Animal Biotechnology Test Exam Critical Questions

Animal biotechnology test exam critical questions
1 why animal cell culture is more complex than plant cell culture?
Answer
 Animal cell has similar to humans cell these can leads to more contamination than plant cell
 Animal cell has 3D structure and needs adherences
2. What is glycosylated (carbohydrate-modified) animal cell culture?

Answer
 Therefore, glycosylation is engineering of animal cells which express cloned

glycoprotein products is an enabling technology for generating molecular and functional
diversity of these products.
3. What is primary cell culture?
Answer
 These cells are obtained directly from tissues and organs by mechanical or chemical
disintegration or by enzymatic digestion.
 low growth rate and are heterogeneous
3. What is relation between Hayflicklimit to telomere length?

4. What is deference and similarity between serum and plasma?
5. What is Disaggregation of Cells?
6. What is contact inhibition of cell culture?
Life Sciences Questions and Answers – Animal Cell Culture
This set of Life Sciences Multiple Choice Questions & Answers (MCQs) focuses on “Animal Cell Culture”.
1. The growth of animal cells in vitro in a suitable culture medium is called___________

a) Gene expression
b) Transgenesis
c) Plant tissue culture
d) Animal cell culture
View Answer
Answer: d
Explanation: Animal cell culture is the process of growing cell in vitro in a suitable culture medium which contains a mixture of nutrients and
growth factors.
2. Cells from kidney tissues cannot survive independently, it requires other surface for attachment and survival.
a) True
b) False
View Answer
Answer: a
Explanation: Cells derived from kidney tissues are anchorage dependent primary cell culture which requires another surface for attachment
and survival.
3. Name the type of culture which is prepared by inoculating directly from the tissue of an organism to culture media?
a) Primary cell culture
b) Secondary cell culture
c) Cell lines
d) Transformed cell culture
View Answer
Answer: a
Explanation: Primary cell culture is the direct inoculation of the tissues of an organism into a culture medium without going prior to cell
proliferation.
4. What is a cell line?

a) Multilayer culture
b) Transformed cells
c) Multiple growth of cells
d) Sub culturing of primary culture
View Answer
Answer: d
Explanation: Cell line is defined as the sub culturing of primary cell culture. It can be a finite cell line or a infinite cell line
5. Name the organism on which first cell line was observed?

a) E.coli
b) Sheep
c) Mouse
d) Drosophila
View Answer
Answer: c
Explanation: First cell line observed was mouse fibroblast L cell which was derived from connective tissues of mouse by exposing it to a
chemical carcinogen.
6. Which of the following is the characteristics of a normal cell?

a) Anchorage independent
b) Continuous cell lines
c) Dependent on external growth factor
d) No contact inhibition
View Answer
Answer: c
Explanation: A normal cell needs external factors for its survival. It is anchorage dependent and have a finite number of cell lines.
7. Name the cell line of the human embryonic lung?

a) HeLa
b) WISH
c) L
d) MRC-5
View Answer
Answer: d
Explanation: MRC-5 is the cell line of the human embryonic lung while HeLa is the cell line of human cervical carcinoma. L represents human
connective tissue and WISH is the cell line of human amnion.
8. Which of the following is NOT the part of growth medium for animal culture?
a) Starch
b) Serum
c) Carbon source
d) Inorganic salts
View Answer
Answer: a
Explanation: Growth medium of an animal cell contains serum (5-20%), nitrogen sources, inorganic salts, carbon sources, growth factors,
buffer in water and vitamins.
9. Which of the following is NOT the major function of the serum?

a) Promotion of tuber and bulb formation
b) Stimulate cell growth
c) Enhance cell attachment
d) Provide transport proteins
View Answer
Answer: a
Explanation: Serum is the primary component of growth medium which helps in cell attachment and growth. It also provides transport protein
for carrying hormone, lipids, and minerals.
Multiple Choice Questions and Answers on Animal Cell Culture and Regulation
Question 1 : Sometimes cell lines can be cultured for such a long time that they apparently develop the potential to be subcultured indefinitely in vitro. Such
cells lines are called
1. established cell lines
2. primary cell lines
3. secondary cell lines
4. propagated cell lines
Answer : 1
Question 2 : Higher dissolved oxygen concentration in the culture media are toxic and lead to
1. DNA degradation
2. lipid peroxidation
3. metabolism of nutrients in culture media at a rate greater than that required for consumption
4. all of the above
Answer : 4
Question 3 : Which of the following is the technique used for the embryo culture?
1. Organ cultures on plasma clots
2. Organ cultures on agar
3. Whole embryo cultures
4. All of these
Answer : 4
Question 4 : The major problem associated with the isolation of free cells and cell aggregates from organs is that of
1. releasing the cells from their supporting matrix
2. inhibiting the cells from their supporting matrix
3. disintegrating the cells from their supporting matrix
4. none of the above
Answer : 1
Question 5 : The technique of organ culture may be divided on the basis of employing
1. solid medium
2. liquid medium
3. both (1) and (2)
4. semi-solid medium
Answer : 3
Question 6 : At low glucose concentration, below 0.25 mmol/litre, large portions of glucose and glutamine is shunted via
1. oxidative pathway
2. anaerobic pathway
3. both (1) and (2)
4. none of these
Answer : 1
Question 7 : An established cell line can be called where it has been subcultured at least
1. 70 times at an interval of 3 days between subcultures
3. 70 times at an interval of 1 day between subcultures
Answer : 1
Question 8 : Specific oxygen consumption rates for mammalian cells are in the range of (where n and prepresents to nano and pico respectively)
1. 0.05-5 nmol of O2/cell/h
2. 5 – 10 nmol of O2 /cell/h
3. 0.05-5 pmol of O2/cell/h
4. 5 – 10 pmol of O2/cell/h
Answer : 3
Question 9 : Which of the following is not a source of energy in active muscle cells?
1. Creatine phosphate
2. ATP
3. Lactic acid
4. Glucose
Answer : 3
Question 10 : In animal cell culture, particularly mammalian cell culture, transformation means
1. uptake of new genetic material
2. phenotypic modifications of cells in culture
3. both (1)and (2)
4. release of genetic information
Answer : 2
Question 11 : How does CO2 help in the cell metabolism during cell culture?
1. It participates in the de novo synthesis of purines and pyrimidines
2. Helps in the cells respiration
3. For monitoring pH of the culture
4. All of the above
Answer : 1
Question 12 : Which of the following is not the explantation technique?
1. Slide culture
2. Carrel flask culture
3. Roller test tube culture
4. Adherent primary culture
Answer : 4
Question 13 : Cells which have undergone transformation frequently become
1. anchorage independent
2. anchorage dependent
3. stable
4. unstable
Answer : 1
Question 14 : During the growth of animal cells in culture, it is noticed that the cells do not look very healthy. After an investigation, this is found that there
is a lot of lactic acid in the culture fluid. What is probably wrong with this culture?
1. Ethyl alcohol is being produced in excess
2. The cells have too much oxygen
3. Glycolysis is being inhibited
4. The cells do not have enough oxygen
Answer : 4
Question 15 : Range of optimum glucose concentration present in the culture media is
1. 5.5 – 55 mmol/litre
2. 55 – 75 mmol/litre
3. 75-105 mmol/litre
4. 105-150 mmol/litre
Answer : 1
Question 16 : Range of optimum glutamine concentration present in the culture media is
1. 1-2 mmol/litre
2. 2-7 mmol/litre
3. 7-15 mmol/litre
4. 15 – 20 mmol/litre
Answer : 2
Question 17 : The human fibroplast is a classical example of
1. stable primary cell lines
2. established cell lines
3. cell transformations
4. none of these
Answer : 1
Question 18 : pH of culture medium is initially controlled by
1. presence of CO2
2. presence of bicarbonate buffer
3. addition of bases
4. none of these
Answer : 2
Question 19 : Which of the following abnormality, resulted from the inheritance of an unbalanced complement of chromosomes can be diagnosed through
karotyping?
1. Down’s syndrome
2. Turner’s syndrome
3. Klinefelter’s syndrome
4. all of these
Answer : 4
Question 20 : What is the concentration of CO2 required for culturing animal cells?
1. 2-5%
2. 1-10%
3. 10-15%
4. 15-20%
Answer : 2
Question 21 : Which of the following is correct?
1. ECL can be established in suspension cultures whereas it is exceptional for primary cell lines (PCL)
2. ECL and PCL can be established in suspension cultures
3. PCL can be established in suspension cultures
4. none of the above
Answer : 1
Question 22 : Which of the followings are the metabolic products of glucose and glutamine?
1. CO2 and NH3
2. CO2 and lactate
3. Lactate and ammonium
4. Lactate only
Answer : 3
Question 23 : To prevent the accumulation of lactate
1. low glutamine concentration is required
2. high glutamine concentration is required
3. low glucose concentration is required
4. high glucose concentration is required
Answer : 3
Question 24 : What are the main constituents of culture for animal cell growth?
1. Glucose and Glutamine
2. Growth factors
3. Cytokines
4. All of these
Answer : 1
Question 25 : When dissolved oxygen is lower than the critical concentration, viable cell concentration declines because of
1. incomplete glutamine oxidation
2. increase in specific lactate production from glucose
3. both (1) and (2)
4. accumulation of ammonia
Answer : 3
Question 26 : According to Eagle, the growth of L-strain and Hela-strain cultures require to have mandatory presence of
1. 6 amino acids
2. 8 amino acids
3. 10 amino acids
4. 12 amino acids
Answer : 4
Question 27 : Excess CO2 suppress cell growth and productivity by
1. inhibiting respiration
2. altering intracellular pH by diffusing across cell membrane
3. both (1) and (2)
4. altering pH of the medium
Answer : 3
Question 28 : Which of the following is incorrect?
1. Established cell lines (ECL) have short doubling time
2. ECL are invariably aneuploid
3. ECL grow in higher density
4. ECL do not show much evidence of spatial orientation
Answer : 3
Question 29 : What is the effect of excess accumulation of metabolite products (lactate and ammonium) on cells?
1. They act as growth promoters
2. They act as growth inhibitors
3. Have no effect on cells
4. Lactate helps in the growth while ammonium inhibits the growth
Answer : 4
Question 30 : For culturing, plasma from the adult chicken is preferred to mammalian plasma because
1. it forms a clear and solid coagulum even after dilution
2. it is too opaque
3. it doesn’t produce solid clots
4. it forms a semi solid coagulum
Answer : 1
Question 31 : Toxicity due to accumulation of ammonia can be overcome
1. by substituting glutamine by glutamate
2. by controlled addition of glutamine at low level
3. by removal of ammonia or ammonium from culture medium
4. all of the above
Answer : 4
Question 32 : Range of osmolarity tolerated/accepted in mOsm/Kg of H2O by mammalian cells is
1. 150-300
2. 280-360
3. 300-325
4. 360-400
Answer : 2
Question 33 : Disaggregating of cells can be achieved by
1. physical disruption
2. enzymatic digestion
3. treating with chelating agents
4. all of the above
Answer : 4
Question 34 : Accumulation of lactate leads to
1. increase in pH
2. no change in pH
3. reduction in the pH of culture hence loss of cell viability
4. no loss of cell viability
Answer : 3
ntroduction to Animal Tissue Culture: MCQ for DBT BET, GPAT, GATE, & CSIR
NET
January 12, 2021 jagir.apc Biotechnology, DBT BET, GATE Exam, GPAT Preparation, How to prepare for
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Questions, Quiz, Study Material, UGC NET JRF Exam Advantages of Animal tissue culture, Animal tissue
culture, Animal tissue culture MCQ for GATE, Animal tissue culture MCQ for GPATE, Animal tissue
culture notes, Application of animal tissue culture, Cell line, Histotypic culture:, Limitations of animal
tissue culture, MCQ of animal tissue culture for CSIR NET, Organ culture, Organotypic culture, What is
animal tissue culture?
What is animal tissue culture?
Animal tissue culture involves the growth and maintenance of animal cells Invitro inappropriate
nutrition media and growth conditions. Thus, when cells are grown and maintained under
laboratory conditions, the process is known as cell culture.
Basics terms used in cell culture
Organ culture: the term refers to the tissue that remains the same as in-vivo histological
features is known as organ culture. Cell Culture: the term refers to the disaggregated cells
obtained from the original tissue or cell line. Histotypic culture: the term refers to the cell
culture for their reaggregation to form a tissue-like structure Organotypic culture: the term
refers to the recombination of different types of cells to form more defined tissue or
organ Primary culture: the term refers to the culture freshly prepared from isolated tissue or
cells from an organism Cell line: the term refers to, when the primary cultures are subcultured
which results in cell lines. Continuous cell lines are the indefinite growth of the cells in
subsequent sub-culturing. Whereas, finite cell lines refers to cell death after multiple
subcultures. Minimum laboratory requirement for animal cell culture technology
1. Infrastructure: animal house, microbial laboratory, clean and quite sterile area, preparation
facilities, storage facilities for glassware, chemicals, liquids, and small equipment.
2. Equipment: equipment like Laminar flow, sterilizer, CO cylinder, refrigerator, ware purifier,
2
pipette washer, deep washing sink, liquid nitrogen freezer, inverted microscope, balance, a slow
cooling device for freezing cells, centrifuge are the minimum requirement for animal cell tissue
culture laboratory.
3. Culture vessels: vessels such as Petri dish, flasks, multiwell plates, stirrer bottles are generally
used for cell culture. They are made of glass or disposable plastics such as polystyrene, polyvinyl
chloride, polycarbonate, mentinex, and thermonex. The material used in culture techniques is
important because the surface of the vessel serves as a substrate for cells to grow.
4. Contamination in tissue culture laboratories: several routes like materials i.e. glassware,
pipettes, types of equipment like incubators, refrigerators, laminar flow hood, reagents like
media, solutions are major factors responsible for the contamination.
5. Aseptic condition: proper aseptic conditions are maintained to reduce contamination from
different microorganisms and viruses.
6. Sterilization: Dry heat sterilization at 160 C for one hour, moist heat of autoclave at 121 C for 15-
o o
20 mins, and sterilization by a filter of 0.1-0.2 µm size is used to kill microorganisms, and
destroying spores.
Advantages of Animal tissue culture
1. Cultured cells are easy to store for long period in liquid nitrogen
2. Cell categorization is easy for immunological and cytological studies
3. Biological studies can be easily done using cell cultures
4. Cell culture techniques can reduce the use of animals in various studies.
Limitations
1. Technical skill and expertise is required to carry tissue culture techniques

