gamete/meiosis

(meiotic arrest at 1st meiotic

prophase to allow

the egg to grow)


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OOGENESIS: Oogenic Meiosis

In amphibians germ cells migrate on fibronectin matrices from pro-larval gut to the

gonads. Similar migration in mammals also occurs. For this migration STEM CELL

FACTOR is critical; germ cells grow as they migrate.

Some species produce thousands of eggs continuously, e.g. sea urchin, frog.

In mammals germ cells known as oogonia divide to form approximately 7

million germ cells of which most die out. Those oocytes which survive enter

first meiotic division. Primary oocytes proceed up to diplotene of first meiotic

prophase and are maintained till the female matures.With the onset of puberty, cohorts of oocytes periodically resume meiosis.

Primary oocytes continue to die even after birth. In human females, only about

400 oocytes reach maturity.


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Oogenic meiosis conserves volume of oocyte cytoplasm

in a single cell rather than dividing it equally.


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Parthenogenetic mechanisms

1) Polar body may act as sperm to fertilize theovum e.g. Drosophila sp.

2) Oogonia may double their chromosome

number before meiosis e.g. some lizards.3) No meiosis, diploid ova- grasshoppers

4) In Hymenoptera (bees, wasps, ants)

unfertilized eggs become males, fertilized turninto females. Haploid males produce sperm

through bypassing 1st meiosis division, thus, 2

sperm cells are produced through 2nd

meiosis.


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Oocytes Maturation:

In Amphibians oocytes cytoplasm contains:

energy sources and energy producing organelles (yolkand mitochondrion)

enzymes and precursors for DNA, RNA and proteinsynthesis.

Stored m-RNAs, structural proteins, morphogeneticregulatory factors to control early embryogenesis.

m-RNAs include those for- cyclin, actin, tubulin,histones, cadherins, metalloproteinases, Hunchback,

Nanos, GLP-1, morphogens, Oskar etc.


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Amphibian vitellogenesis is instructed by

Estrogens

that direct the liver to express and secretevitellogenin- the major component of yolk.


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Completion Of Meiosis:

Progesterone And Fertilization Primary oocytes can remain in diplotene of

meiotic prophase for years. Resumption ofmeiosis in amphibians requires progesterone.

Within 6 hours of progesterone release germinal

vesicle breakdown (GVBD) occurs. Meiosis first iscompleted and the mature ovum is released byovulation. Ovulated egg is in 2nd meioticmetaphase when released.

Resumption of meiosis is regulated by mitosis-promoting factor/ maturation- promoting factor/MPF which consists of two subunits, cyclin B andp34 protein. Progesterone converts pre-MPF intoactive MPF.


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Completion Of Meiosis:

Progesterone And Fertilization

Mediator of progesterone signal is c-mos protein.

Progesterone polyadenylates maternal c-mos

mRNA translated into 39kDa phosphoprotein.

C-mos protein activates phosphorylation cascadefor activation of p34 subunit of MPF.


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M i f M li O


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Maturation of Mammalian Oocytes

Majority of oocytes are maintained in diplotene of meiosis

prophase I known as dictyate stage.Oocyte is enveloped by primordial follicle made of single layer

of epithelial granulosa cells and less organized layer ofmesenchymal thecal cells. Periodically, a group of folliclesenter a stage of follicular growth where the oocytes alsoundergoes a 500- fold increase in volume. Granulosa cellproliferation is mediated by a paracrine factor (produced by

oocyte) GDF9, member of TGF-B family. Throughout thegrowth, the oocyte remains in dictyate state. Follicle cellssecrete growth and differentiation factors for the oocytes togrow and also to bring the blood vessels (TGF-B2, VEGF,

Leptin, FGF2).

(Maturation of Mammalian Oocytes )


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(Maturation of Mammalian Oocytes...)

Pituitary secretes FSH maturing follicles grow and proliferate

further. FSH also induces formation of LH receptors on the granulosacells.

Pituitary secretes LH Breaks dictyate block. Oocyte undergoesfirst meiotic division. One set of chromosomes is retained inside the

oocyte, other in polar body. It is at this stage that ovulation occurs.Physical expulsion of ovum appears to be the result of LH activity.

mRNA for plasminogen is dormant in the oocyte cytoplasm. LH causesits polyadenylation activation and translation into the powerful

protease.LH also increases collagenase activity & also increases prostaglandins

in the follicle. Prostaglandins cause localized smooth musclecontractions in the ovary and increase the permeability of ovarian

capillaries increased fluid pressure in the antrum.

T d F tili ti d I l t ti


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Towards Fertilization and ImplantationGonadotropins (FSH, LH) cause follicle cells to secrete

estrogen proliferation of uterine endometrium, itsenrichment with blood vessels and thinning of cervicalmucus.

CL produces some estrogen and lots of progesterone.

Progesterone completes the preparation of uterine tissuefor implantation, stimulates the growth of uterine wall

and blood vessels.

MOA for Mifepristone (RU486):Blocks progesterone receptors no thickening of uterinewalls no implantation

Used for post-conception birth control


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Progesterone inhibits production of FSH no more

follicles and ova.

Hormone mediated separation of communication

between oocytes and follicular cells may be critical to

resumption of meiosis by the ovum.


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Recognition of egg and sperm


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Recognition of egg and sperm

Steps involved include:

Chemoattraction of sperm to the egg by solublemolecules secreted by the egg.

Exocytosis of acrosomal vesicle to release its

enzymes. Binding of sperm to extracellular envelope

(vitelline layer or zona pellucida) of the egg.

