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Embryo Lab Exercise 1: Gametogenesis

Introduction
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Cells classified into


o Somatic cells
Give rise to all other cells in the body
o Germ cells
Give rise to gametes (egg and sperm)
Primordial germ cells (initial germ cells) journey from where they are formed to the
developing gonad (testis/ovary)
These primordial germ cells divide mitotically to produce a large population of
germ cells Spermatogonia (males) Oogonia (females)
o Spermatogonia and Oogonia Stem cells
Stem cells are undifferentiated cells that divide mitotically
producing 2 types of cells:
May remain as stem cells to keep cell line alive
May be differentiated
Gametogenesis
o First phase in sexual reproduction of animals
o Formation of sex cells for male and female gametes
o Gametes will transform into specialized cells eventually
o Spermatogenesis
Process of forming sperms
o Oogenesis
Formation of ova or eggs

Major Processes involved in Gametogenesis


1. Cell Proliferation
o Primitive germ cells/Primordial sex cells divide by mitosis (identical cell result)
2. Cell Growth
o Cessation of mitosis in favor of acquisition of raw materials for synthesis of protoplasm
3. Cell Maturation
o 2 successive mitosis takes place
o Accompanied by chromosomal divisions
Divisions result in reduction of chromosomes by half

Gametogenesis
o During this stage sperm and eggs develop their unique and distinctive structures
o Sperm
Round cells elongate until they become oval and pointed in one part
Lose their cytoplasm, retain only a little part
Form a flagellum for swimming (motility)
o Eggs
Do not transform much
Only increase in size
Have a large amount of yolk
Immotile
o Sperm and Eggs undergo meiotic divisions that reduce their chromosome number into HALF

SPERMATOGENESIS
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Process where male gametes are formed


Occurs in seminiferous tubules in the testes
Develops with specialized somatic cells

Histology of the Grasshopper Testis


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Development of spermatozoa takes place in male gonads (testes)


Each testis is divided into lobes ***GRASSHOPPER ONLY LOBED IN LE LAB
Lobes pointed end directed to center center opens into sperm duct (vas deferens)
Cysts
o Separate compartments inside the lobes
o Contains clusters of similar cells undergoing same stage of development
o Compartments
Separated by septa composed of connective tissues
While being developed, cyst is pushed towards inner end of lobe
Immature cysts takes its place in the periphery
o Cysts show progressive maturation
Pointed end of organ continuous with sperm ducts contain mature and functional
sperm cells
Slides of grasshopper testis were stained using the Feulgen method
o Specific for DNA
o Used for studying chromosome and nuclear structure
o Cytoplasmic components are yellow/brown

Stages of Development
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Spermatogonia
o Small, crowded and located at blunt end of testis
o Nuclei beaded appearance (chromatin)
o Large nuclei
o Mitotic divisions seen here
o Multiply first before undergoing maturation
Primary Spermatocytes
o Spermatogonium ready to divide
o Located in cysts near spermatogonia
o Very large nuclei
o Stages of prophase I identifiable in this specimen
Interphase
Inconspicuous appears finely granular single pale vascular nucleolus
Leptotene
Finely beaded filaments of chromatin
Chromatin becomes filamentous
Completely filled cell
Chromosomes longest here (UNCOILED)
Zygotene/Synaptene
Thickened regions in chromosomes
Beginning of pairing (synapsis) of homologous chromosomes
Synaptonemal complex
o Point of contact between 2 homologues
Paired chromosomes assumes shape of long loops
o Attached to extremity of nuclear envelop
Drawn to one side in a shape of a bouquet
Double thickness indicates advance pairing
Pachytene
Nuclear volume increases, chromosomes shorten and become thicker
Complete pairing and exchange of chromosome arms here
Cells in haploid condition
Chromatin stays in this condition for a long period

Lobe is half-filled with cyst in this stage


Diplotene
Chromosomes partly split
4 strands in each homologous pair can be discerned due to relaxation of
pairing
Chromosomes thicker
Chiasma
o First seen at diplotene
o Interchanges between homologoes keep them united
X like configuration is observed
Diakinesis
Condensation of chromosomes means coiling
Chromosomes seen at their shortest and thickest
o Nucleus then goes through metaphase, anaphase and telophase of first maturation to yield
Secondary Spermatocytes
Secondary Spermatocytes
o Smaller than primaries
o Chromosomes not as evident as primaries
Spermatids
o Resemble ordinary cells about half the size of the secondary spermatocytes
o Haploid chromosomes; but not functioning male gametes
o Still need to undergo metamorphosis called Spermiogenesis
Spermiogenesis
Transformation of spermatids to spermatozoa
If no spermiogenesis sperms are useless
In the inner regions of lobe
o Major nuclear and cytoplasmic changes that terminate in the formation of the functional sperm
o Spermatid elongates, cytoplasm is sloughed off except for a small amount
Small amount becomes the tail sheath
Tail sheath surrounds a fine tail filament which grows back from the
centrosome
o Chromatin mass becomes condensed and elongated to form the head
o Sperm (ready to fertilize) consists of:
1. Head
a. In grasshopper is 10 or more times longer than it is wide
2. Middle piece
a. Contains centrosome
b. Mitochondria
3. Long Tail
Spermatozoa
o Mature sperm cells
o Long,thin and darkly stained heads with tails
o 7 to 8 times longer than heads
o Abundant at distal region of grasshopper testis

