First Semester

Comparative Vertebrate Embryology - Laboratory

Exercise 1: Gametogenesis

Egg
o
o

Two type of Cell

Germ Cells
o Gives rise to the gametes (Egg and
the sperm cells)
Somatic cells
o Other cells in the Bod

Increase in size
Accumulation of yolk

SPERMATOGENESIS
Formation of male gametes
Occurs in the seminiferous tubule
In association of specialized somatic cells

Primordial Germ Cells

The initial germ cells that form
Must journey to the gonads and divide
mitotically.
o Spermatogonia males
o Oogonia Females

Histology of Grasshoppers Testis

Development happens in the male gonad
Testis divide to several lobes
o Arranged with their blunt ends
facing the surface of the organ.
o The more pointed leads to the vas
deferens
Inside the lobe
o Compartments Cysts
o Similar cells undergoing the same
stage of development
o Separated by septa connective
tissue
o The more immature cysts of the
periphery
o Shows progressive maturation

Stem Cells
Undifferentiated cells that divide mitotically
producing two types of cells.
Some will remain undifferentiated to
maintain cell line alive.
Gametogenesis
First phase of sexual reproduction
Formation of sex cells for male and female
gametes
o Spermatogenesis
o Oogenesis

Spermatogonia
Small, crowded, at the blunt ends of the
testis
Nuclei beaded apperance of chromatin
material
Have large nuclei and conspicuous
chromatin

Major Processes of Gametogenesis

Cell Proliferation
o Primitive germ layer undergo
repetitive mitotic division into
identical cells
Cell Growth
o Cessation of mitosis in favour of
acquisition of raw materials for
synthesis of protoplasm
Cell Maturation
o Two successive cell division
o Accompanied by chromosomal
division that will lead to reduction of
chromosome number to half.

Primary Spermatocyte
Spermatogonium undergone growth, ready
to divide.
Located near the spermatogonia
Very large nuclei
Stages of Prophase 1
o Interphase Fine granular, show
single pair vascular tissue
o Leptotene Chromatin crust finely
beaded filaments, filamentous,
chromosome uncoiled- completely
fill the cell
o Zygotene have thickened regionsynapsis, synaptonemal complex,
paired chromosomes takes the
shape of a bouquet, double

Changes in appearance
Sperm
o Elongation that is more pointed in
one part
o Losses cytoplasm, leaving minimal
amount
o Flagellation
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Comparative Vertebrate Embryology - Laboratory

thickness of chromosomes means

advance pairing.
o Pachytene Complete pairing and
eventual exchange of chromosomal
arm, the cells seems to be haploid,
chromosomes shorten and thicker.
o Diplotene Chromosomes partly
split, relaxation of pairing, presence
of chiama, configuration of x,
cross, or loop
o Diakinesis

progressive
condensation by means of coiling,
chromosomes are in their shortest
and thickest appearance.
The nucleus goes through the metaphase,
anaphase, and telophase first maturation
division.

Histology of Frogs Testis

Mesorchium
Portion of the peritoneum that suspends the
testis to the dorsal body wall.
Tunica Albuginea
Outer connective tissue that covers the
testis
Known as septula or intertubular connective
tissue
Seminiferous tubule
Much-coiled tubule containing the clusters
of spermatogenic cells undergoing the same
stage of development and the sertoli cells
Connected to the vas deferens
Cells going through same stage are found in
a cluster or group within the tubule.
Spermatocysts
Cluster of similar cells undergoing the same
stage of development
Surrounded by follicle cells
Formed from primary spermatogonium and
its associated follicle
Spermatogonia
Small, closely packed cells with granular oval
nucleus.
o Primary Spermatozoa fastened to
the walls of the seminiferous tubule
o Residual Spermatozoid Source of
germ cell proliferation
Active Spermatogenesis short season in
summer, begins with a rapid succession of
mitotic division
The millions of sperm came from
comparatively large number of gonia by
repeated mitotic division, maturation, and
spermatogenesis.
The seminiferous tubules do not contain
permanent population of sperm cells.

Secondary Spermatocyte
Smaller than the primaries
Chromosomes are not that evident
Spermatids
Half the size of the secondary spermatocyte
Possess haploid set of chromosomes but
not capable of functioning as male gamete
SPERMIOGENESIS transformation of
spermatids to spermatozoa.
o May be observed in the inner region
of the lobe
o Major nuclear and cytoplasmic
changes that will eventually
terminate in the formation of
functional gamete.
o Elongation of spermatid cytoplasm
sloughed off.
o Tail Sheath surrounding the tail
filament,
grows
back
from
centrosome
o Head

Condensation
and
elongation of chromatin mass
o Middle Piece contains the
centrosome and mitochondria
o Tail

Primary Spermatocyte
Very large cell, spherical,
with large
vesicular nuclei
Characteristic
meiotic
prophase
configurations of chromosomes can be seen.

Spermatozoa
Exhibit long, thin, darkly staining head with
distinct tails.
Tail measures 7-8 times longer than the
head.
Abundant at the distal region of the testis.