Passage of sperm through the extracellularenvelope.

Fusion of egg and sperm cell membranes.

Ch tt ti


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Chemoattraction

Species-specific

Chemo-attractant

released by the egg e.g. Resact by eggjelly of sea urchin Arbacia punctulata

Sperm swim up the concentration of resact.

Resact

also a sperm activating peptide immediate anddrastic increase in mitochondrial respiration and sperm

motility.

Resact receptor is a transmembrane protein, binding withresact activation of dyenin ATPase and mitochondrial ATP

generating apparatus

Acrosomal reaction


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Acrosomal reactionExocytosis of acrosomal contents

Extension of the acrosomal process

Exocytosis Of Acrosomal Contents

Initiated in sperm by interaction with at least 3 carbohydrates inthe egg jelly (sea urchins).

Carbohydrates bind to receptors on sperm cell membranedirectly above the acrosomal vesicle opening of Ca++

channels in the cell membrane entry of Ca++ in the spermhead

Ca

++

mediated fusion of acrosomal membrane withsperm cell membrane exocytosis of acrosomal contents.E.g.- Jelly- carbohydrates from one species fail to activate

acrosome reaction even in closely related speciesbarrier tointerspecies fertilization.


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Acrosomal activation expression of fertilin on sperm surface must for fertilization


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Extension of the acrosomal process

Involves polymerization of globular actin into actin

microfilamentsInflux of Ca++ activates Rho B proteins located in the acrosomal

region and mid-piece of sperm. Rho B is a GTP- binding

protein, which helps in the organization of the actincytoskeleton into the formation of the acrosomal process.


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Exocytosis of

acrosomal contents

Extension of the

acrosomal process

Capacitation


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CapacitationCapacitation can be mimicked in vitro using tissue culture media

(with Ca++, HCO3- and serum albumin).

Uncapacitated sperms are held up in the cumulus.

Sperms achieve competence in ampulla.

For capacitation the sperm may actively hold on to themembranes of the oviductal cells in the isthmus.

3 functions:

restriction of entry into the ampulla decreased polyspermyslowing down of capacitation storage of spermatozoa

expansion of life span of sperm (functional)

K+ Ca++HCO3

-


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K CaHCO3

K+

Ca++HCO3

-

Mammalian Sperm Capacitation

Hyperactivation


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Hyperactivation

Increased motility when sperm enters the oviduct; increased

velocity and greater force due to activation of cAMP of spermspecific Ca++ channel in tail.


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SPERM BINDING DOES NOT OCCUR OVER

THE ENTIRE EGG SURFACE AREA

Gamete Binding & Recognition In Mammals (Mouse)


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Gamete Binding & Recognition In Mammals (Mouse)

Zona pellucida is a glycoprotein matrix that binds sperm, thus,

conferring some degree of species specificity; also initiatesacrosomal reaction.

Three major glycoproteins- ZP (zona protein)1, 2 & 3.

ZP3 initially binds the sperm. Purified ZP3 can bind to the sperm

and inhibit its binding to the zona pellucida.

Binding of the sperm protein is to the serine and threonine-linked

carbohydrate chains of ZP3.

ZP3 cross links the receptors on sperm cell membrane e.g.galactosyl transferase-I. Cross linking activation of G protein opening of Ca++ channels activation of cytoskeletal fusionprotein (SNARE complex) exocytosis of acrosomal vesicle.


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Variety of proteases are

released lysis of zonapellucida.

Anterior portion of the sperm

cell membrane is shed. Protein

in inner acrosomal membranebind to the ZP2 glycoprotein

needed for maintainingadhesion known as secondary

binding.

Gamete Fusion And Prevention Of Polyspermy


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Gamete Fusion And Prevention Of Polyspermy

Gamete fusion depends on interaction between sperm protein

and integrin - associated CD9 protein on the egg. Female mice

with gene knockout for CD9 are infertile and the sperm fails

to fuse with their egg. Infertility is reversed by microinjection

of CD9 mRNA.

CD9 also critical for fusion of myocytes to form

multinucleated myotube of the striated muscle.


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Gamete Fusion


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Blocks To Polyspermy


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Blocks To Polyspermy....1. Fast- change in electric potential of egg cell membrane

from -70mV to +20 mV.2. Slow- cortical granule reaction

Cortical granules- fuse with egg cell membrane, release

contents. In sea urchin- fertilization envelope.

In mammals- modify zona pellucida sperm receptors so that

they can no longer bind sperm. Cortical granule reaction- Ca++ is the initiator.

Fertilization increase Ca++ in egg fusion of corticalgranules Ca++ wave!


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Ca++ wave Activation Of Egg Metabolism


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Activation Of Egg Metabolism



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Fusion Of Pronuclei


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Fusion Of Pronuclei

Fusion Of Pronuclei


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Fusion Of Pronuclei

Implantation


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Implantation Blastocyst expands within zona pellucida. When in uterus zona

hatcing occurs.

Strypsin- lyses hole in fibrillar matrix of zona.

Trophoblast cells contain integrins that bind uterine collagen,

fibronectin and laminin, and synthesize heparan sulphate

proteoglycan.

Endometrium has heparan sulphate receptors.

Once contact is established the trophoblast secretes anotherset of proteases {collagenase, stromelysin and plasminogenactivator} digestion of extracellular matrix burial ofblastocyst within the uterine wallImplantation


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Molecular Biology of Reproductive Processes

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