HISTOLOGY OF FROG TESTIS


Structures
1. Mesorchium
a. Peritoneum that suspends testis to dorsal body wall
b. Between layers of tubes of vas deferens
2. Tunica Albuginea

3.

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a. Outer connective tissue


b. Covers testis
c. Known as intertubular connective tissue or septula
d. Tough tissue; extends into substance of testis
e. Partitions into many compartments containing seminiferous tubules
Seminiferous Tubules
a. Coiled tubule containing spermatogenic cells
b. Also has sertoli cells nurse cells
c. Tubule connected to vas deferens
d. Spermatogenic cells may undergo maturation
e. Cells going through a particular stage found in a culture/group within the tubule
Spermatocyst or Cell Nest or Follicular Cyst
a. Cluster of similar cells undergoing same stage of devt
b. Surrounded by follicle cells
c. Spermatocyst = primary spermatogonium + associate follicle
d. Spermatocyst forms gonia of the second and third order which undergo maturation to form
SPERMATOZOA
e. Cyst wall eventually breaks down
f. Spermatocysts mature independently of one another
g. Each seminiferous tubule may contain cysts in different stages of maturation
Spermatogonia
a. Closely packed cells with granular nucleus
b. Primary spermatozoa fastened to walls of seminiferous tubule
c. Gonium invested by follicle cells
d. Residual spermatozoid source of germ cell proliferation
e. Active spermatogenesis
i. Short season in summer
ii. Begins rapid succession of mitosis
iii. Daughter cells from spermatogonial divisions retain follicle cells
1. Eventually become sperms
f. Millions of sperms from frog come from small number of gonia by repeated mitosis, maturation
and spermatogenesis
g. Seminiferous tubules
i. Do not contain permanent population of sperm cells
ii. Sperm cells evacuated at time of each spawning
Primary Spermatocytes
a. Very large cells
b. Irregular, large and vesicular nuclei
c. Meiotic prophase can be discerned
d. Divides to form haploid secondary spermatocytes
Secondary Spermatocytes
a. Haploid; result of first meiotic division
b. Half the size of primaries
c. Lie toward lumen of tubule
d. Clearly visible cytoplasm
Spermatids
a. Condensed nucleus
b. Cluster of granules
c. Metamorphosis of spermatids to spermatozoon
i. Inner of 2 spermatid centrioles passes into nucleus while the other one gives rise to
the tail-like flagellum
Spermatozoa
a. Average at 0.30 mm
b. Elongated solid staining head
c. Anterior acrosome pointing outward to seminiferous tubule
d. Tail is a gray filamentous extension of head
e. Groups into 25 to 40 cells for external sources of nutrition
f. Columnar type basal cell known as sertoli cells

i. Sertoli cells; source of nutrition for sperm heads


10. Sertoli or Sustentacular Cell
a. Nurse cell; supplying nutrients to sperm
b. Was associated with the primary spermatogonia and spermatocytes as follicle cells
c. When the tails of immature sperms grow out and follicular cysts (spermatocysts burst open
into lumen of seminiferous tubule)
i. Each bursted follicle cell attracts a bundle of sperm and transforms into a goblet
shape nurse cell
d. Frog sperms attach through winter, as long as 6 months
i. When detached, theyre stored in the mesonephric tubules and in different ducts
ii. In the mesonephric tubules they die agad
11. Interstitial Cells
a. Small clusters of cells interspersed with intertubular connective tissue
b. Undetermined if these cells secrete male sex hormone in the frog
12. Ova
a. Egg cells seen between seminiferous tubules of some male individuals
b. Male organ OVITESTIS
HISTOLOGY OF MOUSE TESTIS (chick testis also applicable here)
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Stained with hematoxylin and eosin


o Hematoxylin
Blue stain that stains nuclei
o Eosin
Pinkish-orange counter stain to contract acidic components
Cytoplasm
Tunica albuginea
o Tissue covering mouse testis
o Thick layer of fibrous connective tissue
o With a covering of mesothelial cells
o Internally, this tissue subdivides the testis into lobules
Lobules contain seminiferous tubules
Inside the testis
o Seminiferous tubules
Filled with spermatogenic cells
Spherical structures
Made up of specialized seminiferous epithelium with 2 types of cells
Sertoli cells (somatic cells)
Male sex cells (undergoing spermatogenesis and spermiogenesis)
Spermatogenic cycle
o In mice it lasts a total of 48 days
o Successive cycles start every 12 days
o Total of 4 overlapping cycles at every site along the tubule