2. The cost factor for tissue culture production is very high
3. It is not easy to control environmental factors like pH. Temperature, dissolved gases, etc.
4. Disposal of biohazards is not easy
5. Genetic instability is seen in continuous cell lines
Application of animal tissue culture
1. Intracellular activities like cell cycle, cell differentiation, etc can be studied
2. Studies on hormonal receptors, signal transduction can be done using cell culture
3. Cell cultures can be used to evaluate environmental interactions like genotoxicity, mutagenesis,
etc
4. Cell culture can be used for the production of vaccines e.g. malaria vaccine
5. Production of high-value therapeutics such as plasminogen, interferons, blood clotting factors,
hormones, monoclonal antibodies, and erythropoietin can be done using animal tissue culture.

The risk associated with Animal tissue culture

Risks category Factors
Maintenance ricks leakage of disposals, age, and condition of the

equipment
Personnel risks lack of interest and concentration and

inadequate training
Physical risks Intense cold, electric shocks, and fire
Chemical risks Exposure to toxic substances like poisons,

carcinogens, mutagen, irritants, and allergens
Biohazards Viruses, pathogenic organisms, culture cells,

and DNA
Radioisotope risks Energy emission its penetrations and ionization
Multiple-choice Questions
1. What is animal tissue culture?

a) Growth and maintenance of animal cells
b) Growth and selling of animal cells
c) Only maintenance of animal cells
d) All
2.Culture freshly prepared from isolated tissue is known as _____
a) Organ culture
b) Primary culture
c) Cell line
d) Histotypic culture
3.Recombination of different types of cells to form more defined tissue or organ is
known as __________________
a) Organotypic culture
b) Primary culture
c) Secondary culture
d) Cell line
4. ______________is the primary equipment required for animal tissue culture
laboratories
a) Glasswares
b) Laminar flow
c) Sterilizers
d) All
6. The material used in culture techniques is important because the surface of the
vessel serves as a substrate for cells to grow.
a) True
b) b) False
7. The following are methods of sterilization EXCEPT:
a) Dry heat sterilization
b) b) Autoclaving
c) c) Sterilization by filters
d) d) Laminar airflow
7. The following are the routes of contamination in tissue culture laboratories EXCEPT:
a) Incubators
b) Refrigerators
c) Laminar flow hood
e) Autoclave
8. _____________is the advantage of animal tissue culture
a) It is cost-effective
b) No skilled personnel is required
c)Tissue cultures can be stored for a long time
d) Maintenance of environmental conditions is easy
9. _________________is one of the limitation of animal tissue culture
a) Disposal of biohazards is not easy
b) Cultured cells are not easy to store
c)Both
d) None
10.Exposure to carcinogens is a contributing factor for _________
a) Chemical risk
b) b) Biohazards
c) c) Physical risk
d) Personnel risk
Answer Key
1. a
2. b
3. a
4. b
5. a
6. d
7. d
8. c
9. a
10. a
11. Cell Biology Questions and Answers – Techniques – Cell Culture
12. This set of Cell Biology Multiple Choice Questions & Answers (MCQs) focuses on “Techniques – Cell Culture”.
13. 1. Trypsin is used for dissociating the tissue into single cells.
a) True
b) False
View Answer
14. Answer: a
Explanation: Trypsin is a proteolytic enzyme and is used for dissociating the tissue into single cells. During the primary culture,
tissue samples are taken generally from the embryos rather than the adult organism because of easy dissociation.
15. 2. In the secondary culture, cells are obtained from _______________________
a) primary culture
b) the organism
c) organ culture
d) phenotypic culture
View Answer
16. Answer: a
Explanation: The culture that is done after the primary culture is known as the secondary culture (the process termed passaging
or subculturing). The primary culture is obtained from the organism itself.
17. 3. EDTA binds the ______________ ions.
a) magnesium
b) iron
c) carbon
d) calcium
View Answer
18. Answer: d
Explanation: EDTA (ethylenediamine tetracetate) is a chelating agent that binds to calcium ions and prevents cellular adhesion. A
chelating agent reacts with metal ions to form a water-soluble complex.
19. 4. HeLa cells are a cell line.
a) True
b) False
View Answer
20. Answer: a
Explanation: If a homogenous cell culture has the ability to grow indefinitely in vitro, it is termed a cell line. Cell lines would never
undergo apoptosis and senescence. HeLa is a cancerous cell line derived from humans and was the first line to be maintained.
21. advertisement
22. 5. Under favorable conditions, the protoplasts can grow into a ____________________
a) callus culture
b) organ culture
c) root culture
d) shoot culture
View Answer
23. Answer: a
Explanation: Under favorable conditions and chemically defined media, the protoplasts can grow into an undifferentiated cell
mass, known as the callus culture. Protoplasts are the cells whose plasma membranes have been removed.
24. 6. The process of dedifferentiation in cell culture can give rise to ________________________
a) induced-pluripotent stem cells
b) carcinoma cells
c) single protoplasts
d) fused protoplasts
View Answer
25. Answer: a
Explanation: Dedifferentiation is the process by which the differentiated cells reprogram and go back to their precursor
counterparts. The cells that are induced to undergo dedifferentiation are termed as induced pluripotent stem cells
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Understanding the in vitro system and factors that affect it
1. Cell culture is in vitro because (choose the best answer)?*
cells are not touching each other. cells have lost some proteins.
interactions with other cell types and 3D interactions are lost. changes occur in the cell membrane
2. For adherent cells, adhesion provides a signal for cells to do which of the following. *
grow and divide transduce differentiation signals
enhance survival all of the above
3. Animal cells grown in culture generally retain their functions even though they are growing in vitro.*
True False
4. Cell surface proteins that promote cell-cell contact cause cells to do which of the following? *
find other cells anchor to plastic surfaces
stop growing due to contact inhibition allow cells to know where they are located
5. Paracrine signals come from which of the following?*
components in the basal media they are added by the researcher depending on their experiment
other neighboring cells unwanted cell culture contaminants
6. Apoptosis is a form of cell death that*
is irreversible is caused by some signal loss

