Developmental Biology Lecture and Laboratory

Midterm

Spermatogenesis

Review on the Histology of the Vertebrate Testis

In mammals, the PGCs are found in the posterior wall of the yolk sac
near the origin of the allantois. These cells reach the future gonads by
migration around the wall of the posterior gut and then through the
dorsal mesentery.

1.Peritubular cells 2. Basal membrane 3. Spermatogonia

4. Tight junction 5. Spermatocyte I 6. Spermatocyte II
7a. spermatid 7b. Spermatid 8. Acrosome 9. Residual bodies
10.Spermatozoas 11. Nucleus of sustentacular cells (Sertoli)
A. Basal zone B. Adluminal zone

Sperm maturation. (A) Cross section of the seminiferous tubule. (B)

Simplified diagram of a portion of the seminiferous tubule, illustrating
relationships between spermatogonia, spermatocytes, and sperm. As
these germ cells mature, they progress toward the lumen of the
seminiferous tubule.

Spermatogenesis

- the developmental pathway from germ cell to mature

sperm—begins at puberty and occurs in the recesses between
the Sertoli cells

Spermatogenesis is divided into three major phases:

Proposed hypothesis for migration of chicken PGCs toward the germinal
crescent. (A) Location of PGCs between EGK stage X and HH stage 4. At EGK 1. A proliferative phase where sperm stem cells
stage X, PGCs are located in the central region of the area pellucida. By HH
stage 4, most of the PGCs have reached the germinal crescent region. (B) Global (spermatogonia) increase by mitosis
movement of embryonic cells, including epiblast and hypoblast cells, between
EGK stage X and HH stage 2. (C) Active migration zone between EGK stage X and 2. A meiotic phase, involving the two divisions that create the
HH stage 2, covering the anterior part of the embryo. (D) Migration pathway of haploid state
PGCs toward the germinal crescent. Once PGCs reach the anterior region
passively by embryonic flow (blue lines), they start actively migrating toward the 3. A postmeiotic “shaping” phase called spermiogenesis,
germinal crescent region (red lines).
during which the round cells (spermatids) eject most of their
cytoplasm and become the streamlined sperm
Some Common Normal Values in Humans Sertoli cells/ Sustentacular cells

❖ The normal volume varies from 1.5 to 5.0 mL per

ejaculation.
❖ The sperm count varies from 20 to 150 million sperm/mL.
❖ At least 60% of the sperm should have a normal shape
and show normal forward movement (motility).

Phases of Spermatogenesis

❖ Spermatocytogenesis- cells from the spermatogonium up

to and including the secondary spermatocyte

Mitosis - Clonal expansion

Meiosis- from spermatocytes to spermatids

❖ Spermiogenesis- (spermiohistogenesis) differentiation of

the sperm cell, starting with the spermatid phase

❖ form the blood testis barrier

- important because sperms are considered as foreign
bodies
- if enter blood, antibodies will kill it
❖ supply nutrients
❖ secrete testicular fluid
❖ phagocytosis

The spermatogenesis generations

The stem cell population of the germinal cells lies on the basal lamina
of the convoluted seminiferous tubules. These are Type A
spermatogonia. These cells undergo mitosis: one of the daughter cells
renew the stock of type A spermatogonia, the other becomes a type B
spermatogonia. These divide and their daughter cells migrate towards
the lumen. In roughly 64 days they differentiate themselves thereby
into sperm cells up to the outer surface of the epithelium (one should
note that in these cellular divisions, the separation of the cytoplasm is
not complete. Whole networks of connected cells arise. So, for
example in the last generation, the spermatids, far more cells are
bound to each other than as shown here).
The developmental stages of meiotic prophase I. Shown is
immunohistochemical analysis of spermatogenic cells with antisera
that recognize SCP3 (synaptonemal complex protein 3; SCs red), MLH1
(Mut-L homolog 1; MLH1 foci, yellow) and CREST antigens
(centromeres, blue). Cells were at the following stages of meiotic
prophase I: ( A ) leptotene; ( B ) early zygotene; ( C ) late zygotene; ( D
) early pachytene, arrowhead indicates the sex chromosomes (X and
Y); ( E ) late pachytene, arrowhead indicates the desynapsed sex
chromosomes and ( F ) diplotene.
Homolog Pairing culminates in the formation of a
Golgi phase – Cap phase -
Synaptonemal Complex
vacuoles with acrosomal
PAS (periodic granules cover
acid-Schiff) nucleus &
positive becomes
granules in acrosomal cap
Golgi

❖ A single bivalent is shown schematically: at leptotene, the

two sister chromatids coalesce, and their chromatid loops
extend out from a common axial core. Assembly of the
synaptonemal complex begins in early zygotene and is
complete in pachytene. The complex disassembles in
Acromosal
diplotene.
phase –
anterior pole
oriented Maturation
towards phase –
basement residual
membrane of cytoplasm is
ST; nucleus shed
becomes
elongated

Spermiogenesis: 3 Major Objectives

1. The safeguard of the male genome within the confines of

Three differing stages of spermiogenesis: on the left: a fresh spermatid on the
right: an immature sperm cell in the middle: an in-between stage. A rotation of
the nucleus causes a repositioning of the acrosomal vesicle to occur. This 2.
inverts itself like a cap over the nucleus that continues to be condensed (dotted
line). The cytoplasm cell components that are no longer needed are discarded
and phagocytized by Sertoli's cells. The mitochondria are packed thickly (tightly) 3.
together around the beginning part of the flagellum (mid-piece).
a compact nucleus.
The accumulation of enzymes in the acrosome to be
released at fertilization.
The development of a sperm propelling tail consisting of
an axoneme surrounded by a scaffold of keratin-
containing outer dense fibers and a fibrous sheath.

