LM 4 and 5

Question 1

Why are spermatogonia referred to as true stem cells?

Answer 1

They are capable of infinite self-renewal

Question 2

Why are spermatogonia excellent target cells for biotechnology?

Answer 2

• Can be genetically engineered in lab

* can be propagated and transferred to produce germ cells and genetically altered offspring

Question 3

Why has it been historically difficult to manipulate spermatogonia in the lab?

Answer 3

• They are embedded in the seminiferous tubules
• Difficult to obtain a pure sample
• Extremely difficult to culture in lab setting

Question 4

What was the objective of the Brinster and Zimmerman experiment in 1994 published in PNAS?

Answer 4

To establish methods for successful spermatogonial cell transfer

Question 5

What were the methods of the Brinster and Zimmerman experiment in 1994 published in PNAS?

Answer 5

• Recipient mice were anesthetized, and the testicles were exteriorized and placed under microscope
• Smeiniferous tubules were injected with the cellular suspension of donor cells in the lumen
• Testicles were re-closed and males recovered
• Mice were then monitored to determine results of donor cell incorporation

Question 6

Describe the recipient male mice in the Brinster and Zimmerman experiment in 1994 published in the PNAS

Answer 6

• W strain male mice that were deficient in spermatogenesis

* They had been treated with Busulfan to induce the deficiency

Question 7

Describe the donor cells in the Brinster and Zimmerman experiment in 1994 published in the PNAS

Answer 7

*Mixture of testicular cells from normal male mice

Question 8

Describe the results of the Brinster and Zimmerman experiment in 1994 published in the PNAS

Answer 8

• 25% of injected testes incorporated donor cells
• Spermatogonia established in ST over time
• Remainder of cells in mixture died out

Question 9

What were the implications of the Brinster and Zimmerman experiment in 1994 published in PNAS

Answer 9

• Can isolate and hold spermatogonia (in cell mixture) in lab for several hours
• Spermatogonia can establish themselves in ST of sterile recipient males

Question 10

Describe the methods of the experiment published by Brinster and Avarbock in 1994 in PNAS

Answer 10

• Used same methods of donor cell mixture injected into lumen of the ST of sterile W strain male mice
• Recipients were then observed through puberty and mating

Question 11

Describe the results of the Brinster and Avabock 1994 PNAS

Answer 11

Recipient sterility was overcome by testicular cell transfer

Question 12

What was the major problem with spermatogonial cell transfer between the years 1994-2002

Answer 12

*ST spermatogonial cell transfer was okay within species, but no cross-species transfers worked
due to species-specific microenvironment

Question 13

What was the solution to the major problem facing ST spermatogonial cell trasnfer between the years of 1994-2002

Answer 13

• Transfer whole testis pieces under skin of nude mice (immunodeficient)
• Able to maintain species-specific microenvironment due to being immunocompromised

Question 14

Describe the experiment done by Honaramooz et al. 2002 in Nature

Answer 14

• Graft a pig testis about 0.5-1mm
• Size expanded to 4-8mm after 10 weeks
• A porcine stem cell was extracted from a 27 week graft
• Intracytoplasmic sperm injection (ICSI) used to produce pig embryos from porcine sperm cells in male mice

Question 15

Describe the experiment done by Snedaker et al. 2004 J Androl

Answer 15

• Kitten testicular fragments were grafted under the back skin of nude mice
• At 54 weeks, 25% of the grafts had full formed cat spermatozoa

Question 16

Describe the experiment of Honaramooz et al. 2004 in Bio repro

Answer 16

• Immature Rhesus monkey testicular fragments were grafted under the back skin of nude mice
• ICSI was performed in monkey ova using sperm from the grafts
• Both morulas and blastocysts were produced

Question 17

What was the major implication of the various testicular fragment grafting experiments done in 2002-2004?

Answer 17

Sperm cells and offspring from one species can be produced in a different species (namely nude mice)

Question 18

What potential applications arose after the various testicular fragment grafting experiments done in 2002-2004?

Answer 18

• Zoological species propagation

* Treatments for rescue of fertility after cancer treatment in humans

Question 19

What limitations were still present after the various testicular fragment grafting experiments done in 2002-2004?

Answer 19

• Donor: immature testicular tissue only could be used

* Recipient mice: Must be immunocompromised

Question 20

Describe the experiment done by Honaramooz et al. in 2003 published in Bio Repro

Answer 20

• AAT transgenic goats used that have an α-1 antitrypsin deficiency
• Testes were removed and testicular cells were prepared
• Cell preps were injected into the rete testis of 5 immature recipient male goats
• The recipients were mated and transgenic offspring were produced

Question 21

Describe the implications of the experiment done by Honaramooz et al. in 2003 published in Bio Repor

Answer 21

• Speramtogonial cell transfer can be performed in domestic animal species
• Immunocompromised recipients not required
• Simple rete testis infusion technique useable
• Offspring from donor’s spermatogonia produced
• Novel, simple method for making transgenic animals
• A recipient male can produce offspring from another, unrelated site

Question 22

Describe the experiment of spermatogonial transfer in fish

Answer 22

• Trout spermatogonia was injected into sterile (triploid) salmon fry
• It was incorporated into the gonads and differentiated into spermatogonia and oogonia
• The salmon produced trout offspring

Question 23

What was the major implication of the experiment involving spermatogonial transfer in fish?

