THE Molecular

Biology of
reproductive
processes
Dr. Bhaskar Ganguly
ZSexual reproduction is favored by
EVOLUTION.

Random recombination of genetic

information increases the chances
of producing at least some
offspring with better adaptability
and survival.
Z Reproductive cycle: An alternation of
Diploid & Haploid states

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

 Diploid cells:
Germ cells {produce gametes}

Haploid cells {fuse to form the zygote which develops into a diploid organism}
 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”.
 DTC: A single, non-dividing cell at the end of
each gonad.

 Cells around the DTC divide mitotically, migrate

away & begin dividing meiotically.

 DTC extends long filaments, containing Lag2, to

the Primordial Germ Cells (PGCs) that have
GLP1, a receptor for Lag2.

 Lag2 maintains the cells in mitosis & inhibits

meiotic differentiation; glp1 mutations inhibit
mitosis and all PGCs begin dividing meiotically.
 SPERM or EGG ?
In Caenorhabditis elegans, a hermaphrodite, the
proximal 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!

 SPERMATOGENESIS (Vertebrates)
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 (contd…)

PGCs A1 spermatogonia A3 A4

Intermediate Spermatogonia

B Spermatogonia

Primary Spermatocytes
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.
 In spermatogonial division,
cytokinesis is incomplete &
cytoplasmic bridges persist through
which ions, nutrients, and signals are
exchanged so that the cohorts mature
synchronously.
 SPERMATOGENESIS (contd…)

Spermatids are yet connected. They are cytologically

haploid but are functional diploids because of sharing
of gene products.

With division & differentiation these cells move farther

& farther away from the basement membrane. Once
near the lumen, spermatids lose their cytoplasmic
bridges & differentiate into spermatozoa.
{~65 days in human}
Spermiogenesis (differentiation of the sperm)

Also known as spermateliosis i.e.

preparation for functions of motility &
interaction.
 Spermiogenesis
(differentiation of the sperm)
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 seminiferous
tubule; tail forming from centriole extends into the lumen.
3. Nucleus flattens & condenses, remaining cytoplasm (residual body/cytoplasmic droplet) is
discarded; mitochondia form a ring around the base of the tail.

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 onion genes.
OOGENESIS: Oogenic Meiosis
Spermatogenic 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)
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.
Oogenic meiosis conserves volume of oocyte cytoplasm
in a single cell rather than dividing it equally.
 Parthenogenetic mechanisms
1) Polar body may act as sperm to fertilize the
ovum 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 turn
into females. Haploid males produce sperm
through bypassing 1st meiosis division, thus, 2
sperm cells are produced through 2nd meiosis.
Oocytes Maturation:
In Amphibians oocytes cytoplasm contains:
 energy sources and energy producing organelles (yolk
and mitochondrion)
 enzymes and precursors for DNA, RNA and protein
synthesis.
 Stored m-RNAs, structural proteins, morphogenetic
regulatory factors to control early embryogenesis.
 m-RNAs include those for- cyclin, actin, tubulin,
histones, cadherins, metalloproteinases, Hunchback,
Nanos, GLP-1, morphogens, Oskar etc.
Most of this accumulation occurs in meiosis prophase I:
pre-vitellogenesis
vitellogenesis (diplotene of prophase I)
As yolk is deposited, mitochondria replicate forming
„mitochondrial cloud‟.
These millions of mitochondria are distributed to different
blastomeres during cleavage. Cortical granules are formed from
Golgi apparatus.
As vitellogenesis nears an end, oocyte cytoplasm becomes
stratified.
Cortical granules, mitochondria and pigment granules
accumulate at periphery in actin rich cortex.
Yolk platelets are heavily concentrated at the vegetal pole;
glycogen granules, ribosomes, lipid vesicles, endoplasmic
reticulum concentrate towards animal pole.
Specific m-RNAs localize according to specific needs.
 Amphibian vitellogenesis is instructed by
Estrogens
that direct the liver to express and secrete
vitellogenin- the major component of yolk.
 Completion Of Meiosis:
Progesterone And Fertilization
 Primary oocytes can remain in diplotene of
meiotic prophase for years. Resumption of
meiosis in amphibians requires progesterone.
 Within 6 hours of progesterone release germinal
vesicle breakdown (GVBD) occurs. Meiosis first is
completed and the mature ovum is released by
ovulation. Ovulated egg is in 2nd meiotic
metaphase when released.
 Resumption of meiosis is regulated by mitosis-
promoting factor/ maturation- promoting factor/
MPF which consists of two subunits, cyclin B and
p34 protein. Progesterone converts pre-MPF into
active MPF.
 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 cascade
for activation of p34 subunit of MPF.
Later......
The oocyte is again arrested in meiosis of metaphase
II. Metaphase block is due to combined action of c-
mos and cdk-2 which are the subunits of cytostatic
factor (CSF). CSF prevents the degradation of cyclin.
Metaphase block is removed by fertilization.
Fertilization Ca++ flux activation of calmodulin
calmodulin dependent protein kinase II is activated 
x cdk-2
calpain II  x c-mos.

i) cyclin can be degraded and meiosis can be

completed.
ii) calmodulin dependent protein kinase II – allows
centrosome to duplicate and spindle fibers to form.
 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 of
mesenchymal thecal cells. Periodically, a group of follicles
enter a stage of follicular growth where the oocytes also
undergoes a 500- fold increase in volume. Granulosa cell
proliferation is mediated by a paracrine factor (produced by
oocyte) GDF9, member of TGF-B family. Throughout the
growth, the oocyte remains in dictyate state. Follicle cells
secrete growth and differentiation factors for the oocytes to
grow and also to bring the blood vessels (TGF-B2, VEGF,
Leptin, FGF2).
 (Maturation of Mammalian Oocytes...)