in many cases is preventable all of the above
7. The cell culture environment includes*
media and media components the gas composition
the plastic the cells are attached to all the above
8. Confluency refers to*
the area that each cell occupies the viable cells per ml.
the ratio of area occupied by the cells and the total area available the area below the line of a growth curve
9. When we grow cells in culture*
we can only grow one cell type at a time cells with a growth advantage can outgrow a cell culture given enough time
cells supply other cells everything they need to grow serum must always be heat-inactivated
10. When animal cells attach to each other or a matrix, cell signaling occurs by (choose all that apply) *
direct uptake of other cells proteins into the cells activation of catalytic domains in accessory proteins
direct binding of cell adhesion receptors to specific attachment proteins direct binding surface proteins to actin in the medium
Media Use and Care
1. Cells need to be refed only if the cells have reached confluency.*
True False
2. The function of phenol red is to (check all that apply) *
indicate the relative pH of the media help protect the media from light damage
is used to help the cell membrane stay permeable activate estrogen pathways in animal cells
3. Basal media the following components in it (check all that apply) *
fetal bovine serum trace elements
water soluble vitamins amino acids
4. Complete media is (choose the best answer)*
sometimes, also called kitchen sink media necessary for some animal cells
supports the growth and proliferation of animal cells facilitates detachment of adherent animal cells
5. L-glutamine is an important amino acid because (choose the best answer) *
it is an amino acid cells get much of their energy from the catabolism of L-glutamine
it is not important since it is made by some cells it is an amine donner

6. Switching media types from classical to serum-free media (choose the best answer) *
is easy and can be quickly may be possible given care
cannot be done once you grow animal cells in a classical medium there is no reason to switch media
7. Phenol Red is a mimic of which of the following?*
tryptophan testosterone
estrogen histidine
8. GlutaMAX™-I is alternative to L-glutamine because?*
GlutaMAX™-I is double the strength of L-glutamine L-glutamine is less stable
it is a newly discovered amino acid other amino acids are added to L-glutamine
9. Freshly prepared complete media will last years when stored at -20°C. *
this it true since many biologicals last a long time when stored at -20°C this is false since -80°C is known to work better
this is false since basal media should not be frozen because components will
this is true since my serum comes from the vendor frozen
precipitate out of solution
10. Sodium bicarbonate is added to cell culture media for the following reason *
to help keep cells stuck to the plastic to promote the uptake of CO2 into animal cells
to help maintain the correct pH when CO2 is present it helps keep iron soluble
Contamination and prevention
1. Types of contamination that you cannot see with a standard inverted microscope include the following (choose all that apply) *
bacteria viral
mycoplasma fungal
2. When growing animal cells it is important to*
use continuous vigilance to guard against contamination wear sturdy work shoes
have lots of computer memory drink plenty of water
3. When looking for bacterial contamination, the best place to look is*
on the upper surface of the dish or flask since that is where the original
in an area of the dish that is highly confluent
contamination event occurred
between cells where there is a lighter background over the whole dish at the lowest magnification possible
4. The atmosphere inside a biological safety cabinet is*
full of CO2 gas very clean but not absolutely sterile
absolutely sterile lower in oxygen then the air outside

5. When working in the biological safety cabinet it is important to (choose all that apply) *
make sure the cabinet is on and working correctly Wipe the cabinet down with 70% alcohol
minimize quick movement not introduce contamination
6. Of all the contaminations possible, probably the easiest to detect is*
cross-contamination yeast and fungi
mycoplasma chemical
7. Mycelium is a form of*
viral waste fungi
antibiotic mycoplasm
8. In the event of a bacterial contamination the easiest thing to do is *
discard your cells and thaw a frozen vial of fresh cells keep culturing like nothing has happened
simply add antibiotics to kill the contaminant wash the cells several times in D-PBS
9. Antibiotics should*
always be used used, only when necessary
never be used only during a cross-contamination event
10. Following a bacterial contamination your media may turn yellow if there is *
L-Glutamine in the media phenylalanine present
phenol red in the media low CO2 in the media
Cell culture best practices
1. Conditioned medium made from*
components in serum expired basal media
used media that still has nutritional value complete filtered media
2. Which benchmark cell line verification is not recommended by ATCC*
STR analysis growth curve analysis
DNA sequencing mycoplasma detection
3. When freezing animal cells it is important to (check all that apply) *
freeze as rapidly as possible freeze slowly, at about 1degree per minute
it is not really necessary to freeze animal cells keep your -20°C freezer calibrated
4. Cross-contamination is*
not to much of a problem and never happens the result of working with multiple cells at one time
can be controlled with antibiotics cannot really happen if you follow good protocols
5. Conditioned media is useful for (check all that apply)*
as part of a freezing medium for freezing animal cells overcoming difficult growth periods
helping grow very low density animal cells only the cell type that was used to make the conditioned media
6. Cell line authentication is*
the sum of studies demonstrating the lineage or identity of an animal cell and
having a written record of how you have grown your animal cells
a lack of contamination
having some record of where your animal cells came from not necessary if your cells came from a primary culture
7. Currently, cell analysis using short tandem repeats (STRs) is useful*
for mycoplasma detection for human cell line detection
when studying media components only if you are studying gene regulation
8. When thawing animal cells you should*
thaw slowly as you would during freezing thaw as rapidly as possible in a very hot water bath
not worry as long as you get your cells thawed thaw rapidly in 37°C water
9. Growth curve analysis is important because (check all that apply) *
they provide a baseline of growth characteristics they allow the researcher to see changes in growth patterns over time
you can establish a cell doubling time they give a record of growth
10. Isosyme analysis ( choose all that apply)*
is an old technique that is not used much anymore measures specific proteins in a cell that allow one to identify a species
uses isoelectric focusing to migrate proteins to known positions is also called zymography
Cell Culture Basics Video Questions
1. Cell culture media is complex and can be stored under which of the following conditions? *
on the bench top and out of direct sun light always store media in the biological safety cabinet
at 37°C in the dark at 4°C in the dark
2. The sash of the biological safety cabinet must be set*
at the indicated level when in use where it is comfortable for the user
up high for ease of use at the indicated level when not in use
Sterile Technique
1. When working with animal cells in a biological safety cabinet*
never open any item outside the biological safety cabinet otherwise it will not
never stop until all you work is completed, then exit the biological safety cabinet
be sterile
you don't have to worry about sterile technique use quick movements to enter and exit the biological safety cabinet
2. The basics of sterile technique dictate that the researcher does which of the following (check all that apply). *
washes hands after completion of work uses only sterile reagents, pipettes and media
maintains a very clean environment uses serological pipettes only once
Passaging Cells
1. During the growth of animal cells it is important to keep cells in which phase of the growth curve? *
stationary phase lag phase
log phase decline phase
2. The purpose of a disassociation medium is to*
wash the cells to disassociate any spent medium from them help supply additional nutrients
to cause adherent cells to detach from one another and the substratum reduce further enzymatic activity
Freezing Cells
1. Cryopreservation of animal cells is important because (choose all that apply) *
it provides a back-up if your cells get contaminated animal cells can change over time
your cells may die by accident well maintained cell seed stocks are critical to reproducibility
2. When freezing cells it is best to have cells that are (choose all that apply) *
low passage and well fed cells resistant to high-speed centrifugation
high passage and un-fed cells low viability cells
Thawng Cells
1. During the thawing process it is important to transition the cells from the vapor phase of liquid nitrogen to 37°C *
slowly, so the cells have time to acclimate to 37°C as quickly as possible in a 37°C waterbath
by warming the vial in your hand as you spray 70% alcohol onto the vial
2. PPE, Personal protection equipment should be worn when thawing cells what have been cryopreserved in liquid nitrogen. Choose which of the following should be used when
thawing frozen animal cells.*
a full face shield a lab coat and rubber apron
Insulted gloves steel toed shoes
Question 1
Complete the sentence by selecting the correct option from the three options below.
Plotting a semi-logarithmic graph of the rate of cell proliferation over time produces a
______ _____.
Your answer:
a) growth curve
Feedback:
Figure 15.2 shows how cell density increases over time. The growth forms a characteristic
curve and consists of a lag, log, plateau and death phase. A growth curve is used to
determine doubling time of a population of cells and is necessary for calculating how many
cells to seed in advance of an experiment.
Question 2
The following are a list of essential components of cell culture media. Match them to the
requirements for effective cell culture which they fulfil.
Phenol red
Correct. Your answer: pH indicator
You did not match any answer.
Glutamine
Correct answer: Glucose and amino acids for respiration
Inorganic salts
Correct answer: Regulation of osmotic pressure and membrane potential
Bicarbonate
Correct answer: pH buffer
Overall incorrect answer.
Feedback:
Section 15.2.2 describes the essential requirements for optimal cell growth. Glutamine is
used to provide glucose and amino acids to respiring cells at a final concentration of 2mM
and inorganic salts (such as K, Ca, and Na) help to maintain cell membrane potential and
osmotic pressure. Bicarbonate is often used as a buffering system to keep the pH of the
solution between 7.2 and 7.4 and this is monitored by the addition of phenol red to many
commercially available mediums.
Question 3
The following oxygen tensions are commonly encountered in cell culture when replicating in
vivo conditions. Match them to the concentrations they describe.
Hypoxic
Correct answer: ≤5 %
Normoxic
Incorrect. Your answer: ≤5 %
Correct answer: ~20%
Anoxic
Correct answer: ≤1%
Overall incorrect answer.
Feedback:
Normally mammalian cell cultures require around 20% O content and around 5% CO - a
2 2
condition referred to as normoxia. Hypoxia (≤5% O ) and anoxia (≤1% O ) are occasionally
2 2
used for mimicking certain in vivo conditions. Altering the oxygen tension in the cell culture
environment results in epigenetic changes.
Question 4
Subculturing a cell line always increase the passage number. Is this true or false?
You did not answer the question.

