SCITAMA

Embryology Lab
Exercise 1- Gametogenesis
Introduction
Gametogenesis
Germ cells give rise to gametes
Primordial germ cells divide mitotically to produce a large population of germ
cells
o Spermatogonia in males
o Oognia in females
o Both are usually stem cells
First phase of the sexual reproduction of animals
Process of the formation of sex cells for the male and female gametes
o Spermatogenesis is males
o Oogenesis in females
Involves 3 major processes:
1. Cell proliferation
Primitive germ cells repeatedly divide by mitosis
2. Cell growth
Mitosis stops
Raw materials are acquired for protoplasm synthesis
3. Cell maturation
2 successive cell divisions (mitosis) takes place
Accompanied by chromosomal divisions
Results in the reduction of chromosomes by half
Sperms and eggs develop their unique and distinctive structures
o Sperm
A round cell that starts to elongate until it becomes oval in shape
that is more pointed in one part
Most of the cytoplasm is lost and only a few is retained
Flagellum is formed
o Egg
Do not transform much
Increase in size
Start to have a large amount of yolk
Immotile

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Spermatogenesis
Spermatogenesis
Process by which male gametes are formed
Occurs in the seminiferous tubules in the testes
Grasshoppers Testis
Histology
Lobes
o Testis is divided into several lobes
o Has a blunt and pointed end
Blunt end facing the surface of the organ
Pointed end is directed towards the center, opening to the sperm
duct (vas deferens)
o Not all stages of development can be found in one lobe
Cysts
o Each lobe has separate compartments called cysts
o Contains clusters of similar cells undergoing the same stage of
development
o Separated from each other by connective tissue (c.t.) called septa
Stages of maturation are arranged progressively from the blunt end to the
pointed end
o Pointed end contains mature and functional sperm cells
Feulgen method
o Staining method specific for DNA
o Useful for studying chromosomes and nuclear structure
o Cytoplasmic components are stained yellow or brown

Stages of Development
1. Spermatogonia
Small and crowded cells
Located at the blunt end
Nuclei shows beaded chromatin material
Large nuclei containing masses of chromatin
Mitotic division may be seen
2. Primary Spermatocytes
Spermatogonium that has grown and ready to divide
Located in cysts nearest those containing spermatogonia
Have very large nuclei
Makes it easier to identify substages prophase I
3. Substages of Prophase I
Chromosomes become progressively thicker and shorter from stages a to f.
a. Interphase
o Appears finely granular and may show a single pale vascular nucleolus
b. Leptotene
o Chromatin becomes filamentous
o Chromosomes are at their thinnest and longest uncoiled states and fill
the cell
c. Zygotene/Synaptene
o Thickened regions in chromosomes that indicate the beginning of
pairing (synapsis) of homologous chromosomes
Point of contact between two homologues is the synaptonemal
complex
o Chromosome assumes the shape of long loops attached by their
extremity on one given area of nuclear envelop
Drawn to one side of the cell until they take the shape of a
bouquet
Polarization
d. Pachytene
o Chromosomes become thicker and shorter
o Pairing up is complete
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o Exchange of chromosomal ares happen

o Cell APPEARS to be haploid
o Chromatin stays in this condition for a long period of time
o Easy to identify
e. Diplotene
o Chromosomes party split
o Four strands in each homologous pair can be discerned due to
relaxation of pairing
o Chromosomes are thicker
o Chiasma can be first seen
o Configurations (X, cross, loop, etc.) may be observed
f. Diakinesis
o Condensation and coiling of chromosomes is even more evident
o Chromosomes are at their shortest and thickest
o Configurations (X, cross, loop, ring, bar etc.) are more observable
4. Metaphase I, Anaphase I, Telophase I
o Stages are also observable
o Yield secondary spermatocyte in the end
5. Secondary Spermatocyte
Smaller than primary spermatocyte
Haploid
Chromosomes not as evident in primary spermatocyte
6. Spermatids
First resemble ordinary cells smaller than secondary spermatocytes
Haploid
Spermiogenesis
o Metamorphosis spermatids undergo to become spermatozoa
o Can be observed in the inner regions of a lobe
o Involved major nuclear and cytoplasmic changes
o Spermatid primarily elongates
o Most of the cytoplasm is sloughed off
o Small amount of cytoplasm that is left becomes the tail sheath
surround a fine tail filament that grows back from the centrosome
o Chromatin mass becomes more condensed and elongated to form the
darkly stained head
o Definitive sperm consists of the head, middle piece (contains
centrosome and mitochondria) and tail
7. Spermatozoa
Mature sperm cells
Have long, thin and darkly staining heads with distinct tails
Abundant at the distal region