Answer 23

It can be used as a method of species expansion/preservation such as using the blue fin tuna as a target and salmon as the recipient

Question 24

Why is the dairy industry interested in sexed semen?

Answer 24

• Production of heifers preferred because primary income is fluid milk sales
• Value of the bull is minimal and replacement heifers are always needed

Question 25

Why is the beef industry interested in sexed semen?

Answer 25

• Production of steers generally preferred

* Same sexed lots for marketing are preferable

Question 26

Why is sexed semen important to human medicine?

Answer 26

• Can eliminate the potential for X-linked diseases

* Gender balancing within families

Question 27

What is the difference between X and Y bearing sperm?

Answer 27

• Less DNA in Y bearing sperm

* This is the only consistently measurable difference that has ever been found

Question 28

What is FACS and what is it used for?

Answer 28

Fluorescence Activated Cell Sorting used to sex semen

Question 29

Describe how FACS works

Answer 29

• Sperm are dyed with a fluorescent dye that intercolates in DNA strands
• The sperm enter a flow chamber where it is diluted to one sperm cell per droplet
• They are then released from the flow chamber one at a time where they pass a laser beam that excites the dye so it fluoresces and produces a light scatter
• X bearing sperm fluoresce more due to having more DNA and the light scatter ensures proper shape of the sperm
• The sperm then pass through a charging plate which ionizes the sperm to possess a charge (X:+, Y:-)
• The charged sperm pass through deflection plates to be sorted based upon charge (Y are attracted to the positive plate and vice versa)
• Any sperm that don’t meet fluorescent or light scatter requirements don’t receive a charge and fall through the deflection plates
• ratio is X 49% and Y 51%

Question 30

What is the percent difference between X and Y sperm in the bull, chinchilla, possum, and human?

Answer 30

• Bull: 3.8%
• Chinchilla: 7.5%
• Possum: 2.3%
• Human: 2.8%

Question 31

Describe the sorting windows for Y and X bearing sperm and how much this limits sperm sorting

Answer 31

• Sperm on the left side of the Y-peak and right side of the X-peak can be sorted and are referred to as the X and Y windows
• Almost half of X and Y sperm can’t be sorted due to these windows overlapping

Question 32

What improvement was made to semen sorting in 1999?

Answer 32

• Originally had a spherical beveled needle to move semen out of flow chamber, but now has an orienting nozzle
• The nozzle ensures a shorted distance to drop before contact with the laser in addition to allowing 70% of sperm to be in the correct orientation for light scatter as opposed to the previous 25%.

Question 33

What is the sort rate of sexed semen and what did it max out to in 2008?

Answer 33

• Sort Rate=Sperm/Second

* Maxed out at 8000/sec

Question 34

What is the sorting efficiency of sexed semen and what is the range of this efficiency

Answer 34

• Sort Efficiency=Sorted Sperm/Total Sperm

* Fluctuated between 25-40%

Question 35

In 2008, about how long did one sort take?

Answer 35

4 minutes

Question 36

How many sexed doses were sold in 2008 and how many are there today?

Answer 36

• 2008: about 6 million

* Today: 10s of millions

Question 37

What are the pregnancy rates for conventional semen, sexed semen, and sexed semen as a percent of conventional?

Answer 37

• Conventional: 56%
• Sexed: 45%
• Sexed as a percent of conventional: 80%

Question 38

What is the percent of desired calving for sexed semen, conventional semen, and herd bull breeding?

Answer 38

• Sexed: 89%
• Conventional: 50%
• Herd Bull: 50%

Question 39

What are the recommendations for success when using frozen sexed semen?

Answer 39

• Breed to heifers (45% vs 27% in cows)
• Use optimized estrus synchronization methods
• The insemination dose of 2*10^6 motile cells is only 5-10% of a standard insemination dose so use deep uterine insemination with careful semen handling techniques

Question 40

What were the dolphin pregnancy rates using control frozen-thawed, sexed chilled, and sexed frozen thawed semen under all AI conditions and optimal AI conditions?

Answer 40

• Control frozen thawed: 66.7% in all AI conditions, 75% under optimal conditions
• Sexed chilled: 54.5% in all AI conditions, 71.4% under optimal conditions
• Sexed frozen thawed: 37.5% in all AI conditions, 50% under optimal conditions

Question 41

What are the desired human gender rates of sexed semen and why?

Answer 41

• X sorted: 92% desired
• Y sorted: 83.6% desired
• Due to narrow difference in DNA