Correct answer:
a) True
Feedback:
Each time subculturing is carried out this increases the passage number. For example, a
primary culture established from dissociated tissue is referred to as P-0, however when this
flask of cells is harvested and then distributed between new culture vessels each new flask of
cells becomes a P-1.
Question 5
A cell line always requires enzymatic dissociation before it can be distributed between new
culture vessels. Is this true or false?
Your answer:
a) True
Correct answer:
b) False
Feedback:
Cell cultures are either grown as monolayers or in suspension. Cells grown in suspension are
simply centrifuged to obtain a cell pellet before passage whereas cells grown in monolayers
will always require some form of dissociation although this may not always be enzymatic.
For example, cells that are sensitive to trypsin or trypsin substitutes may be scraped from the
culture vessel.
Question 6
Senescence refers to the process by which a cell line is found to contain chromosomes
characteristics of another cell line due to cross-contamination. Is this true or false?
Your answer:
b) False
Feedback:
Senescence occurs when a mortal cell line has undergone the maximum number of cell
population-doublings. The cell line has reached its Hayflick limit and the telomeres have
reached the critical minimum length rendering the cell no longer able to divide. Whereas
cross-contamination is an area of concern for laboratories handling large numbers of cell
lines and cell lines should be authentificated for species origin using PCR for STRs.
Question 7
The total number of cells in a culture is counted using the trypan blue exclusion assay and is
found to be 2.7 x 10 cells/ml. The culture is diluted 1:27 and then 100μl seeded per well into
6
a 96 well plate. What is the final cell density per well?

Your answer:
d) 1 x 10 4
Feedback:
Figure 15.4 describes how cell counts may be obtained. Diluting the cell culture 1:27 results
in a solution containing 1 x 10 cells/ml, by seeding 100μl per well you are effectively
5
diluting the solution by a further 1:10 - this means each well will contain 1 x 10 cells.
4
Question 8
The total number of cells in a culture is counted using the trypan blue exclusion assay and is
found to be 6.8 x 10 cells/ml. Each well in a 6 well plate requires 2 x 10 cells. How should
6 5
the solution be diluted so that 1ml can be added to each well?

Your answer:
c) 1:3.4 then 1:10
Feedback:
Figure 15.4 describes how cell counts may be obtained. Diluting 6.8 x 10 cells/ml by 2 x
6
10 (the number of cells/ml you require in the end) results in a dilution factor of 34. However,
5
it is much easier in practice (and uses less precious media!) to dilute your cell suspension
1:3.4 (to give 2 x 10 cells/ml) and then 1:10 (to give 2 x 10 cells/ml). You can then add 1ml
6 5
to each well to give a cell density of 2 x 10 cells/well in a volume of 1ml.

5
 Gametogenesis is the production of sex cells (gametes) through meiosis.

 Spermatogenesis involves the following transitions: primordial germ cells ->
spermatogonia -> type B spermatogonia -> primary spermatocyte -> secondary
spermatocyte -> spermatids. Spermatogonia also produce type A spermatogonia which
are recycled to replenish the spermatogonia supply.
 Spermiogenesis is the transition of spermatids into mature sperm cells, with spermiation
ending with mobile sperm.
 Sperm capacitation is the final step to allow for fertilisation. It involves the removal of
glycoproteins and cholesterol and activating signalling pathways.
 The Spermatogenic cycle is the time taken for the same stage of spermatogenesis to
appear in the same segment of the seminiferous tubule.
 The Spermatogenic wave is the appearance of the different stages of spermatogenesis in
the seminiferous tubules in a sequential and ‘wave like’ motion.
 Formation of oocytes begins before birth, arresting in prophase I, becoming primary
oocytes surrounded by follicular cells, called primordial follicles. Most of these undergo
atresia before puberty. 15-20 primordial follicles mature each month, developing
cuboidal epithelium with granulosa and theca cells in the preantral stage. In the antral
stage, spaces between granulosa cells coalesce to form the antrum. Only one oocyte
reaches the preovulatory phase, finishing meiosis I and forming a secondary oocyte.
 Ovulation occurs, releasing the oocyte into the fimbriae of the fallopian tubes, passing
down it by peristalsis towards the uterus. Because ovulation is a traumatic action,
increased ovulation may be related to increased ovarian cancer incidence.
 The corpus luteum is formed from the theca interna and granulosa cells, secreting
progesterone and oestrogen causing the uterus to enter the proliferative phase. If β-hCG is
released by the implanted zygote, the corpus luteum persists and releases progesterone
for 4 months until the placenta takes over. If no β-hCG is released, the corpus luteum
forms a mass of scar tissue called the corpus albicans, stopping progesterone release and
causing menstruation.
Core
Gametogenesis – the process of making sex cells (gametes) through meiosis. This occurs in
the testes in males, and in the ovaries (and the fallopian tubes) in females.
The Male Gamete
Spermatogenesis
Spermatogenesis is the process that creates sperm from primordial germ cells in the testicles.

It has a number of steps, which are listed below and included in the diagram:
1. The primordial germ cell divides by mitosis to produce spermatogonia.

2. The spermatogonia divides by mitosis to produce two types of cells:
o Type A Spermatogonium – replace the spermatogonia in a loop, will divide into

type A and type B spermatogonia in the future.
o Type B Spermatogonium – continue along the spermatogenesis pathway to
produce sperm.
3. Type B spermatogonium divide by mitosis to form primary spermatocytes.
4. Primary spermatocytes divide by meiosis to form secondary spermatocytes.
5. Secondary spermatocytes go through meiosis to turn into spermatids.
6. Spermatids continue through spermiogenesis to produce sperm cells.
Spermiogenesis
Spermiogenesis is the final stage in the production of sperm cells and involves
the transition of spermatids into motile sperm cells.
Spermiation is the outcome of spermiogenesis. Spermiation occurs when the spermatids are
released into the lumen of the seminiferous tubules. They then pass through the rete
testis and ductuli efferentes and into the epididymis. Spermatids are not mobile until they pass
through the epididymis. Before this, they are transported by Sertoli cells
secretion and peristalsis, and once they undergo spermiation, they are mobile and are mature
sperm cells.

Diagram - Spermatogenesis and spermiogenesis
SimpleMed original by Maddie Swannack
Sperm Capacitation
Capacitation of sperm is the final step before sperm becomes able to fertilise an oocyte.