Frogs Testis
Histology
Have similar organization in seminiferous tubules as grasshoppers

Structures
Mesorchium
o Portion of peritoneum that suspends the testis to dorsal body wall
immediate ventral to the kidney and between the layers which pass the
tubes of the vas deferens
Tunica albuginea
o Outer connective tissue
o Covers the testis
o Intertubular connective tissue or septula
o Extends into the substance of the testis
o Partitions testis into many compartments that contain seminiferous tubules
Seminiferous tubules
o Contains clusters of spermatogenic cells

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Each cluster in the seminiferous tubules undergo the same stage of

development, nurse cells and sertoli cells
o Connected with the vas deferens
o May contain spermatocysts undergoing different stages of maturation
Spermatocyst
o AKA cell nest of follicular cysts
o Cluster of similar cells undergoing the same stage of development
o Surrouded by follicle cells
o Formed from a primary spermatogonium and its associate follicle
o Cyst wall break down eventually
o Mature independently from one another
Sertoli Cells
o AKA substantacular cell
o Nurse cell that supplies nutrients to clusters of sperm cells until they can
be liberated
o Goblet-shaped, columnar basal cell
o Associated with the primary spermatogonia and spermatocytes as follicle
cells at first
Interstitial cells
o Small clusters of cells found interspersed with the tunica albuginea
Ova
o Egg cels seen between seminiferous tubules of some males
o Ovatestis

Stages of Development
1. Spermatogonia
Small, closely packed cells
Granular oval nucleus
Invested by a follicle cell
Some remain spermatogonia forever to proliferate
2. Primary Spermatocyte
Very large cells that may be seen in LPO
Irregularly spherical
Possess large and vesicular nuclei
3. Secondary Spermatocyte
About half the size of primary spermatocytes
Lie towards the lumen
Have a darkly staining nucleus
Cytoplasm clearly visible and prominent
4. Spermatids
Even smaller than secondary spermatocytes
Condensed nucleus of irregular shape
Clusters of spermatids undergoing metamorphosis appear as a cluster of
granules with a dark nucleus
Inner centriole passes into the nucleus, the other centriole gives rise to a tail-
like flagellum
5. Spermatozoa
Average 0.33mm
Elongated, solid staining head
Anterior acrosome pointing towards the periphery of the seminiferous tubule
Tail appears to be a lightly stained filamentous extension
Heads group together or converge to sertoli cell
Mouses Testis
Histology
Tunica albuginea covers the testis and subdivides it into lobules that each
contain seminiferous tubules
Seminiferous tubules are filled with spermatogenic cells and sertoli cells
Spermatogenic cycle lasts a total of 48 days, but successive cycles start every
12 days
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Structures
Seminiferous Tubules
o Have small blood vessles filled with RBC, leydig cells and connective
tissue cells with elongated nuclei in between
Leydig Cells
o Intersittial cells
o Produce testosterone
o Clumps of cells with conspicuous nuclei that contains clumps of blue-
stained chromatin
Sertoli Cells
o Sustentacular/nurse cells
o Spaced at intervals against the basement membrane and around the
circumference of the seminiferous tubule
o Distinct outlines
o Large, oval and often indented nuclei with compound nucleolo
o Surrounded by spermatozoa
o Form the blood-testes barrier
Nutrients and circulating substances do not directly reach germ
cells
o Determines what substances reaches germ cells
o Produce antigen-binding proteins necessary for spermatogenesis
o Two Types:
Active Sertoli Cells
Pyramidal in shape
Apex directed towards the lumen
Nucleus perpendicular to the tubule wall
Inactive/Resting Sertoli Cells
Polygonal in shape
Nucleus parallel to tubule wall
Spermatogenic cells
o Arranged in a layer
o Includes spermatogonia to spermatozoa

Stages of Development
1. Spermatogonia
Most primitive of the male sex cells
Undergo meiosis
Progenies are translocated
Mature in groups around the circumference of the tubule towards the lumen
Rests on the basement membrane and interrupted at intervals by sertoli cells
Thin layers of ells with compact, deeply staining nulei
Small nuclei
Three Types:
o Type A Spermatogonia
Chromatin particles appear fine and evenly dispersed
Single promineny chromatin nucleolus
Germ cell line
o Type B Spermatogonia
Coarse granules or flakes of heavily stained chromatin
associated with nuclear membrance and nucleolus
Most common type
Last stage before transformation to primary spermatocyte
o Intermediate Spermatogonia
Chromophilic chromatin in the form of delicate crusts or flakes
attached to nuclear membrane
Prominent nucleoli appearing in the center
2. Primary Spermatocyte
Large size
Nuclei larger than spermatogonia
Larger than Sertoli cells
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Chromosomes are very much visible