Nuclear Compaction (Human Sperm)

❖ Histone synthesis stops during

spermiogenesis
❖ Histones are eventually replaced
by protamines
❖ Many nuclear proteins are
expressed systematically during
the nuclear condensation period.
❖ Almost all of these proteins are
derived from histone H1
❖ H1 undergo complex processes
of post- translational modification
in mammals.
❖ In the human sperm, protamines
and histones are present at
The mature sperm cell is slender; in the middle part, the mitochondria are thick ca. 85:15 ratio
and ring-shaped. The DNA in the nucleus is maximally condensed.
Further Maturation
❖ Chemical maturation -epididymis - bathed in a caput to
cauda gradient of ribonucleases, glycosidases, and
proteases, including the 26S proteasome.
❖ Sperm motility -activated motility (epidydimis) and
hyperactivated (female reproductive tract)
❖ Ubiquitin surveillance system
Fertilizing capacity of sperm
❖ The tripeptide FPP (fertilization promoting peptide)
produced by the male is essential for capacitation
- High levels of FPP prevent capacitation
- The proper concentration occurs after ejaculation in the
female reproductive tract where the concentration drops
after mixing with vaginal secretions and/or becomes less
active due to the pH of the vagina).
- Synergistic effect with adenosine that increases
adenylyl cyclase activity in the sperm.
- FPP - in the seminal fluid; produced in prostate
- Comes into contact with the spermatozoa upon
ejaculation.
Antoni van Leeuwenhoek’s (1632‐1723) observation of small
animals, animaculae, in the sperm of different animals; from
Philosophical Transactions of the Royal Society (1679).
The cycle of the seminiferous epithelium
Spermatogenic cycles of rat and mouse. The diagrams show the histological
relationship of spermatogenic cells in the rat (A) and mouse (B) seminiferous
tubules of the testis during spermatogenesis. In each diagram, the lower row of
cells is generally closer to the basement membrane and the upper row of cells
is generally closer to the lumen of the seminiferous tubule. In the rat, there are
14 stages (indicated by the Roman numerals) that repeat at the same location of
the tubule at a 12.9-day interval. In the mouse, there are 12 stages (indicated by
the Roman numerals) that repeat at an 8.6-day interval
Duration of 1 Cycle/Duration of Spermatogenesis
❖ Mouse: 8.6/ 34.5
❖ Hamster: 8.7/35
❖ Sprague-Dawley rat: 12.9/51.6
❖ Wistar rat: 13.3/ 53.2
❖ Bull: 13.5/ 49
❖ Man: 16 /70 to 74 (4.5 cycles)
The Sertoli Cells
❖ Structure
- Do not divide during reproductive period; are resistant
to infection, effects of X ray irradiation, malnutrition;
- Involved in maintenance of blood-testis barrier
❖ Functions
- Support. protection and nutritional regulation of
developing spermatozoa
- Phagocytosis
- Secretion
• ABP under influence of testosterone and FSH from
anterior pituitary
• Aromatase enzyme that converts testosterone to
estradiol
• Inhibin, a peptide which suppresses FSH release
• MDIF - promotes regression of Mullerian duct
Interstitial Cells

❖ Connective tissues, nerves, blood and lymphatic vessels

❖ Leydig cells appear during puberty; have characteristic of
steroid secreting cells; secrete testosterone responsible
for development of secondary sex characteristics
❖ Triphasic appearance in life of human

Gene transcription during spermatogenesis

❖ Transcription at diplotene Y chromosome of Drosophila

transcribed to ensure sperm viability but not to determine
sex; XY and XO both male but latter is sterile)
❖ Isoform for B tubulin (necessary for motility) transcribed
during spermatogenesis (responsible for meiotic
spindles, axoneme and microtubules associated with the
lengthening mitochondria)
❖ mRNA for bindin (sea urchin) transcribed late in
spermatogenesis; necessary for binding of sperm and egg

Interaction between the hypothalamus and the testes

❖ Akap 82- transcribed at spermatid stage; an 82-kDa

protein as the major protein of the fibrous sheath of
mouse sperm transcribed from X chromosome
❖ mRNA for sperm protamine protein transcribed by
spermatid (necessary for compacting chromatin during
spermiogenesis)
❖ β1-4 galactosyltransferase which binds sperm to zona
pellucida also transcribed at spermiogenic phase.

Hormonal Control of Spermatogenesis

❖ Spermatogenesis at puberty depends on hypothalamus,

Pulsaltile discharge of FSH is much less evident due to slower
pituitary (LH) and functional Leydig cells to produce
clearance. LH and FSH secretion are controlled by
testosterone
testosterone: estradiol ratio and inhibin secreted by Sertoli
❖ FSH ->promotes proliferation of spermatogonia; for
cells
secretion of ABP and to develop BTB and other functions
of Sertoli; once Sertoli cells are developed, testosterone
alone can maintain spermatogenesis
Smoking and other environmental hazards
BTB = Blood-testis barrier
❖ Has negative effects on penile functions
ABP = Androgen binding protein ❖ Has ill effects on spermatogenesis

❖ Yield of spermatozoa will increase with FSH; prevents

atresia of Type A spermatogonia; normally 50% of type A
spermatogonia will degenerate.
❖ Increased sexual activity increases FSH level and will
reduce loss of A spermatogonia; inhibin decreases FSH
Environmental/lifestyle effects on spermatogenesis
Richard M. Sharpe (27 May 2010)
https://doi.org/10.1098/rstb.2009.0206
varicocele. A varicocele happens when the veins of the
scrotum are swelled and dillated, it can be corrected with
surgery.
❖ Other conditions such as retrograde ejaculations
(ejaculating sperm back up into the bladder instead of
ejaculating outward). Retrograde ejaculations may be
treated as simply as using a decongestant
❖ Hormonal imbalances can also be a cause of male
infertility. Blood tests can be used to check for hormonal
imbalances. It is important to discuss any and all medical
treatments with your doctor.

Smoking and spermatogenesis

❖ Development of the sperms was mildly reduced in rats in

the case and control groups, respectively (P <001). mean
number of Sertoli cells were 9.2 +/- 1.2 and 13.3 +/- 1.8 in
the case and control groups, respectively (P <. 001).
❖ A concurrent reduction in the number of germ cells and
Leydig cells with the decrease in the number of Sertoli
cells was seen in the rats of the case group.
Effect of cigarette smoke on spermatogenesis in rats
Ahmadnia H, Ghanbari M, Moradi MR, Khaje-Dalouee M. (2007)
https://pubmed.ncbi.nlm.nih.gov/17987579/
❖ The stem cells arise from division and differentiation of
primordial germ cells in the embryonic testes. In mature
testes, they divide mitotically to form spermatogonia,
which in turn generate spermatocytes by mitosis. Each
spermatocyte gives rise to four spermatids through
meiosis, reducing the chromosome number from diploid
(2n = 46 in humans) to haploid (n = 23). Spermatids
undergo extensive changes in differentiating into sperm.