Capacitation occurs in the female reproductive tract following ejaculation. It involves
the removal of glycoproteins and cholesterol from the
sperm membrane and activation of signalling pathways inside the sperm cells. Removal of the
glycoproteins allows the sperm to bind to the zona pellucida of the oocyte and initiate
the acrosome reaction. This is the process that allows the sperm to penetrate the egg cell and
begin fertilisation.
Capacitation is an important consideration in in vitro fertilisation (IVF). As the sperm never

encounters the female reproductive tract before meeting the egg, the sperm must be treated in a
capacitation medium to trigger the process artificially.
Spermatogenic Wave
Not all the stages of spermatogenesis are visible histologically in a single cross-section of a
seminiferous tubule. This observation confirms that sperm is produced continuously rather than
batch produced.
The spermatogenic cycle is the time taken for the same stage of spermatogenesis to appear in

the same segment of the seminiferous tubule. Simply put, how long it takes to
produce one fully mature sperm cell from a spermatogonium. In humans this takes
around 16 days.
The spermatogenic wave is the appearance of the different stages of spermatogenesis in

the seminiferous tubules in a sequential and ‘wave like’ motion. This wave-like movement is
the spermatogenic wave.
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The Female Gamete
Oocyte
The oocyte (pronounced o-o-cyte not oo-cyte) is the female gamete and is also known as
an egg cell. A woman is born with all her oocytes formed but not fully developed in her ovaries.
This stock declines after birth with age due to atresia (cell death).
Formation of oocytes begins before birth. Germ cells differentiate

into oogonia and proliferate by mitosis. The oogonia then begin meiosis,
but arrest in prophase I (meaning they have replicated their chromosomes but not commenced
division). At this point, the oogonia become primary oocytes. These primary oocytes number
approximately 2 million, and each one is surrounded by a flat layer of epithelial cells
called follicular cells. The primary oocytes surrounded by follicular cells are
named primordial follicles.
Most of these primordial follicles undergo atresia during childhood, leaving approximately

40,000 in the ovaries by the start of puberty. From puberty onwards, 15-20 oocytes
mature each month, passing through the following three stages:
Preantral Stage
As primordial follicles grow, the epithelium changes from flat to cuboidal. A number of new

cell types develop: granulosa cells secrete a glycoprotein layer named zona pellucida, and
the theca interna cells become paracrine tissue, going on to produce testosterone under the
influence of LH so granulosa cells can turn it into oestrogen.
Antral Stage
As development continues, fluid filled spaces appear between granulosa cells, which coalesce to

form the antrum (the name for the largest fluid filled sac). Several follicles (15-20) begin
development every menstrual cycle but only one progresses to the preovulatory stage.

Preovulatory Stage
An increase in FSH and LH induce growth. The primary oocyte then enters meiosis I.

However, in division the cytoplasm is not evenly distributed. The majority is given to
the secondary oocyte and the minority to the first polar body. The polar bodies never become
an egg and in effect are dustbins for unneeded genetic material. The secondary oocyte then
enters meiosis II and halts until it is fertilised.
Ovulation
Before ovulation occurs, the fimbriae of the fallopian tube sweep the surface of the ovary, and
the peristaltic action of the fallopian tube begins. This means that as soon as ovulation occurs,
the fallopian tubes are doing everything they can to make sure the oocyte progresses into the
uterus.
Ovulation occurs when the graafian/ovarian follicle (the follicle containing the secondary

oocyte) becomes approximately 2.5cm in size. The LH surge increases collagenase activity (an
enzyme that breaks down collagen), and prostaglandin release causes ovarian
wall contractions. This causes the oocyte to be extruded from the ovary in a traumatic release.
The fallopian tube then acts to move the oocyte into the uterus through peristaltic
contractions and fimbriae movements. The fallopian tubes are also lined with cilia which help
to waft the oocyte towards the uterus.
Ovarian Cancer
As the oocyte leaves the ovary through a traumatic rupture, repeated repair is needed. This

means that there is an increase in cellular proliferation is needed at these sites, which can
increase the risks of some types of ovarian cancer. This means that increased ovulation can lead
to an increased risk of ovarian cancer.
Preventing ovulation can therefore be a protective factor for ovarian cancer, meaning that

multiparous women (women who have had more than one child, ovulation pauses during
pregnancy) and women who use birth control methods that prevent ovulation (such as the
combined oral contraceptive pill) are at a lower risk of ovarian cancer.
Mittelschmerz
Mittelschmerz or ovulation pain which occurs on ovulation. It is unknown what specifically

causes the pain. One theory is that fluid or blood from the follicle irritate the abdomen.
Alternatively it is though that if the epithelium of the ovary is too well innervated when the
oocyte traumatically ruptures from the ovary it can cause pain. The term gets its name from the
fact that the painful ‘schmertz’ ovulation occurs on the 14th day of the cycle, in
the middle ‘mittel’ of the month. It is important, however, that abdominal pain in a woman is
not dismissed to be Mittelschmerz pain.
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Diagram - Processes from meiosis to fertilisation

The Corpus Luteum
The corpus luteum (lit. ‘yellow body’) is formed from the remains of the ovarian follicle (graafian) including the
remaining granulosa and theca interna cells. As the cells become vascularised they change into yellow coloured lutein cells. The corpus
lutetium secretes oestrogen and progesterone, which stimulates the uterine mucosa to enter the proliferative stage to prepare for
implantation of the fertilised oocyte.
If fertilisation occurs, β-hCG release prompts the corpus luteum to grow and form the corpus luteum graviditatis, which continues to
secrete progesterone until the 4th month of pregnancy, when the placenta takes over progesterone production. The progesterone released
maintains the thick lining of the endometrium.
If the corpus luteum does not receive β-hCG signals from the implanted zygote (oocyte and sperm combo) within 14 days, it dies and turns
into the corpus albicans.

The Corpus Albicans
The corpus albicans (lit. ‘white body’) is essentially scar tissue formed from the regressed corpus luteum and does not release
progesterone. As a result the endometrial lining breaks down which is observed as menstruation.

Table - Differences between spermatogenesis and oogenesis


Edited by: Ben Appleby
Reviewed by: Thomas Burnell
Quiz
7. Gametogenesis
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Learning Objectives
 Distinguish between spermatogenesis and oogenesis
Key Points
 Gametogenesis, the production of sperm (spermatogenesis) and eggs (oogenesis), takes
place through the process of meiosis.
 In oogenesis, diploid oogonium go through mitosis until one develops into a primary
oocyte, which will begin the first meiotic division, but then arrest; it will finish this
division as it develops in the follicle, giving rise to a haploid secondary oocyte and a
smaller polar body.
 The secondary oocyte begins the second meiotic division and then arrests again; it will
not finish this division unless it is fertilized by a sperm; if this occurs, a mature ovum
and another polar body is produced.
 In spermatogenesis, diploid spermatogonia go through mitosis until they begin to
develop into gametes; eventually, one develops into a primary spermatocyte that will go
through the first meiotic division to form two haploid secondary spermatocytes.
 The secondary spermatocytes will go through a second meiotic division to each produce
two spermatids; these cells will eventually develop flagella and become mature sperm.
Key Terms
 spermatocyte: a male gametocyte, from which a spermatozoon develops
 oocyte: a cell that develops into an egg or ovum; a female gametocyte
 polar body: one of the small cells that are by-products of the meiosis that forms an
egg
 mitosis: the division of a cell nucleus in which the genome is copied and separated into
two identical halves. It is normally followed by cell division
 meiosis: cell division of a diploid cell into four haploid cells, which develop to produce
gametes
Gametogenesis (Spermatogenesis and Oogenesis)
Gametogenesis, the production of sperm and eggs, takes place through the process of meiosis.
During meiosis, two cell divisions separate the paired chromosomes in the nucleus and then
separate the chromatids that were made during an earlier stage of the cell’s life cycle, resulting
in gametes that each contain half the number of chromosomes as the parent. The production of
sperm is called spermatogenesis and the production of eggs is called oogenesis.
Oogenesis
Oogenesis occurs in the outermost layers of the ovaries. As with sperm production, oogenesis
starts with a germ cell, called an oogonium (plural: oogonia), but this cell undergoes mitosis to
increase in number, eventually resulting in up to one to two million cells in the embryo.
Figure 43.3C.143.
3C.1: Oogenesis: The process of oogenesis occurs in the ovary’s outermost layer. A primary
oocyte begins the first meiotic division, but then arrests until later in life when it will finish this
division in a developing follicle. This results in a secondary oocyte, which will complete meiosis
if it is fertilized.
The cell starting meiosis is called a primary oocyte. This cell will begin the first meiotic division,
but be arrested in its progress in the first prophase stage. At the time of birth, all future eggs
are in the prophase stage. At adolescence, anterior pituitary hormones cause the development
of a number of follicles in an ovary. This results in the primary oocyte finishing the first meiotic
division. The cell divides unequally, with most of the cellular material and organelles going to
one cell, called a secondary oocyte, and only one set of chromosomes and a small amount of
cytoplasm going to the other cell. This second cell is called a polar body and usually dies. A
secondary meiotic arrest occurs, this time at the metaphase II stage. At ovulation, this
secondary oocyte will be released and travel toward the uterus through the oviduct. If the
secondary oocyte is fertilized, the cell continues through the meiosis II, completing meiosis,
producing a second polar body and a fertilized egg containing all 46 chromosomes of a human
being, half of them coming from the sperm.
Spermatogenesis
Spermatogenesis occurs in the wall of the seminiferous tubules, with stem cells at the periphery
of the tube and the spermatozoa at the lumen of the tube. Immediately under the capsule of
the tubule are diploid, undifferentiated cells. These stem cells, called spermatogonia (singular:
spermatagonium), go through mitosis with one offspring going on to differentiate into a sperm
cell, while the other gives rise to the next generation of sperm.
Figure 43.3C.143.
3C.1: Spermatogenesis: During spermatogenesis, four sperm result from each primary
spermatocyte, which divides into two haploid secondary spermatocytes; these cells will go
through a second meiotic division to produce four spermatids.
Meiosis begins with a cell called a primary spermatocyte. At the end of the first meiotic division,
a haploid cell is produced called a secondary spermatocyte. This haploid cell must go through
another meiotic cell division. The cell produced at the end of meiosis is called a spermatid.
When it reaches the lumen of the tubule and grows a flagellum (or “tail”), it is called a sperm
cell. Four sperm result from each primary spermatocyte that goes through meiosis.
Stem cells are deposited during gestation and are present at birth through the beginning of
adolescence, but in an inactive state. During adolescence, gonadotropic hormones from the
anterior pituitary cause the activation of these cells and the production of viable sperm. This
continues into old age.
Gametogenesis occurs when a haploid cell (n) is formed from a diploid cell (2n) through meiosis. We call
gametogenesis in the male spermatogenesis and it produces spermatozoa.
In the female, we call it oogenesis. It results in the formation of ova. This article covers both oogenesis
and spermatogenesis.
Spermatogenesis
In the beginning
Males start producing sperm when they reach puberty, which is usually from 10-16 years old. Biological
males continually produce sperm in large quantities (~200 million a day). This maximises the likelihood
of sperm reaching the egg following ejaculation.
Sperm production occurs in the testes of the male, specifically in the seminiferous tubules. In the
testicles, a blood-testis barrier forms to keep the tubules separate from the systemic circulation.
Protecting the sperm
Sertoli cells form the blood-testis barrier. This is important in preventing substances found in blood from
affecting the developing sperm. These products might include hormones or waste products.
It is also important as it prevents the immune system of the male from recognising the sperm as foreign
– the sperm are genetically different from the male and will express different surface antigens.
Forming functional sperm
Spermatogonia are the initial pool of diploid cells that divide by mitosis to give two identical cells. One of
these cells will be used to replenish the pool of spermatogonia – these cells are A1 spermatogonia. This
replenishment of spermatogonia means that males are fertile throughout their adult life. The other cell
– type B spermatogonium – will eventually form mature sperm.
Type B spermatogonia replicate by mitosis several times to form identical diploid cells linked by
cytoplasm bridges, these cells are now known as primary spermatocytes. Primary spermatocytes then
undergo meiosis.
Meiosis I produces two haploid cells, known as secondary spermatocytes.
Meiosis II produces four haploid cells, known as spermatids.
Maturation
The cytoplasmic bridges break down and the spermatids are released into the lumen of the seminiferous
tubule – a process called spermiation. The spermatids undergo spermiogenesis (remodelling and
differentiation into mature spermatozoa) as they travel along the seminiferous tubules until they reach
the epididymis.
From the seminiferous tubule, cells will travel to the rete testis. This acts to “concentrate” the sperm by
removing excess fluid. Then, cells move to the epididymis where the sperm is stored and undergoes the
final stages of maturation.
Spermatogenesis takes approximately 70 days, therefore in order for sperm production to be
continuous and not intermittent, multiple spermatogenic processes are occurring simultaneously within
the same seminiferous tubule, with new groups of spermatogonia arising every 16 days (spermatogenic
cycle). Each of these populations of spermatogenic cells will be at different stages of spermatogenesis.
Following ejaculation
Note that once sperm leave the male body and enter the female reproductive tract, the conditions there
cause the sperm to undergo capacitation, which is the removal of cholesterol and glycoproteins from
the head of the sperm cell to allow it to bind to the zona pellucida of the egg cell.
By OpenStax College - Anatomy & Physiology, Connexions Web site.