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Nuclear membrane are disintegrated

Refer to PMAT in the manual (page 11 of lab manual)
3. Secondary Spermatocyte
Smaller than primary spermatocyte
Pale stained granular chromatin
Several globules of chromophilic chromatin are free
Grossly resemble spermatids but are larger and fewer
4. Spermatids
Initially possesses round nuclei
Abundant and small
Elongate
Nucleus will decease in size due to the elimination of nuclear sap
Nucleus staining intensifies
Golgi apparatus becomes the acrosomal rudiment; both appear as an intense
red granule near the nucleus
Midpiece is a pink-stained thickening along the first half of the length of the
flagellum
5. Spermatozoa
Deeply stained short structures
Form the innermost layer of the tubule
Heads are buried in Sertoli cells
Tails extend to lumen
Made up of head, middle piece and tail
Chicks Testis
Histology
Similar to mouse
Does not stain clearly

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Oogenesis
Oogenesis
Process of producing egg cells
Polar body is produced after meiotic division
o Due to unequal cytokinesis
o Provides ovum with more cytoplasm and stored food
o All polar bodies are nonfunctional
Takes place within the follicles of the ovaries
Ovaries are covered by a continuous sheet called the germinal epithelium
Mesovarium is the mesentery that attached the ovary to the body wall and is the
route for blood vessels
Ovary is divided into a central medulla and peripheral cortex
o Medulla consists of connective tissue and blood vessels
o Cortex is the business part of the ovary
Frogs Ovary
Histology
Large sac of yolk
Heavier and larger yolk granules concentrated towards the animal hemisphere
Coat necessary for retaining the shape of the egg and aids in cleavage and
gastrulation surrounds the egg
Coat contains pigments

Structures
Mesovarium
o Double-layered extension of the peritoneum
o Attached ovary to dorsal wall
Ovarian Follicle
o Collective term for composed of the ff:
Theca externa
Outermost connective tissue surrounding the ovary
Continuous with the mesovarium
Region of rupture during ovulation
Contains blood vessels and nerves
Theca interna
Inner connective tissue surrounding the ovary and each egg
Contains blood vessels and nerves
Follicle cells
Derived from the oogonia
Contain oval and granular nuclei
Surround the developing oocyte
Found in close association with the oocyte throughout the
process of maturation
Oogonia
o Located at the periphery of the ovary
o Much smaller than oocytes
o Have no pigment
o Contain little to no yolk
o Each cluster represents a future ovarian unit
o Most will become follicle cells; only a few will become ova
Oocyte
o Potential ova
o Bigger than oogonia
o Has a germinal vescicle
Immature nucleus
Lightly stained
o Nucleus may be exluded during sectioning
o Distinction between primary and secondary oocyte is impossible
Vitelline membrane
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o Non-cellular, transparent membrance between follicle cells and each

mature egg
o Not seen in early oogonia stage
Perivitelline membrane
o Fluid-filled space formed between egg and vitelline membrane after
fertilization
Cats Ovary
Histology
Germinal epithelium forms outer surface of ovary
Tunica albuginea is a layer of connective tissue under the germinal epithelium
Stroma is another layer of connective tissue under tunical albuginea
o Early follicles surrounded by a single layer of squamous follicle cells are
embedded in the stroma
o Stroma cells are unspecialized intermediate mesoderm cells
Some become modified as thecal cells
Others become interstitial cells

Structures
Germinal epithelium
o Epithelial lining of the ovary
o Composed of cuboidal cells
Tunica albuginea
o Fibrous connective tissue under germinal epithelium
Stroma
o Connective tissue under tunica albuginea
o Penetrates the substace of the ovary
o Consists of connective tissue fibers and spindle shaped cells
Oogonium
o Potential ovum
o Vesicular nucleus with prominent nucleolus
o Embedded in stroma
o Occurs in clusters
o Those without follicle cells surrounding them are in their early stages
Corpus luteum
o Mass of granular tissue in the ovary resulting from the ovulation of egg
cells from follicles
o Produces progesterone
o Lightly stained
o Follicle converge at the middle portion
o The following structures can be found in the corpus luteum:
Lutein cells
Large, pale staining cells
Large vesicular nucleus
Crom from stratum granulosum of ovulated Graafian follicle
Located at the periphery of corpus luteum and within its folds
Not seen
Capillaries
Network of small blood vessels
Single endothelial lining
Empty spaces
May has dark masses of blood cospuscles
Capsule
Layer surrounding corpus luteum
Consists of connective tissue fibers and fusiform cells
derived (from theca externa), lutein-like cells (from theca
interna) and blood vessels
Corpus albicans
o Usually found in the central portion of ovary
o Non-functional corpus luteum
o Formed when fertilization does not take place
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o Stroma forms a capsule around regressing corpus luteum and invades the
regressing luteal cells
o Repaces luteal cells as they degenerate
o Leaves a white hyalinized scar
o Pale stain in its central portion
Periovarial membrance
o Peritoneum of ovary
o Continues into oviduct
o Consists of squamous epithelial cells and adipocytes
Periovarial space
o Space between the periovarial membrance and ovary
Oviduct
o Convoluted structure
o Found alongside of the ovary
o Attached to the periovarial membrane