Causes of low sperm count – abnormal sperm count

❖ Medications: Tagamet, ketoconazole, and erythromycin.

❖ Illness or infection: Low sperm count can be caused by
something as simple as a virus or infection.
❖ Anatomical problems: There are certain conditions that
can cause male factor fertility problems. One concern is a
 Oogenesis The number of germ cells in the human ovary changes over
the lifespan

Differences of oogenesis from spermatogenesis
❖ GAMETE: Product cell is immotile
❖ SIZE: Cell is big & contains all factors needed for early
development of future embryo: enzymes, mRNAs,
organelles, & metabolic substrates.
❖ MEIOTIC ARRESTS: 1° ooctyte at diplotene; 2° oocyte at
prometaphase
❖ NUMBER OF FUNCTIONAL GERM CELL: 1
❖ Presence of cell coverings in the final product.
Meiosis in the mouse oocyte. The tubulin of the microtubules is stained green;
the DNA is stained blue. (A) Mouse oocyte in meiotic prophase. The large diploid
nucleus (the germinal vesicle) is still intact and actively transcribing genes
whose mRNAs will be stored in the egg as maternal mRNA. (B) The nuclear
envelope of the germinal vesicle breaks down as metaphase begins. (C) Meiotic
anaphase I, wherein the spindle migrates to the periphery of the egg and
releases a small polar body. (D) Meiotic metaphase II, wherein the second polar
body is given off (the first polar body has also divided).

Peculiarities of oogenesis

❖ Some species produce millions of eggs in a lifetime,

mammals produce fewer ova.
❖ Oocyte accumulates materials to regulate future
developmental processes
- prepares enzymes and precursors for DNA, RNA and
protein synthesis
- stores mRNA, structural proteins and morphogenetic
factors
Chromosomal nondisjunction and meiosis. (A) Maternal age affects the
- take place in previtellogenic and vitellogenic phases incidence of trisomies in human pregnancy. (B,C) Reduction of chromosome-
associated cohesin in aged mice. DNA (white) and cohesin (green) stained in
oocyte nuclei of (B) 2-month-old (young) and (C) 14-month-old (aged, for a
mouse) ovaries. A significant loss of cohesin can be seen (especially around the
kinetochores) in aged mice.

Cellular components stored in the mature oocyte of Xenopus

laevis
The nuclear envelope (NE) contains much more nuclear pore
complexes (NPCs) - structures mediating the transport
between cytoplasm and nucleus

Oocyte maturation in amphibians

3 phases

❖ Previtellogenesis (meiotic prophase 1)

❖ Vitellogenesis (meiotic prophase 1)
❖ Maturation

Previtellogenesis Vitellogenesis
Animal/Vegetal Axis Established

❖ Yolk platelets (membrane bound) are in vegetal

mRNA, tRNA, rRNA,
hemispheres)
nucleus increase in ❖ Glycogen granules, lipid inclusions, ER, ribosomes in
size, nucleoli and
Yolk formation animal pole
mitochondria
❖ Specific RNAs are localized to certain regions of the
increase in number,
cortical granules in cytoplasm (localization credited to cytoskeleton)
CB, follicle ❖ Cortical granules found in actin rich cortex
cells/nurse cells
Specific localizations important for development as proven by
centrifugation experiments

Oocyte maturation in amphibians

Vitellogenesis

❖ Occurs when oocyte is in diplotene stage- active

transcription takes place
❖ Autosynthesis and heterosynthesis of yolk

❖ Maturation
- vitellogenin is split into two smaller proteins
• phosvitin
• lipovitellin.
- Both proteins are packaged together into yolk
platelets

A Remarkable Form of Chromosome That Exists During the

Extended Diplotene Phase of Meiosis in The Growing Ovarian
Eggs of All Animals Except Mammals

❖ Discovered in the late 19th Century in the eggs of an

amphibian and a fish
Oocyte Meiotic Maturation and Egg Activation Oogenesis leads to the production of an immature oocyte arrested in prophase
of meiosis I. After resumption of meiosis in response to hormonal stimulation,
the oocyte progresses until the second arrest point in meiosis II (CSF arrest).
Fertilization releases the oocyte from this arrest and triggers exit from meiosis
II. The lower panel shows a simplified scheme illustrating the cytoplasmic
injection experiments that led to the identification of MPF and CSF in frog
oocytes. Small amounts of cytoplasm taken from mature oocytes were injected
into immature oocytes or one blastomere of two-cell embryos. After injection
immature oocytes resume meiosis whereas injected blastomeres arrest in
metaphase. These observations led Masui and Markert to postulate that
cytoplasm of mature oocytes contains two distinct biochemical activities, MPF
and CSF, that regulate the oocyte maturation process

How eggs arrest at metaphase II: MPF stabilisation plus

APC/C inhibition equals Cytostatic Factor

Madgwick, S., Jones, K.T. (2007)

https://doi.org/10.1186/1747-1028-2-4
The oocytes of most animal species arrest in meiotic prophase I (reviewed
by Masui and Clarke, 1979; Masui, 2001). In response to a hormonal stimulus (1-
methyladenine in starfish; progesterone in Xenopus; MSP in C. elegans), oocytes
begin meiotic maturation: the nuclear envelope breaks down (GVBD), as the
oocyte enters M-phase from prophase. The point of fertilization is species-
specific. In the case of C. elegans (I) fertilization occurs after maturation but
prior to completion of meiosis I. In C. elegans, fertilization is required for
completing both meiotic divisions. For most insects (II), fertilization occurs at
metaphase or anaphase I. In most vertebrates (III), fertilization occurs at
metaphase II. Sea urchins complete meiosis before fertilization (IV).

germinal vesicle breakdown (GVBD) due to progesterone and

C-mos - 39kDa phosphoprotein

Early frog life cycle; the appearance of frog egg and oocyte
and the level of MPF activity are shown during meiosis,
fertilization and early development of zygote. Points of cell Model for meiosis II exit after fertilization-induced destruction
cycle arrest and the stimuli that release these arrests are of XErp1
also indicated.