http://cnx.org/content/col11496/1.6/, Jun 19, 2013., CC BY 3.0,
https://commons.wikimedia.org/w/index.php?curid=30132982
Figure 1 – Diagram showing the overall process of spermatogenesis
Oogenesis
Oogenesis differs from spermatogenesis in that it begins in the foetus prior to birth. Primordial germ
cells (which originate in the yolk sac of the embryo) move to colonise the cortex of the primordial gonad.
Replication by mitosis peaks at approximately 7 million by mid-gestation (~20 weeks).
Cell death occurs after this peak to leave 2 million cells. Meiosis I begins before birth and forms primary
oocytes. There is therefore a finite supply of ova.
Primary oocytes are arranged in the gonads as clusters. They have flattened epithelial cells surrounding
them, and this is called the primary follicle.
During childhood, further atresia (cell death) occurs, leaving ~40,000 eggs at puberty.
Once puberty begins, a number of primary oocytes (15-20) begin to mature each month, although only
one of these reaches full maturation to become an oocyte.
The primary oocytes undergo 3 stages:
Pre-antral.
Antral.
Preovulatory.
Pre-Antral Stage
The primary oocyte is still in meiosis I, but will grow dramatically in this stage. The follicular cells grow
and proliferate to form a stratified cuboidal epithelium. Now, we call these granulosa cells and they
secrete glycoproteins. These chemicals form the zona pellucida around the primary oocyte.
Surrounding connective tissue cells also differentiates to become the theca folliculi, a specialised layer of
surrounding cells that is responsive to LH and can secrete androgens under its influence.
Antral Stage
Fluid filled spaces form between granulosa cells, these eventually combine together to form a central
fluid filled space called the antrum.
We now call the follicles secondary follicles. In each monthly cycle one of these secondary follicles
becomes dominant and develops further under the influence of FSH, LH and oestrogen. (See article on
the menstrual cycle).
Pre-Ovulatory Stage
The LH surge induces this stage and meiosis I is now complete. Inside the follicle, 2 unequally sized
haploid cells form. One of the daughter cells receives far less cytoplasm than the other and forms the
first polar body, which will not go on to form an ovum.
Another haploid cell is also formed, known as the secondary oocyte. Both daughter cells then undergo
meiosis II.
An initial polar body will replicate to give two polar bodies but the secondary oocyte arrests in
metaphase of meiosis II. This happens 3 hours prior to ovulation.
Ovulation
Now, the follicle has grown in size and is mature – it is called a Graafian follicle.
An LH surge occurs and increases collagenase activity. This is an enzyme that disrupts collagen.
Therefore, there is weakening of the follicular wall. This, combined with muscular contractions of the
ovarian wall, results in the ovum being released from the ovary. The ovum is then taken up into the
fallopian tube via the fimbriae (finger-like projections of the fallopian tube).
Fertilisation – the final stage of female gametogenesis

The secondary oocyte will only complete meiosis II following fertilisation. Here, it gives off a third polar
body. Following meiosis II, a fertilised egg results. If fertilisation doesn’t occurs, the oocyte degenerates
24 hours after ovulation, remaining arrested in meiosis II.
If fertilisation does occur, peristaltic movements of the fallopian tube move the egg to the uterus where
it can implant into the posterior uterine wall.
By Henry Vandyke Carter - Henry Gray (1918) Anatomy of the Human Body (See "Book" section
below)Bartleby.com: Gray's Anatomy, Plate 5This is a retouched picture, which means that it has been
digitally altered from its original version. Modifications: vectorization (CorelDraw). The original can be
viewed here: Gray5.png. Modifications made by Mysid., Public Domain,
https://commons.wikimedia.org/w/index.php?curid=1415394
1. What are gametes?

Gametes are cells specialized in sexual reproduction. They
contain half of the maximum number of chromosomes of the
species and unite with another gamete to give birth to a zygote
with double of number of chromosomes of the gametic cells.
In humans, gametes are formed by meiosis; male gametes are
sperm cells and female gametes are egg cells.
 Gametogenesis Review - Image Diversity: sperm cells egg

cells
Meiosis and Gametogenesis
More Bite-Sized Q&As Below
2. What type of cell division permits
sexual reproduction? What is
gametogenesis?
Meiosis is the type of cell division that allows sexual
reproduction, since it reduces the number of chromosomes of
the species to a half, making the combination of two gametes to
form a new individual possible. (In some organisms, meiosis
creates haploid gametophytes that by means of mitosis
generate gametes. Even in this case, the function of meiosis is
the same: to provide cells with half of the number of
chromosomes of the species, with the separation of
homologous.)
Gametogenesis is the name given to the process of gamete
production.
 Gametogenesis Review - Image Diversity: meiosis

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Gonads and Germ Cells
3. What is the name of the cells capable
of making gametes? What is the ploidy
of these gamete-forming cells?
The cells that form gametes are germ cells, as opposed to
somatic cells. The ploidy (number of chromosomes) of germ
cells is the same as somatic cells (only during the formation of
gametes does meiosis occur, reducing the number of
chromosomes to half).
 Gametogenesis Review - Image Diversity: germ cells
4. What are gonads? What are the male

and the female gonads in humans?
Gonads are the organs that produce gametes. They contain
germ cells that undergo division and generate gametes. In
males, the gonads are the testicles. In females, the gonads are
the ovaries.
 Gametogenesis Review - Image Diversity: gonads

Spermatogenesis
5. Indicating the name and respective
ploidy of each cell involved, how can the
formation of sperm cells from germ cells
be described?
The formation of sperm cells, or spermatogenesis, begins with a
germ cell called the spermatogonium (2n), which undergoes
mitosis and gives birth to the spermatocyte I (2n). The
spermatocyte I undergoes meiosis I and generates two
spermatocyte II (n) cells, which then undergo meiosis II and
produce four spermatids (n). Each spermatid undergoes a
maturation process called spermiogenesis and four sperm cells
are produced.
 Gametogenesis Review - Image