Secondary Spermatocyte
Result of the first meiotic division
Half of the size of the primaries and lie
towards the lumen of the tubule.
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First Semester

Comparative Vertebrate Embryology - Laboratory

Have darkly staining nucleus, and clear and

visible cytoplasm.

Spermatids
Even smaller than the preceeding cells
Possess a condensed nucleus of irregular
shape.
During spermiogenesis, the inner of the two
spermatid centrioles passes into the nucleus
while the other one give rise to the
flagellum.
Spermatozoa
Average length of 0.03mm.
Has an elongated solid staining head with an
anterior chromosomes
The tail appears like gray filamentous
extension
The maturation depends upon the external
sources of nutrition, grouped into 25-40 cells
converging into the relative columnar type
basal cell sertoli cells.
Sertoli Cells
Supplying nutrient to the cluster of sperm
until they are ejaculated.
Associated with the primary spermatogonia
and spermatocyte as follicle cells.
In frogs, sperms remained attached
throughout the winter, as long as six
months.
Interstitial Cells
Small clusters of cells found interspersed
with the intertubular connective tissue
Ova
Egg cells seen between the seminiferous
tubule.
Making the organ ovotestis

Spherical
structures
filled
with
spermatogenic cells.
In mammalian, convoluted tubules and
made up of specialized seminiferous
epithelium
o Sertoli cells
o Male
Sex
cells
undergoing
spermatogenesis
and
spermiogenesis
Contains the cells with conspicuous nuclei
call the Interstitial or Leydig cells
o Responsible for the production of
testosterone.

Spermatogenic cycle
Last a total of 48 days, successive cycles start
every 12 days
Total of 4 overlapping cycles at every site of
the tubule.
Sertoli Cells
Located near the spermatogonia
Has distinct outline and large, oval, often
indented nuclei with compound nucleoli
Surrounded by spermatozoa
Two types of sertoli cells
o Active- fulfilling the normal function,
pyramidal in shape, nucleus
perpendicular to the tubule wall
o Inactive (resting)- polygonal in
shape, nucleus parallel to the dorsal
body wall.
SPERMATOGENIC CELLS:
Spermatogonia
Most primitive of the male sex cells
Three criteria:
o Rests on the basement membrane
and interrupted at intervals of
Sertoli cells
o Appear as thin layer of cells with
compact deeply staining nuclei
o Nuclei tend to be quite small,
comparable with the size of the
spermatids.
Three types
o Type A- chromatin particles appear
fine and evenly dispersed, in
eccentric position, usually in the
germ cell line
o Type B- chromatin is identified with
the presence of coarse granules or

HISTOLOGY OF MOUSE TESTIS

Stained in haematoxylin and eosin
o Nucleus blue (basic, stained by
haematoxylin)
o Cytoplasm pinkish orange (acidic
by eosin)
Tunica Albuginea
Thick layer of fibrous connective tissue with
a covering of mesothelial cells
Subdivides the testis into a number of
lobules each containing seminiferous
tubules
Seminiferous tubule
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Comparative Vertebrate Embryology - Laboratory

flakes, most common gonia, last

stage before primary spermatocyte
Intermediatechromophilic
chromatin in the form of delicate
crustor flake, has prominent nuclei
appearing in the center.

Products of meiotic division which is

responsible for haploid condition
Formation of Polar bodies
o Produced after first meiotic division
o Smaller than egg
o Due to unequal cytokinesis
o Provides the ovum a much greater
amount of cytoplasm and store
food.
o Non functional
After meiosis, the ootid quickly change into
the mature egg cell or ovum
It takes place within the follicles of the ovary
o The ovary is covered by continuous
sheet of epithelium called the
germinal epithelium
Mesovarium
The mesentery that attached the ovary to
the body wall
Route for blood vessels to and from the
ovary.

Primary Spermatocyte
Significantly large in size
Nuclei larger than the spermatogonia
The chromosomes are very much visible and
the nuclear membrane is disintegrated
already.
Visible stages:
o Interphase Similar to gonia B,
finely granular nucleus with pale
nucleolus, exist for about 6 hours.
o Prophase I
o Metaphase I
o Anaphase I
o Telophase I
Secondary Spermatocyte
Result of first meiotic division of the primary
spermatocyte
Pale stained granular chromatin
Twice the diameter of spermatids.

Ovary
Divided into two parts
o Central Medulla consist largely of
connective tissue and blood vessels,
appear more or less continuous with
the mesovarium
o Peripheral cortex business part of
the ovary

Spermatids
Result of second meiotic division
Lie next inward beyond the spermatocyte
layer in the seminiferous tubule
Nuclei quite round, small size (2/3 of primary
oocyte), pale staining properties, and
position above the level of primary
spermatocyte IMPORTANT CLUES
DNA undergoes progressive condensation
Decrease of nuclear size due to the
elimination of nuclear sap.