MPF and CSF key activities in vertebrate oocytes

 During CSF arrest, the APC/C is kept inactive by XErp1. Fertilization The Human Menstrual Cycle
leads to the production of intracellular Ca 2+ transients that activate
CaMKII. CaMKII phosphorylation on Thr195 directs Plx1 to its substrate
XErp1. Plx1 phosphorylation then triggers SCF-TRCP-dependent
degradation of XErp1 by the ubiquitin/proteasome system. As a result,
the APC/C is liberated from its repression and triggers meiosis II exit.

Oogenesis in mammals

❖ Ovulation patterns
1. Egg is ovulated during sexual intercourse; egg is
ovulated while in MII
2. Periodic type of ovulation (ovulates during estrus
period)
- Hormonally regulated and starts with environmental
cues eg. daylight -hypothalamus releases gonadotropin
releasing hormones->pituitary>gonadotropins- The coordination l (B) ovarian and (D) uterine cycles is
>ovary>estrogen> causes mating specific behavior controlled by (A) the pituitary and (C) the ovarian hormones.
(gonadotropins stimulate follicular growth and initiation of During the follicular phase, the egg matures within the follicle,
ovulation thus estrus and ovulation can occur) and the uterine lining is prepared to receive the blastocyst.
3. Cyclic ovulation as in humans & other primates The mature egg is released around day 14. If a blastocyst docs
❖ Menstrual cycle entails the periodic shedding of blood and not implant into the uterus, the uterine wall begins to break
cellular debris from the uterus at monthly intervals. down, leading to menstruation.
❖ Represents 3 different activities:

(1) the ovarian cycle - functions to mature and release an

oocyte Hormonal events leading to ovulation

(2) the uterine cycle - functions to provide the appropriate

environment for the developing blastocyst, and

(3) the cervical cycle - functions to allow sperm to enter the

female reproductive tract only at the appropriate time.

These three functions are integrated through the hormones of

the hypothalamus, pituitary, and ovary.

Follicle Cycle in Humans

Note: During ovulation, secondary oocyte is not directly released into the
fallopian tube. The occyte is first released into the peritoneal cavity, which then
gets sucked into the fallopian tube. If somehow, the oocyte fails to be sucked up
into oviduct and remains in the peritoneal cavity – there are chances that that it
may be fertilized in the peritoneal cavity. This may give rise to ectopic abdominal
pregnancy. The placental separation from such pregnancies result in massive
blood loss as there are no muscles to contract and stop bleeding like in uterine
cavity.
 Avian oogenesis

❖ Early ovum 50 um and grows gradually till 6 mm; then it

grows at 2.5 mm daily
❖ By ovulation time-> 35 mm due to accumulation of yolk
from the liver
❖ Young ova lie deep within ovary; mature follicles protrude
into the lumen
❖ Avian follicles- oocyte with zona radiata and follicle cells
with no antrum; oocyte ruptures through stigma during
ovulation, muscular contraction in follicular stalk is
observed
❖ As in mammals, completion of MI coincidental with
ovulation; 2nd maturation division resumes at fertilization

Chicken Egg Fertilization

Estrogen ↑, FSH ↓, LH ↑

❖ Days 7-10, increasing production of estrogen, the

granulosa cells continue to grow.
❖ Day 10, estrogen secretion rises sharply. This rise is
followed by surge of LH and a smaller burst of FSH.

Experiments with female monkeys have shown that exposure

of the hypothalamus to greater than 200 pg of estrogen per
milliliter of blood for more than 50 hours results in the
hypothalamic secretion of gonadotropin-releasing factor. This
factor causes the subsequent release of FSH and LH from the
pituitary. Within 10 to 12 hours after the gonadotropin peak, the
egg is ovulated.

Blocking the progesterone receptor with the synthetic steroid

mifepristone (RU486) stops the uterine wall from thickening
and prevents the implantation of a blastocyst.

❖ Ovulation done
- Luteal phase follows
- Ruptured follicle (still under the influence of LH)
becomes corpus luteum
- Secretes some amount of estrogen
- Secretes mainly progesterone General hormonal control in the female
- Prepares uterine wall (uterine wall
thickens) for implantation of the
blastocyst

Normally, only 1 oocyte is ovulated

 Fertilization: Beginning a New Organism

Fertilization

❖ The process whereby the gametes – sperm and egg –

fuse together to begin the creation of a new organism
❖ Accomplishes two separate ends: sex (the combining of
genes derived from two parents) and reproduction (the
generation of a new organism).

Sperm

❖ Each sperm cell consists of a haploid nucleus, a

❖ Oogenesis begins in the female embryo with the
propulsion system to move the nucleus, and a sac of
production of oogonia from primordial germ cells. The
enzymes that enable the nucleus to enter the egg.
oogonia divide by mitosis to form cells that begin meiosis,
❖ Acrosomal vesicle, or acrosome - derived from the cell’s
but stop the process at prophase I before birth. These
Golgi apparatus and contains enzymes that digest
developmentally arrested cells, which are primary
proteins and complex sugars
oocytes, each reside within a small follicle, a cavity lined
❖ Sperm head – the acrosome and nucleus
with protective cells. At birth, the ovaries together contain
❖ Axoneme - a structure formed by microtubules emanating
about 1–2 million primary oocytes, of which about 500
from one of the two centrioles at the base of the sperm
fully mature between puberty and menopause.
nucleus and the major motor portion of the flagellum

Modification of a germ cell to form a mammalian sperm. (A) The centriole duplicates, using
one centriole to organize a long flagellum at what will be the posterior end of the sperm; the
other centriole will enter the egg at fertilization. The Golgi apparatus forms the acrosomal
vesicle at the future anterior end. Mitochondria collect around the flagellum near the base of
the haploid nucleus and become incorporated into the midpiece (“neck”) of the sperm. The
remaining cytoplasm is jettisoned, and the nucleus condenses. The size of the mature sperm
has been enlarged relative to the other stages. (B) Mature bull sperm. The DNA is stained
blue, mitochondria are stained green, and the tubulin of the flagellum is stained red. (C) The
acrosomal vesicle of this mouse sperm is stained green by the fusion of proacrosin with
green fluorescent protein (GFP).