Diversity: spermatogenesis
6. What is the difference between

spermatogonium and spermatocyte I
cells?
The male germ cells are spermatogonia (diploid cells, 2n),which
are located in the testicles. They mature and by means of
mitosis give birth to spermatocytes I (2n), which will undergo
meiosis.
spermatocyte I and spermatocyte II
cells?
The spermatocyte I (2n) undergoes the first division of meiosis
(meiosis I), producing two spermatocyte II (haploid, n) cells.

spermatocyte II and spermatid cells?
Spermatids (n) are the products of the second division of
meiosis (meiosis II) during male gametogenesis. Each
spermatocyte II produces two spermatids, totaling four
spermatids for each spermatocyte I that undergoes meiosis.
Spermiogenesis
spermatids and sperm cells? What is the
name given to the transformation of
spermatids into sperm cells?
Sperm cells (male gametes) are mature spermatids that have
already undergone differentiation (the appearance of the
flagellum, the reduction of the cytoplasm, the formation of the
acrosome, the increase in the number of mitochondria). This
differentiation process is called spermiogenesis.
 Gametogenesis - Image Diversity: spermiogenesis
10. What is the acrosome of the sperm

cell? How is it formed?
The acrosome is a structure that contains a large number of
digestive enzymes. It is located at the anterior end of the sperm
cell and is formed through the union of Golgi apparatus
vesicles. The function of the acrosome is to release its enzymes
when the sperm cell meets the egg cell to break the external
covering of the female gamete, thus making fertilization
possible.
11. What is the function of the flagellum

of the sperm cell? How is it formed?
The flagellum of the sperm cell is formed of the centrioles that
migrate to the region posterior to the nucleus. Its function is to
promote locomotion towards the egg cell.
12. Why is the cytoplasm of sperm cells

very small? Why do the mitochondria of
sperm cells concentrate at the base of
the flagellum?
The reduced cytoplasm of sperm cells decreases the cell weight
and provides a more hydrodynamic shape for its locomotion in
fluids.
The high concentration of mitochondria at the base of the
flagellum of the sperm cell is necessary for supplying energy to
the flagellum (for it to vibrate and move the sperm cell).
Oogenesis
13. Concerning events during the
periods of life, how different is
gametogenesis in women and in men?
The formation of spermatogonia in men takes place during the
embryonic period. However, the formation of sperm cells is a
continuous process that begins in puberty and goes on until old
age, and sometimes during the whole life of the man.
In women, all oogonia are formed before birth. The oogonia turn
into oocytes I, which enter the first division of meiosis (meiosis
I). However, this division is interrupted at prophase and
continues only in puberty. After the beginning of menses, an
egg cell is released during each period and, if fertilized, it
finishes its meiotic division. Oogenesis stops after menopause
(cessation of menstrual activity) and the climacteric period of life
begins.
14. Indicating the name and respective
ploidy of each cell involved, how can the
formation of egg cells from germ cells be
described?
The formation of egg cells begins with a germ cell called an
oogonium (2n), which undergoes mitosis and gives birth to the
oocyte I (2n). The oocyte I undergoes meiosis I, but this is
interrupted at prophase. After puberty, during each menstrual
cycle, an oocyte I finishes meiosis I and generates one oocyte II
(n) and the first polar body (n). With fertilization, the oocyte II
then undergoes meiosis II and produces the mature egg cell (n)
and the second polar body (n).
 Gametogenesis Review - Image Diversity: oogenesis
15. What is the first polar body? How

different is it from an oocyte II?
In oogenesis, the oogonium differentiates into an oocyte I (2n)
and this cell undergo meiosis. After finishing the first meiotic
division (meiosis I), the oocyte I forms two cells: the oocyte II (n)
and the first polar body. The oocyte II is bigger because it
receives almost all the cytoplasm and the cytoplasmic
structures of the oocyte I as a strategy for metabolite and
nutrient storage. The oocyte II cell then undergoes the second
meiotic division. The first polar body is very small and has
almost no cytoplasm; it either disintegrates or stays attached to
the oocyte II.
16. What is the relationship between
fertilization and the end of the meiotic
process during oogenesis?
The oocyte II only completes the second meiotic division
(interrupted at metaphase) if fertilization by a male gamete
occurs. (Therefore, it can be said that the female gamete is the
oocyte II).
17. What is the second polar body?

After the end of the second meiotic division of the oocyte II, two
cells are generated: the egg cell and the second polar body.
The second polar body is a very small cell that has almost no
cytoplasm and which stays adnexal to the egg cell. The entire
cytoplasmic content of the oocyte II passes on to the egg cell.
Ovulation
18. What is the relationship between the
menstrual cycle and ovulation?
Ovulation is the releasing of the female gamete from the ovary.
Ovulation is a periodical event that occurs during each
menstrual cycle. Considering the day when menses begins the
first day of the menstrual cycle , ovulation occurs around the
14th day, when the concentrations of the hormones LH and
FSH reach high levels.
Fertilization
19. How does the male gamete
penetrate the egg cell? How does the
female gamete protect itself from the
entrance of more gametes after the
entrance of the first sperm cell?
The sperm cell that reaches the egg cell first triggers the
acrosome reaction, a process in which hydrolytic enzymes of
the acrosome are released on the external surface of the zona
pellucida (the protective layer that surrounds the egg cell). A
portion of this layer is digested by the acrosomal enzymes,
allowing the sperm cell to reach the plasma membrane of the
egg cell, thus fertilizing it.
At the moment that the sperm cell makes contact with the egg
cell membrane, a chemical alteration of this membrane occurs.
Enzymes secreted by exocytosis (a cortical reaction) make it
impossible for the zona pellucida to bind to other sperm cells
(zonal reaction) and, as a result, other male gametes cannot
enter the egg cell.
 Gametogenesis Review - Image Diversity: acrosome

reaction
20. What are the female pronucleus and
the male pronucleus?
The female pronucleus is the haploid nucleus of the egg cell.
the male pronucleus is the haploid nucleus of the sperm cell
that has fertilized the egg cell. After fertilization, both pronuclei
fuse, forming the nucleus of the diploid zygote.
21. Concerning their size and basic

morphology, how and why are male and
female gametes different from each
other?
Female gametes are large cells full of vitellus (nutritional
materials). Male gametes are small, mobile and agile flagellate
cells.
These features are related to their respective biological
functions. While female gametes have the basic functions of
receiving the sperm cell nucleus and storing nutrients for the
zygote, male gametes have the function of active movement
towards the egg cell.
AMETOGENESIS
Professor Alfred Cuschieri

Department of Anatomy
University of Malta
Objectives
o Explain the significance and importance of meiosis in
sexual reproduction
o Identify the different stages of gametogenesis in males

and females in micrographs of testis and ovary
o Name the stages at which the first and second meiotic

divisions take place
o Outline the stages of spermiogenesis
o Name the functions of Sertoli cells and granulosa cells
o Define the stages at which meiotic arrest in oocytes

normally occurs
o Identify the components of developing ovarian follicles
and of the oocyte at the time of ovulation
o Discuss the mechanism of non-disjunction in numerical

chromosome aberrations.
Sexual Reproduction
Sexual reproduction involves the formation of male and female

gametes and the the mechanisms necessary for the gametes
to come together and fuse to form one cell that represents the
beginning of a new individual with a distinct genetic identity.
Preparation for pregnancy involves two main programs of

events:
1. Gametogenesis
o the process of formation of the male and female gametes
o occurs in the gonads (ovary or testis)
2. Cyclic changes in the female genital tract

o the ovarian cycle
o the uterine cycle
Prepration for pregnancy has three important practical

applications
1. Controlling undesired pregnancies - Contraception
2. Treating Infertility - Assisted conception
3. Transmission of genetic disease
- transmission of inherited traits
- disorders of meiosis
All these applications have profound medical, social and ethical

implications
One Essential Question: Why is sexual reproduction
necessary?
The main purpose of sexual reproduction is the formation of
offspring who are genetically different from one another and
from their parents.
Meiosis is the fundamental process underlying sexual

reproduction. It involves two essential outcomes:
1. Reduction Division the process in which each gamete

receives a haploid set (n) of chromosomes and genes.
The diploid number (2n) is restored on fusion of two gametes.
2. Rearrangement of genes on the maternal and paternal

chromosomes.
This ensures that the offspring are genetically different from

one another.
Meosis involves four main events, and two cell divisions. (In the
following diagram only one pair of homologous chromosomes is
shown, to represent 23 pairs in humans).
DNA replication precedes meiosis, and occurs in the S phase,
as in all cell divisions. Recall that the chromosomes in the
parent cell contains a diploid set of chromosomes (2n
chromosome number and 2c amount of DNA). Following
replication, there are still 2n chromosomes, but each
chromosome consists of two chromatids, and has 2c DNA.
2. Pairing of homologous chromosomes and crossing over of

chromosome segments occur during prophase of meiosis. They
are crucial event in meiosis.
The complex of a homologous chromosome pair consisting of
four chromatids is a tetrad. The result of crossing over is that
there are now four recombinant chromatids.
3. Separation of chromosomes occurs as a result of the first

meiotic division. The two resulting daughter cells each have one
of a pair of replicated chromosomes, or a haploid set (n) with a
total of 2c DNA.
4. Separation of chromatids occurs as a result of the second