HISTOLOGY OF FROG OVARY

A large sac of yolk with heavier and larger
granules concentrated toward the animal
hemisphere
Surrounded by a coat necessary for retaining
the shape, aids in cleavage and gastrulation
Mesorvarium
Double-layered extension of the peritoneum
Attachment to the dorsal body wall

Spermatozoa
Deeply stained when matured
The heads are buried at the Sertoli cells
Matured spermatozoa becomes free of the
nurse cells and pass into the ducts

Theca externa
Outermost connective tissue surrounding
the ovary
Continuous with the mesovarium
Ruptures during ovulation

*The histology of Chicks Testis is also the same as

mouses testis

Theca interna
Inner connective tissue surrounding the
ovary and each egg

OOGENESIS
Process of producing egg
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First Semester

Comparative Vertebrate Embryology - Laboratory

Follicle Cells
Derived from oogonia
Contains oval and granular nuclei
Surrounds the developing oocyte

Another layer of connective tissue below the

Tunical albuginea
Made up of connective tissue fibers and
spindle shaped cells.

Oogonium
Potential ovum with vesicular nucleus with a
prominent nucleolus
Embedded at the stroma and in clusters.

Ovarian Follicle
Collective term for theca externa, theca
interna, follicle cells
Oogonia
Located at the periphery of the ovary and
much smaller than the oocyte
No pigment and contains a little yolk
Each cluster of oogonia represents a future
ovarian unit.
The majority of oogonia never mature into
ova.

Primary Follicle Stage

Surrounded with single layer of flattened
follicular cells.

Oocytes
Bigger than the oogonia
Lightly stained at the center is the germinal
vesicle or immature nucleus.
Distinction to primary and secondary oocyte
is not possible

Structures of Follicle Stage Cells

Follicles
o Single layer of cells that surrounds
the developing germ cell
o Have dark stained nuclei and maybe
squamosal or low cuboidal.
Oogonium
o Potential ovum surrounded by single
layer of follicle cells
o Spheroid and has a vesicular nucleus
with a prominent nucleolus

Vitelline Membrane
Non-cellular
transparent
membrane
between follicle cells and each mature egg
cells.
Derived from ovum and follicle cells
Developed during maturation process.

Growing Follicle Stage

Immediately succeeds the primary follicle
stage
Proliferated by mitotically dividing cells and
are now several layers thick.
Increased in size and complexity.

Perivitelline Space
Fluid-filled space formed between the egg
and the vitelline membrane after
fertilization
Shows compensatory shrinkage.

Structures
Follicle Cells
o Several layers thick, low columnar or
cuboidal cells enclosing the oocyte
Antrum
o Follicular cavity
o Formed by the union of spaces
between the follicle cells
o Precipitation of proteins in the fluid
may give the cavity a granular
appearance.
Liquor Folliculi
o Viscous follicular fluid secreted by
follicle cells.
Oogonium or Primary Oocyte
o The probable stage of development
of the cell in the growing follicle

HISTOLOGY OF CAT OVARY

Germinal epithelium
Forms the outer surface
Epithelial lining of the ovary
Cuboidal cells
Tunica albuginea
Layer of fibrous connective tissue below the
germinal epithelium
Stroma

First Semester

Comparative Vertebrate Embryology - Laboratory

Hardly distinguishable from each

other
Zona Pellucida
o Deeply
staining
membrane
surrounding the egg cell and
separating it from the follicle cells

Graafian follicle
Large follicle that bulges on the free surface
of the ovary
Structures
Antrum
o Fluid-filled cavity, now it is bigger.
o Lined by stratum granulosum, thick
layer of follicle cells
Cumulus Oophorus
o Eccentric mound of follicle cells
surrounding egg cells
o Includes the oocyte, zona pellucida,
amd surrounding granulose cells
Primary oocyte
o Stage of development at this point
o Has a heavy nuclear envelope
Stratum Granulosum
o Layers of follicle celss boredering the
Graafian, excluding the cumulus
oophorus
Corona Radiata
o Layer of follicle cells of the cumulus
oophorus that lie nearest to the
oocyte
Zona Pellucida
Liquour Folliculi
Theca folliculi
o The stroma immediately adjacent to
the follicle
Theca interna
o Inner layer between the theca
interna and the granulose cells
o Richly vascularized
o Cells are secretory
Theca Externa
o Outer layer of the capsule composed
of spindled shaped dense connective
tissue fibers
Atretic Follicles
o Unsuccessful follicles undergoing
degenerative process
o Shows antrum scattered follicle cells

Exhibited by the presence of

collapsed zona pellucida
o Corpus atretica mass of tissue
regressing corpus luteum
Corpus lutea
o Masses of granular tissue
o Result from the ovulation of egg
cells from follicles.
o Responsible for the production of
progesterone
o Lightly stained, follicles converging
at the middle portion
o Lutein Cells, Capillaries, Capsule
Corpus Albicans
o Found in the central portion of the
ovary.
o Non-functional corpus luteum when
fertilization does not take place
o Pale stain in its central portion
Periovarial Membrane
o Peritoneum of the ovary which
continues into the Oviduct
Periovarial Space
o Space between the periovarial
membrane and the ovary
Oviduct
o Convoluted structure that is found
alongside of the ovary
o Attached
to
the
periovarial
membrane