Capacitation of Sperm in Mammals

❖ Sperm acquires the capacity to fertilize the egg (enables

it to go through acrosome reaction in the female
reproductive tract)
❖ Involves increased intracellular Ca2+
❖ Phosphorylation of proteins by tyrosine kinases mediated
by cAMP

Capacitation
❖ Clearly demonstrated in human spermatozoa that only a
small fraction of the sperm population is capacitated at
any given time
❖ Capacitated state is transient (1-6 hours of life span), that
it occurs only once in the sperm's lifetime
❖ Spermatozoa are in capacitated stage at different time
points, -- >continuous replacement of capacitated cells
within the sperm population.
❖ Maximizes the prospects for an ovulated egg to meet
spermatozoa in the best functional state
Molecular changes during capacitation mammals
Effects of Capacitation on Sperm
❖ Hyperactivation of sperm in reproductive tract
• Increased rate of metabolism
• Flagellum beats more rapidly;
• Result: sperm is more motile
❖ Changes in sperm glycoproteins allow sperm-egg binding
Structure of the sea urchin egg at fertilization
Sperm can be seen in the jelly coat and attached to the vitelline envelope. The
female pronucleus is apparent within the egg cytoplasm.
All the material necessary to begin growth and development
must be stored in the egg, or ovum.
❖ Nutritive proteins - the early embryonic cells must have a
supply of energy and amino acids.
❖ Ribosomes and tRNA - the early embryo must make many
of its own structural proteins and enzymes, and in some
species, there is a burst of protein synthesis soon after
fertilization
❖ Messenger RNAs - the oocyte accumulates not only
proteins but also mRNAs that encode proteins for the
early stages of development.
❖ Morphogenetic factors - molecules that direct the
differentiation of cells into certain cell types are present
in the egg.
❖ Protective chemicals - the embryo cannot run away from
predators or move to a safer environment, so it must be
equipped to deal with threats.
Stages of egg maturation at the time of sperm entry in
different animal species
Sea urchin egg cell surfaces
❖ vitelline envelope 2. Species - specific activation (sea urchin)
- an extracellular matrix that forms a fibrous mat around
the egg and is often involved in sperm-egg recognition • Activation by egg jelly induces acrosome reaction -
❖ cortex acrosome membrane fuses with sperm membrane →
- a thin layer (about 5 μm) of gel-like cytoplasm lying exocytosis of acrosomal vesicles →extension of
immediately beneath the cell membrane of most eggs acrosomal filament
❖ microvilli • Activation is calcium - mediated initiated by sulfated
- small projections which may aid sperm entry into the polysaccharides from the egg jelly
cell
❖ cortical granules
- membrane-bound, Golgi-derived structures located in
the egg cortex; contain enzymes and other components
❖ zona pellucida
- glycoprotein coat (extracellular matrix) around the
mammalian egg, synthesized and secreted by the
growing oocyte
❖ cumulus
- layer of cells which is made up of the ovarian follicular
cells that were nurturing the egg at the time of its release
from the ovary
❖ corona radiata
- the innermost layer of cumulus cells, immediately B. Recognition of sperm and egg (sea urchin)
adjacent to the zona pellucida
• Bindin is the acrosome protein which mediates
specific recognition between egg and the sperm;
Mammalian eggs immediately before fertilization located specifically on the acrosomal process
• Receptor to bindin on the vitelline envelope is a
large glycoprotein (1300 amino acids)

Summary of events leading to the fusion of egg and sperm cell

membranes in sea urchin fertilization, which is external

Main Events

1. Contact and recognition of sperm and egg

2. Regulation of sperm entry into the egg

3. Fusion of male and female pronuclei

4. Activation of egg metabolism

5. Cytoplasmic rearrangement

Contact and recognition of sperm and egg

A. The approach of the sperm (invertebrates

1. Species - specific attraction (chemotaxis)

• Cnidarians secrete chemotactic factors and regulate

time of release (oocyte can attract sperms only after
2nd meiotic division)
• Arbacia punctulata (sea urchin) secrete RESACT - a
14AA peptide acting as chemoattractant; it is also a
sperm activating agent
(1) The sperm is chemotactically attracted to and activated by the egg. (2,3)
Contact with the egg jelly triggers the acrosome reaction, allowing the
acrosomal process to form and release proteolytic enzymes. (4) The sperm
adheres to the vitelline envelope and lyses a hole in it. (5) The sperm adheres to
the egg cell membrane and fuses with it. The sperm pronucleus can now enter
the egg cytoplasm.
Acrosome reaction in sea urchin sperm (A) Contact of a sperm acrosomal process with an egg microvillus. (B) Bindin
(stained black by antibodies against it) is seen to be localized to the acrosomal
process after the acrosome reaction. (C) In vitro model of species-specific
binding. The agglutination of dejellied eggs by bindin was measured by adding
bindin particles to a plastic well containing a suspension of eggs. After 2–5
minutes of gentle shaking, the wells were photographed. Each bindin bound to
and agglutinated only eggs from its own species.

Aberrant development in a dispermic sea urchin egg

(A-C) The portion of the acrosomal membrane lying directly beneath the sperm
cell membrane fuses with the cell membrane to release the contents of the
acrosomal vesicle. (D) The actin molecules assemble to produce microfilaments,
extending the acrosomal process outward. Photographs of the acrosome
reaction in sea urchin sperm are shown below the diagrams.

Species-specific induction of the acrosome reaction by

sulfated polysaccharides characterizing the egg jelly coats of
three species of sea urchins that co-inhabit the intertidal
around Rio de Janeiro

Membrane potential of sea urchin eggs before and after

fertilization

(A) The histograms compare the ability of each polysaccharide to induce the
acrosome reaction in the different species of sperm. (B) Chemical structures of Formation of the fertilization envelope and removal of excess
the acrosome reaction-inducing sulfated polysaccharides reveal their species- sperm
specificity.