meiotic division, and give rise to four daughter cells, each
containing a haploid set of chromosomes (1n; 1c) amount of
Meiosis occurs in the germ cells. As a result of meiosis, four

daughter cells or gametes, are produced, each containing one
of a pair chromosomes and all containing different
chromosomes. Although the above diagram illustrates only
crossing over between the adjacent chromatids, in fact crossing
over also occurs between the two “outer” chromatids, and at
different sites from the other pair, so that all four daughter cells
are different from one another and from their parents. Pairing of
the two “outer” chromatids is possible because, in 3 dimensions
these would also be adjacent to one another.
Genetic Imprinting
In all diploid cells of an individual the chromosomes occur in

homologous pairs. One chromosome of each pair is derived
from the mother and the other from the father. The maternal
and paternal chromosomes are morphologically
indistinguishable but have important functional implications
because the expression of some genes is dependent on
whether they are on the maternal or the paternal chromosome.
This is termed genetic imprinting
Gametogenesis
Gametogenesis is the process of formation of gametes from the
germ cells in the testes and ovaries. Many principles of
gametogenesis are the same in both males and females, and
will be considered first. Gametogenesis is divided into four
phases:
1. Extra-gonadal origin of primordial germ cells
2. Proliferation of germ cells by mitosis
3. Meiosis
4. Structural and functional maturation of the ova and

spermatozoa
Primordial Germ Cells
o Are the earliest precursors of all germ cells
o Are formed in the early stages of embryonic development
o Are first recognizable close to the hindgut as large cells

with high alkaline phosphatase
o Proliferate and migrate into the gonad (testis or ovary)
o Differentiate into male or female germ cells (determined

by sex chromosomes)
The nomenclature of the developmental stages of

gametogenesis is similar in male and female germ cells. It is
summarised in the following diagram.
In males the spermatids, the products of the second meiotic
division, undergo morphological changes, known as
spermiogenesis, during which they become motile
spermatozoa. There is no corresponding stage in females.
The timing of the developmental stages of gametogenesis, and

the number of gametes produced are very different in male and
female germ cells.
In the male spermatogenesis occurs from puberty to old age,

producing immense numbers of spermatozoa at an average
rate of 1.5 million spermatozoa per minute.
o In females oogenesis begins in early foetal life. All oocytes

ever to be formed in females are produced during foetal life.
Many of them degenerate with time and at birth the ovaries
contain about 2 million oocytes. All the oocytes go into meiotic
arrest when they reach the first meiotic division during foetal
life. The primary oocytes remain in the prophase of the first
meiotic division until the time of puberty, when they are
gradually released to complete meiosis at regular intervals
known as the ovarian cycle. On the average only one oocyte
matures during each cycle, which occurs at approximately
monthly intervals, so that the total amount of oocytes to be
ovulated is about 500 oocytes in a lifetime.
Spermatogenesis
Spermatogenesis is the process of formation of the male germ
cells. It occurs in the seminiferous tubules of the testes. The
developmental stages of the male germ cells can be observed
sequentially from basement membrane to lumen.
The sertoli cells are supporting cells that have several functions.
They form the blood-testes barrier: nutrients, and circulating

substances do not directly reach the germ cells; the Sertoli cells
determine which substances reach the germ cells. The
spermatogonia are outside the blood-testis barrier. They form
invaginations surrounding the spermatocytes, spermatids and
developing spermatozoa and are nutritive to them. They also
produce antigen-binding proteins, which are necessay for
spermiogenesis.
The figure below is a light micrograph of a seminiferous tubule
Spermiogenesis is morphological development of spermatids to

spermatozoa. It involves:
a. Nuclear changes - The nucleus becomes condensed and
heterochromatic -histones are replaced by protamines, which
allow a high degree of DNA compaction and makes DNA
inaccessible for transcription
Cytoplasmic changes – These are directed to the formation of

a motile sperm cell capable of penetrating the ovum and
involves the following changes:
o The Golgi apparatus at one pole of the cell forms an

acrosome containing proteolytic enzymes
o The centrriole at the opposite pole organises the formation

of microtubules to form a flagellum
o Alignment of mitochondria in a spiral around the base of

the flagellum – this forms the mid-piece of the spermatozoon
o The excess residual cytoplasm accumulates at one side of

the cell and becomes detached to form a residual body
Oogenesis
At birth the ovary contains primordial follicles. They consist of a
primary oocyte surrounded by granulosa cells.
Gap junctions connect the oocyte to surrounding granulosa

cells.
The gap junctions permit passage of amino acids, glucose and
metabolites for growth of the oocyte.
The follicular cells secrete a meiotic inhibitory factor that is
responsible for the first meiotic arrest.
The developing oocyte undergoes two meiotic arrests:

The Ovarian follicle consists of the following:
The zona pellucida:
o Is secreted by the oocyte
o Consists of glycoprotein and glycosaminoglycans
o Contains sperm receptors necessary for fertilization
The oocyte cytoplasm contains:

- numerous ribosomes (produced by r-DNA amplification in
nucleolus)
- Yolk droplets ( nutritive)
- Cortical granules (formed in the Golgi apparatus).
Cortical granules are released on penetration of the vitelline

membrane by a sperm. They cause a change in the zona
pellucida to prevent double fertilization
The Graafian follicle (tertiary follicle) is distended with liquor and

points at the surface of the ovary like a blister.
Rupture of the follicle occurs at ovulation releasing the
secondary oocyte. At the time of ovulation the second meiotic
division is still not completed. After ovulation
the secondary oocyte is surrounded by a corona radiata.
Abnormalities in meiosis may give rise to numerical
chromosome abnormalitites. Non-disjunction is the usual
mechanism by which abnormalities in chromosome number
may occur. The following diagram illustrates chromosome 21 in
meiosis I and II in (a) normal gametogenesis and (b) non-
disjunction in meiosis I. Failure of separation of the
chromosomes results in a secondary oocyte or a secondary
spermatocyte with two chromosomes 21, which will form
trisomy 21 after fertilisation. The other secondary oocyte or
spermatocyte has does not contain a chromosome 21, and after
fertilization, will result in monosomy 21. This is incompatible
with survival and this condition is therefore not seen. Non-
disjunction also occurs in common trisomies e.g. trisomy 18 or
trisomy 13.
Non-disjunction may also occur in meiosis II, resulting from
failure of separation of the chromatids, as shown in the
following diagram. This type of non-disjunction is rare.

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Animal biotechnology test exam critical questions

2. What is glycosylated (carbohydrate-modified) animal cell culture?

 Therefore, glycosylation is engineering of animal cells which express cloned

3. What is relation between Hayflicklimit to telomere length?

1. The growth of animal cells in vitro in a suitable culture medium is called___________

4. What is a cell line?

5. Name the organism on which first cell line was observed?

6. Which of the following is the characteristics of a normal cell?

7. Name the cell line of the human embryonic lung?

9. Which of the following is NOT the major function of the serum?

What is animal tissue culture?

Advantages of Animal tissue culture

1. Technical skill and expertise is required to carry tissue culture techniques

Application of animal tissue culture

Maintenance ricks leakage of disposals, age, and condition of the

Personnel risks lack of interest and concentration and

Physical risks Intense cold, electric shocks, and fire

Chemical risks Exposure to toxic substances like poisons,

Biohazards Viruses, pathogenic organisms, culture cells,

Radioisotope risks Energy emission its penetrations and ionization

1. What is animal tissue culture?

Not me? Clear form

First name* aw oke Email* yihuneaw oke@gmail.com

Last name* yihun Confirm email* yihuneaw oke@gmail.com

Understanding the in vitro system and factors that affect it

1. Cell culture is in vitro because (choose the best answer)?*

grow and divide transduce differentiation signals

enhance survival all of the above

find other cells anchor to plastic surfaces

5. Paracrine signals come from which of the following?*

other neighboring cells unwanted cell culture contaminants

6. Apoptosis is a form of cell death that*

is irreversible is caused by some signal loss

7. The cell culture environment includes*

media and media components the gas composition

the plastic the cells are attached to all the above

8. Confluency refers to*

9. When we grow cells in culture*

Media Use and Care

1. Cells need to be refed only if the cells have reached confluency.*

2. The function of phenol red is to (check all that apply) *

3. Basal media the following components in it (check all that apply) *

fetal bovine serum trace elements

water soluble vitamins amino acids

4. Complete media is (choose the best answer)*

5. L-glutamine is an important amino acid because (choose the best answer) *

it is not important since it is made by some cells it is an amine donner

is easy and can be quickly may be possible given care

7. Phenol Red is a mimic of which of the following?*

8. GlutaMAX™-I is alternative to L-glutamine because?*

GlutaMAX™-I is double the strength of L-glutamine L-glutamine is less stable

Contamination and prevention

2. When growing animal cells it is important to*

have lots of computer memory drink plenty of water

4. The atmosphere inside a biological safety cabinet is*

full of CO2 gas very clean but not absolutely sterile

absolutely sterile lower in oxygen then the air outside

minimize quick movement not introduce contamination

6. Of all the contaminations possible, probably the easiest to detect is*

cross-contamination yeast and fungi

7. Mycelium is a form of*

viral waste fungi

8. In the event of a bacterial contamination the easiest thing to do is *

always be used used, only when necessary