Pituitary secretes FSH → maturing follicles grow and proliferate

further. FSH also induces formation of LH receptors on the granulosa
cells.
Pituitary secretes LH → Breaks dictyate block. Oocyte undergoes
first 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 causes
its polyadenylation → activation and translation into the powerful
protease.
LH also increases collagenase activity & also increases prostaglandins
in the follicle. Prostaglandins cause localized smooth muscle
contractions in the ovary and increase the permeability of ovarian
capillaries → increased fluid pressure in the antrum.
 Towards Fertilization and Implantation
Gonadotropins (FSH, LH) cause follicle cells to secrete
estrogen  proliferation of uterine endometrium, its
enrichment with blood vessels and thinning of cervical
mucus.
CL produces some estrogen and lots of progesterone.
Progesterone completes the preparation of uterine tissue
for implantation, stimulates the growth of uterine wall
and blood vessels.

MOA for Mifepristone (RU486):

Blocks progesterone receptors  no thickening of uterine
walls  no implantation
Used for post-conception birth control
 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.
 Recognition of egg and sperm
Steps involved include:
• Chemoattraction of sperm to the egg by soluble
molecules 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 extracellular
envelope.
• Fusion of egg and sperm cell membranes.
 Chemoattraction
• Species-specific
• Chemo-attractant – released by the egg e.g. Resact by egg
jelly of sea urchin Arbacia punctulata
• Sperm swim up the concentration of resact.
• Resact – also a sperm activating peptide → immediate and
drastic increase in mitochondrial respiration and sperm
motility.
• Resact receptor is a transmembrane protein, binding with
resact → activation of dyenin ATPase and mitochondrial ATP
generating apparatus
 Acrosomal reaction
Exocytosis of acrosomal contents
Extension of the acrosomal process
Exocytosis Of Acrosomal Contents
Initiated in sperm by interaction with at least 3 carbohydrates in
the egg jelly (sea urchins).
Carbohydrates bind to receptors on sperm cell membrane
directly above the acrosomal vesicle  opening of Ca++
channels in the cell membrane  entry of Ca++ in the sperm
head  Ca++ mediated fusion of acrosomal membrane with
sperm cell membrane  exocytosis of acrosomal contents.
E.g.- Jelly- carbohydrates from one species fail to activate
acrosome reaction even in closely related species barrier to
interspecies fertilization.
Acrosomal activation  expression of fertilin on sperm surface
 must for fertilization
 Extension of the acrosomal process
Involves polymerization of globular actin into actin
microfilaments
Influx 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 actin
cytoskeleton into the formation of the acrosomal process.
 Exocytosis of
acrosomal contents

Extension of the
acrosomal process
 Capacitation
Capacitation 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 the
membranes of the oviductal cells in the isthmus.
3 functions:
• restriction of entry into the ampulla  decreased polyspermy
•slowing down of capacitation storage of spermatozoa
•expansion of life span of sperm (functional)
 +
K HCO3- Ca
++

K+ HCO3- Ca++

Mammalian Sperm Capacitation

 Hyperactivation
Increased motility when sperm enters the oviduct; increased
velocity and greater force due to activation of cAMP of sperm
specific Ca++ channel in tail.
Species Specific Recognition (in Sea Urchins)
Contact of sperm with egg jelly
Acrosomal protein bindin – 30,500D
Even at saturating number of sperm, sperm binding does not occur
over the entire egg surface area
A 350 kD glycoprotein isolated from sea urchin eggs has some
properties expected of bindin receptors

Species specific recognition occurs at the levels of

Attraction
Activation and
Adhesion of sperm
SPERM BINDING DOES NOT OCCUR OVER
THE ENTIRE EGG SURFACE AREA
Gamete Binding & Recognition In Mammals (Mouse)
•Zona pellucida is a glycoprotein matrix that binds sperm, thus,
conferring some degree of species specificity; also initiates
acrosomal 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 fusion
protein (SNARE complex)  exocytosis of acrosomal vesicle.
•Variety of proteases are
released  lysis of zona
pellucida.
•Anterior portion of the sperm
cell membrane is shed. Protein
in inner acrosomal membrane
bind to the ZP2 glycoprotein
 needed for maintaining
adhesion known as secondary
binding.
 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.
Gamete Fusion
Gamete Fusion
 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 cortical
granules  Ca++ wave!
Ca++ wave
Activation Of Egg Metabolism
Activation Of Egg Metabolism
Fusion Of Pronuclei
Fusion Of Pronuclei
 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 another
set of proteases {collagenase, stromelysin and plasminogen
activator}  digestion of extracellular matrix  burial of
blastocyst within the uterine wall Implantation