Species-specific binding of the acrosomal process to the egg

surface in sea urchins

(A) At 10 seconds after sperm addition, sperm surround the egg. (B,C) At 25 (B)
and 35 (C) seconds after insemination, a fertilization envelope is forming around
the egg, starting at the point of sperm entry. (D) The fertilization envelope is
complete, and excess sperm have been removed.
Cortical granule exocytosis and formation of the sea urchin Probable mechanisms of egg activation
fertilization envelope

In both cases, a phospholipase C (PLC) is activated and makes IP3 and

diacylglycerol (DAG). (A) Ca2+ release and egg activation by activated PLC
directly from the sperm, or by a substance from the sperm that activates egg
PLC. This may be the mechanism in mammals. (B) The bindin receptor (perhaps
acting through a G protein) activates tyrosine kinase (TK, an Src kinase), which
activates PLC. This is probably the mechanism in sea urchins.

Roles of inositol phosphates in the release of Ca2+ from the

(A) Schematic diagram of events leading to the formation of the fertilization
endoplasmic reticulum and the initiation of development
envelope. As cortical granules undergo exocytosis, they release cortical granule
serine protease (CGSP), an enzyme that cleaves the proteins linking the vitelline
envelope to the cell membrane. Glycosaminoglycans released by the cortical
granules form an osmotic gradient, causing water to enter and swell the space
between the vitelline envelope and the cell membrane. The enzyme Udx1 in the
former cortical granule membrane catalyzes the formation of hydrogen peroxide
(H2O2), the substrate for soluble ovoperoxidase (OVOP). OVOP and
transglutaminases (TG) harden the vitelline envelope, now called the fertilization
envelope.

Calcium release across a sea urchin egg during fertilization

Phospholipase C splits PIP2 into IP3 and DAG. IP3 causes the release
of Ca2+ from the endoplasmic reticulum, and DAG, with assistance
from the released Ca2+, activates the sodium-hydrogen exchange
pump in the membrane.

G protein involvement in Ca2+ entry into sea urchin eggs.

The egg is preloaded with a dye that fluoresces when it binds Ca2+. When a
sperm fuses with the egg, a wave of Ca2+ release is seen, beginning at the site
of sperm entry and propagating across the egg. Images are arranged in 3-
second intervals, from left to right in the top row, and continuing from left to
right in the bottom row. The wave does not simply diffuse but travels actively,
taking about 30 seconds to traverse the egg
 (A) Mature sea urchin egg immunologically labeled for the cortical granule
protein hyalin (red) and the G protein Gαq (green). The overlap of signals
produces the yellow color. Gαq is localized to the cortex. (B) A wave of Ca2+
appears in the control egg (computer-enhanced to show relative intensities,
with red being the highest) but not in the egg injected with an inhibitor of the
Gαq protein. (C) Possible model for egg activation by the influx of Ca2+.

(A) Sequential photographs show the migration of the egg pronucleus and the
sperm pronucleus toward each other in an egg of Clypeaster japonicus. The
sperm pronucleus is surrounded by its aster of microtubules. (B) The two
pronuclei migrate toward each other on these microtubular processes. (The
pronuclear DNA is stained blue by Hoechst dye.) The microtubules (stained
green with fluorescent antibodies to tubulin) radiate from the centrosome
associated with the (smaller) male pronucleus and reach toward the female
pronucleus. (C) Fusion of pronuclei in the sea urchin egg.

Sperm associations can occur in species in which females

mate with several males in a brief time span

EARLY RESPONSES - contact or fusion of a sea urchin sperm

and egg activates two major blocks to polyspermy: the fast
block, mediated by sodium influx into the cell; and the cortical
granule reaction, or slow block, mediated by the intracellular
release of Ca2+.

LATE RESPONSES: RESUMPTION OF PROTEIN AND DNA

SYNTHESIS - Calcium release activates a series of metabolic
reactions that initiate embryonic development.

Postulated pathway of egg activation in the sea urchin

Recent model for the recognition of sperm by the mouse zona

pellucida

Nuclear events in the fertilization of the sea urchin

Entry of sperm into a golden hamster egg Cleaved ZP2 is necessary for the block to polyspermy in
mammals

Eggs and embryos were visualized by fluorescence microscopy (to see sperm
nuclei; top row) and brightfield microscopy (differential interference contrast, to
see sperm tails; bottom row). Sperm bound normally to eggs containing a
mutant ZP2 that could not be cleaved. However, the egg with normal (i.e.,
cleavable) ZP2 got rid of sperm by the 2-cell stage, whereas the egg with the
mutant (uncleavable) ZP2 retained sperm and permitted polyspermy.

Izumo protein and membrane fusion in mouse fertilization

The “zinc spark” at fertilization

After the artificial activation of a human egg with a calcium channel opener, the
release of zinc ions (starting with the arrowhead) can be seen in increasing and
then diminishing intensity. Extracellular zinc concentrations were detected using
a zinc-sensitive fluorescent dye and are represented using color to show
relative intensities, with red being the highest and green the lowest.

Pronuclear movements during human fertilization

(A) Diagram of sperm-egg cell membrane fusion. During the acrosome reaction,
Izumo localized on the acrosome becomes translocated to the sperm cell
membrane. There it meets the complex of Juno and other egg membrane
proteins on the egg microvilli, initiating membrane fusion and the entry of the
sperm into the egg. (B) Localization of Izumo to the inner and outer acrosomal
membrane. Izumo is stained red, acrosomal proteins green.

Microtubules - green, DNA - dyed blue. Arrows point to the sperm tail. (A) The
mature unfertilized oocyte completes the first meiotic division, budding off a
polar body. (B) As the sperm enters the oocyte (left side), microtubules
 condense around it as the oocyte completes its second meiotic division at the Early Animal Development
periphery. (C) By 15 hours after fertilization, the two pronuclei have come
together, and the centrosome splits to organize a bipolar microtubular array.
The sperm tail is still seen (arrow). (D) At prometaphase, chromosomes from
the sperm and egg intermix on the metaphase plate, and a mitotic spindle
Characteristics
initiates the first mitotic division. The sperm tail can still be seen.