STRUCTURES
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Seminiferous tubules
o Structures found in seminiferous t.
Small blood vesses filled with red-stained erythrocytes
Clumps of cells with conspicuous nuclei that contains blue-stained chromatin
Interstitial cells/Leydig cells
o Produce testosterone
Connective tissue cells with elongated nuclei
Sertoli Cells
o Nurse/sustentacular cells
o Spaced at regular intervals against basement membrane and around seminiferous tubules

Near spermatogonia
Have distinct outlines and large,oval
With indented nuclei with compound nucleoli
Surrounded by spermatozoa
Functions as:
Form blood-testes barrier
Nutrients and other substances do not directly reach cells
Determines which substances reach the germ cells
Spermatogonia are outside blood-testes barrier
Produce antigen-binding proteins
Necessary for spermiogenesis
o Two types of Sertoli Cells
Active Sertoli Cells
Normal function of supporting spermatozoa
Pyramidal shape with apex towards lumen and nucleus perpendicular to tubule
wall
Inactive (resting) Sertoli Cells
Polygonal in shape
Nucleus is parallel to wall of tubule
Spermatogenic Cells
o Arranged in a layer
o Vary in number with the phase of spermatogenic activity
Does not take place in simultaneously in all tubules
o The spermatogenic cells are:
Spermatogonia
Primary spermatocytes
Secondary spermatocytes
Spermatids
Spermatozoa
Spermatogonia
o Most primitive of the male sex cells
o Some undergo meiosis; progenies are translocated as they mature
Translocation mediated by sertoli cells
o Circumferential zones of more advanced cells inside zones of less advanced cells
o Criteria to identify spermatogonia
Rests on basement membrane and interrupted at intervals by sertoli cells
Gonial layer rapid mitosis (thin layers)
Nuclei is small, compared to spermatids
o 3 types of spermatogonia
1. TYPE A Spermatogonia
a. Chromatin appears fine and evenly dispersed
b. Single prominent chromatin nucleolus
c. Germ cell line (eccentric position where u see the chromatin nucleolus)
2. Type B Spermatogonia
a. Chromatin is coarse granules or flakes of heavily stained chromatin
b. Most common gonia
c. Last stage before they transform into primary spermatocytes
3. Intermediate Spermatogonia
a. Chromophilic chromatin in the form of delicate crusts or flakes attached
to membrane
b. Nucleoli in central position
Primary Spermatocytes
o Large size; large nuclei than the spermatogonia
o Visible chromosomes; nuclear membrane are disintegrated already
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Phases that can be identified on this level


Interphase
Granular nucleus with pale nucleolus
Primary spermatocytes have migrated inward
o Formation of new gonial layer multiplication of Type A Gonia
o Griffen and MacMahon
Estimated time of duration for this stage to be 3% of the total
time of the spermatogenic cycle as determined by Oakberg
o Oakberg
Cycle is 212 hours
Interphase exists for 6 hours
Metaphase I
Most easily recognized stage
20 discrete tetrads are visible
Tetrads are highly condensed
Aggregated at center (tetrads)
Anaphase I
Occasionally seen in section
Short duration
Spindle is distinct; present as dyads
Late anaphase shows cleavage furrow
Telophase I
Dyads aggregate at poles after anaphase
Cytoplasm constricts to form 2 cells
Nuclear membranes become distinct and dyads uncoil to form second meiotic
interphase
Secondary Spermatocytes
o Spermatogenic cells resulting from 1st meiotic division of primary spermatocyte
o Smaller than primaries
o Contain granular chromatin, globules of chromophilic chromatin free in the nucleoplasm or
associated with the nuclear membrane
o Resemble spermatids
o Twice the diameter of spermatids and half as numerous
Spermatids
o Resulting from division II lie inward beyond spermatocyte layer in seminiferous tubules
o Haploid
o Small nuclei
o Pale staining properties and above primary spermatocytes
o Nuclei elongates in time
o DNA undergoes condensation
o Nucleus decreases in size due to elimination of nuclear sap
o DNA increases in staining intensity as DNA becomes more and more condensed
o Early Spermatids
Golgi apparatus (or the acrosomal rudiment) appears as a red granule near nucleus
Flagellum appears early
Midpiece is a pink-stained thickening along first half of length
o Lie close to sertoli cells during transformation to spermatozoa
Spermatozoa
o Mature spermatozoa appear in sections as deeply stained short structures
o Innermost layer spermatogenic tubule
o Heads of most mature spermatozoa are buried in sertoli cells
o Tails extend into lumen
o Fully mature spermatozoa becomes free of nurse cells and pass into ducts
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Mature spermatozoa found in lumina of testis tubules and ducts is made up of:
Head (circle shaped and flattened)
Middle piece
Tail (long and flagellated)