Stages of Maturation
1. Primordial Follicle
Oogonia without follicle cells
2. Primary Follicle
Oogonia surrounded by a single layer of flattened follicular cells
Consists of the ff:
o Follicles
Single layered
Surround the developing germ cell
Have dark nuclei
May be squamosal or cuboidal
o Oogonium
Potential ovum surrounded by a single layer of follicle cells
Spheroidal and has a vesicular nucleus
Prominent nucleolus
Moves deeper into the ovary as it matures and starts to enlarge
3. Growing Follicle
Succeeds the primary follicle stage
Follicle cells have proliferated by mitotic division
Several layers thick
Increased in size and complexity
Consists of the ff:
o Follicle Cells
Several layers thick
Low columnar or cuboidal cells enclosing oocyte
o Antrum
AKA follicular cavity
Cavity within the growing follicle
Formed by the union of spaces between follicle cells
May not be that evident in the growing follicle stage
o Liquor folliculi
Viscous follilcular fluid secreted by the follicle cells
Appears as a light pink coagulum within the antrum
May not be that evident in the growing follicle stage
o Oogonium or Primary Oocyte
Probably stage of development of the cell in a growing follicle
Hardly distinguishable from each other
Separated from granulose by zona pellucida
o Zona Pellucida
Deeply staining membrane
Surrounds the egg cell
Separates egg from follicle cells
4. Graafian Follicle
Large follicle

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Bulges on the free side of the ovary

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Consists of the ff:

o Antrum
Fluid-filled cavity
Larger than before
Lined by granulose cells (stratum grnulosum)
Thick layer of follicle cells
o Cumulus Oophorus
Mound of follicle cells surround egg cells
Includes the ff:
Primary oocyte
o Heavy nuclear envelop
o Granular nucleus
o Large nucleolus
o Cytoplasm filled with yolk granules
Zona pellucida
o Deeply staining membrane
o Surrounds the egg cell
o Separates egg from follicle cells
o Stratum granulosum
Layers of follicle cells bordering the Graafian follicle
Excludes the cumulus oophorus
o Corona radiata
Layer of follicle cells of the cumulus oophorus
Lie nearest the oocyte
Separated from the oocyte by the zona pellucida
o Liquor folliculi
Viscous follilcular fluid secreted by the follicle cells
Appears as a light pink coagulum within the antrum
May not be that evident in the growing follicle stage
o Theca folliculi
Stroma adjacent to the follicle that forms a capsule around the
follicle
Includes the ff:
Theca interna
o Inner layer between theca externa and granulose
cells
o Consists of large stromal cells
o Oval nuclei and capillaries
o Richly vascularized
o Secretory cells
Theca externa
o Outer layer
o Composed of spindled-shaped dense connective
tissue fibers
o Continous with the surrounding ovarian stroma
Ovulation involves rupture of the theca interna, theca externa,
membrane propria and layer of granulose cells
5. Atretic follicle
Unsuccessful follicles undergoing degenerative process
Atresia occurs in the ovum followed by degeneration of follicle cells
Antrum with scattered follicles
Unorganized stratum granulosum
Fragments of the germ cells
Presence of collapsed zona pellucida
Cavity invaded by thickened and vascularized theca interna
Mass of tissue resembles a regressing corupus luteum and is called a corpus
atretica

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Humans Ovary
Histology
Most parts are the same as the cats ovary
Degenerating follicles are many
Different stages of follicular development are less observed in humans

Structures
Tunica albuginea and stroma
o More prominent in humans
o Description is similar to description in cats ovary
Corpora lutea
o May be in various stages of regression
o Active corpus luteum may not be seen
o Has a very large cavity filled with loose connective tissue and remnant of
lutein cells
Blood vessels
o Vein vs artery

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