Blocks to Polyspermy (sea urchin)

❖ Polyspermy is a problem for mammals, just as it is for

sea urchins
❖ Fast block (1-3 sec)-depolarization of the egg membrane;
only membranes with -70mV resting potential can fuse
❖ No change in shape; no change in size
with sperm
❖ Ratio of nucleus to cytoplasm progressively increases
❖ Slow block (1 min)- raising of fertilization membrane
❖ Qualitative changes in chemical composition is minimal
traced to release of cortical granules
❖ Cytoplasmic displacement minimal
❖ Increase nuclear material at expense of cytoplasm;
❖ Limited RNA synthesis
Polyspermy occurs more frequently in mouse eggs bearing
mutant ZP2 that cannot be cleaved by ovastacin:

❖ A second slow block to polyspermy comes from the so- Mid-blastula transition
called zinc spark, the release of billions of zinc ions that
is induced by the entry of the first sperm
– bind to the zona pellucida
❖ A third slow block to polyspermy occurs at the level of the
egg cell membrane and involves Juno
– bind sperm in the perivitelline space between the zona
pellucida and the oocyte

How can fertilization go awry?

❖ Too many sperm = dispermy or triploidy

- Leads to spontaneous abortion in most cases.
❖ Infertility
❖ Bad timing:
- The sperm can only survive 48 hours within the female
genital tract.
- In vitro studies show the ovulated egg cannot be Nuclear division is slowed down
fertilized after 24 hours
❖ Cycles of division become longer
• 1-10 cycles are each 8 min long
• Cycle 13 (the last cycle in the syncytial
Non-equivalence of male and female pronuclei
blastoderm) takes 25 minutes
❖ Proven by experiments: • Cycle 14 (in which the Drosophila embryo forms),
- When sperm activates an enucleated egg, nucleus - asynchronous, that is, some groups even reach
replicates, "zygote' divides but development is abnormal 75 mín - 175 min to complete the cycle
- Parthenogenetic egg, DNA replicates, 'zygote" develops
but development is aborted due to abnormalities
❖ Certain genes are active only if they come from either Chemical Changes: Limited during Cleavage
sperm or egg and may not be active in both
❖ The presence of the other homologue is thus a necessary ❖ DNA Synthesis: DNA is synthesized at the expense of
complement in either male or female cytoplasmic RNA

❖ RNA Synthesis: very limited

❖ Protein synthesis
• in sea urchin, the increase is drastic; in the frog the
increase is not so marked
- Nuclear histones*
- Tubulin*
 - Ribonucleotide reductase* Spiral Cleavage Pattern
- DNA polymerase is already present in necessary
quantities

*Important for cleavage

Patterns of Embryonic Cleavage

Looking down on the animal pole of left-coiling (A) and right-coiling (B) snails.
The origin of sinistral and dextral coiling can be traced to the orientation of the
mitotic spindle at the third cleavage. Left- and right-coiling snails develop as
mirror images of each other.

Holoblastic (unequal)

Meroblastic cleavage

Radial Holoblastic Cleavage

Discoidal

Cleavage in the sea urchin. (A) Planes of cleavage in the first

three divisions, and the formation of tiers of cells in divisions
3–6
Early nuclear division and migration during Drosophila
embryogenesis

(B) Confocal fluorescence micrograph of the unequal cell

division that initiates the 16-cell stage, highlighting the
unequal equatorial cleavage of the vegetal blastomeres to
produce the micromeres and macromeres.

Cleavage in an echinoderm embryo

Cycle 1 is initiated after fusion of the male and female pronuclei.

During divisions 1–3, nuclei divide in a sphere at the anterior of the
embryo. During divisions 4–6, nuclei divide and spread out along the
anterior–posterior axis (axial expansion). Nuclei migrate to the cortex
of the embryo during divisions 8–10 (corticalmigration). Pole cells
form at the posterior end of the embryo (cycle 9), while yolk nuclei
remain in the interior. After most of the cortical nuclei complete four
mitotic divisions, they are surrounded by membranes that invaginate
from the surface and the cellular blastoderm is formed.

Cleavage is a series of mitotic cell divisions that transform the

fertilized egg into a blastula, a hollow ball composed of cells
called blastomeres. These light micrographs show the
cleavage stages of a sand dollar embryo, which are virtually
identical to those of a sea urchin.
Micrographs of cleavage in live embryos of the sea urchin
Lytechinus variegatus, seen from the side
(A) Normal development of the 60-cell sea urchin embryo, showing
the fates of the different layers. (B) An isolated animal hemisphere
becomes a ciliated ball of undifferentiated ectodermal cells called a
Dauerblastula (permanent blastula). (C) When an isolated animal
hemisphere is combined with isolated micromeres, a recognizable
pluteus larva is formed, with all the endoderm derived from the
animal hemisphere.

Ability of micromeres to induce a secondary axis in sea urchin

embryos

(A) The 1-cell embryo (zygote). The site of sperm entry is marked with a black
arrow; a white arrow marks the vegetal pole. The fertilization envelope
surrounding the embryo is clearly visible. (B) 2-Cell stage. (C) 8-Cell stage. (D)
16-Cell stage. Micromeres have formed at the vegetal pole. (E) 32-Cell stage. (F)
The blastula has hatched from the fertilization envelope. The vegetal plate is
beginning to thicken.

Fate map and cell lineage of the sea urchin

Strongylocentrotus purpuratus.

(A) Micromeres are transplanted from the vegetal pole of a 16-cell embryo into
the animal pole of a host 16-cell embryo. (B) The transplanted micromeres
invaginate into the blastocoel to create a new set of skeletogenic mesenchyme
cells, and they induce the animal-pole cells next to them to become vegetal
plate endoderm cells. (C) The transplanted micromeres form skeletal rods while
the induced animal cap cells form a secondary archenteron. Meanwhile,
The 60-cell embryo is shown, with the left side facing the viewer. Blastomere gastrulation proceeds normally from the original vegetal plate of the host.
fates are segregated along the animal-vegetal axis of the egg.
Role of the Disheveled and β-catenin proteins in specifying the
vegetal cells of the sea urchin embryo

Ability of micromeres to induce presumptive ectodermal cells

to acquire other fates
 (A) Localization of Disheveled (arrows) in the vegetal cortex of the sea urchin Ingression of skeletogenic mesenchyme cells
oocyte before fertilization (left) and in the region of a 16-cell embryo about to
become the micromeres (right). (B) During normal development, β-catenin
accumulates predominantly in the micromeres and somewhat less in the veg2
tier cells. (C) In embryos treated with lithium chloride, β-catenin accumulates in
the nuclei of all blastula cells (probably by LiCl blocking the GSK3 enzyme of the
Wnt pathway), and the cells of the animal pole become specified as endoderm
and mesoderm. (D) When β-catenin is prevented from entering the nuclei (i.e., it
remains in the cytoplasm), the vegetal cell fates are not specified, and the entire
embryo develops as a ciliated ectodermal ball.

Simplified illustration of the double-negative gated “circuit” for

micromere specification

“Logic circuits” for gene expression

(A) Depiction of changes in the adhesive affinities of the skeletogenic

mesenchyme cells (pink). These cells lose their affinities for hyalin and for their
neighboring blastomeres while gaining an affinity for the proteins of the basal
lamina. Nonmesenchymal blastomeres retain their original high affinities for the
hyaline layer and neighboring cells. (B–D) Skeletogenic mesenchyme cells
breaking through extracellular matrix. The matrix laminin is stained pink, the
mesenchyme cells are green, and cell nuclei are blue. (B) Laminin matrix is
uniformly spread throughout the lining of the blastocoel. (C) A hole is made in
blastocoel laminin above the vegetal cells, and the mesenchyme begins to pass
through it into the blastocoel. (D) Within an hour, cells are in the blastocoel.

Entire sequence of gastrulation in Lytechinus variegatus Positioning of skeletogenic mesenchyme cells in the sea
urchin

(A) Positioning of the micromeres to form the calcium carbonate skeleton is

determined by the ectodermal cells. Skeletogenic mesenchyme cells are stained
green; β-catenin is red; skeletogenic mesenchyme cells appear to accumulate in
those regions characterized by high β- catenin concentrations. (B) Nomarski
videomicrograph showing a long, thin filopodium extending from a skeletogenic
mesenchyme cell to the ectodermal wall of the gastrula (arrows), as well as a
shorter filopodium extending inward from the ectoderm. Mesenchymal filopodia
extend through the extracellular matrix and directly contact the cell membrane
of the ectodermal cells. (C) Seen in cross section through the archenteron (top),
the surface ectoderm expresses FGF in the particular locations where
skeletogenic micromeres congregate. Moreover, the ingressing skeletal
micromeres (bottom; longitudinal section) express the FGF receptor. When FGF
signaling is suppressed, the skeleton does not form properly.
Formation of syncytial cables by skeletogenic mesenchyme
cells of the sea urchin

The embryonic development of fish and amphibians must be carried

out in moist environments. The evolution of the shelled amniote egg
permitted development to proceed on dry land for the reptiles and
their descendants.

(A) Skeletogenic mesenchyme cells in the early gastrula align and fuse to lay
down the matrix of the calcium carbonate spicule (arrows). (B) Scanning
electron micrograph of the syncytial cables formed by the fusing of skeletogenic Reorganization of the cytoplasm and cortical rotation produce
mesenchyme cells. the gray crescent in frog eggs

Invagination of the vegetal plate

(A) Vegetal plate invagination in Lytechinus variegatus, seen by scanning

electron microscopy of the external surface of the early gastrula. The blastopore
is clearly visible. (B) Fate map of the vegetal plate of the sea urchin embryo,
looking “upward” at the vegetal surface. The central portion becomes the non-
skeletogenic mesenchyme cells. The concentric layers around it become the
foregut, midgut, and hindgut. The boundary where the endoderm meets the
ectoderm marks the anus. The non-skeletogenic mesenchyme and foregut come
from the veg2 layer; the midgut comes from veg1 and veg2 cells; the hindgut and
the ectoderm surrounding it come from the veg1 layer.

Extension of the archenteron in sea urchin embryos

Cleavage of a Xenopus egg.

(A) The first three cleavage furrows, numbered in order of appearance. Because
Phylogenetic tree of the chordates showing the relationship of the vegetal yolk impedes cleavage, the second division begins in the animal
the vertebrate groups region of the egg before the first division has divided the vegetal cytoplasm
completely. The third division is displaced toward the animal pole. (B) As
 cleavage progresses, the vegetal hemisphere ultimately contains larger and Early movements of Xenopus gastrulation
fewer blastomeres than the animal hemisphere. The final drawing shows a
cross section through a mid-blastula stage embryo. (C) Fate map of the Xenopus
embryo superimposed on the mid-blastula stage. (D) Scanning electron
micrographs of the first, second, and fourth cleavages. Note the size
discrepancies of the animal and vegetal cells after third cleavage

Key movements during Xenopus gastrulation

(A) At the beginning of gastrulation, the involuting marginal zone (IMZ) forms.
Pink represents the prospective head mesoderm (goosecoid expression).
Chordamesoderm (Xbra expression) is red. (B) Vegetal rotation (arrows) pushes
the prospective pharyngeal endoderm (orange; specified by hhex and cerberus
expression) to the side of the blastocoel. (C,D) The vegetal endoderm (yellow)
movements push the pharyngeal endoderm forward, driving the mesoderm
passively into the embryo and toward the animal pole. The ectoderm (blue)
begins epiboly.

Differential cell migration drives vegetal rotation in Xenopus

gastrulation
Radial intercalation of ectoderm in part drives epiboly

Formation of the blastopore lips in Xenopus laevis

(A) Depiction of epiboly of the ectoderm layer (blue) progressively moving

toward the vegetal pole to completely enclose the endoderm. (B) Scanning
electron micrographs of the Xenopus blastocoel roof (black box in A), showing
the changes in cell shape and arrangement during radial intercalation. Stage 8
(S 8) is a blastula; stages 10 and 11 (S 10 and S 11) represent progressively later
gastrulae. (C) Diagrammatic representations of the blastocoel roof at the same
stages shown in (B). SL, superficial layer; DL, deep cell layer

Cell movements during frog gastrulation Fibronectin and amphibian gastrulation

Collective cell migration of the involuting mesendoderm

Xenopus gastrulation continues

Neurulation