Douglas T. Carrell and Kenneth I. Aston (eds.), Spermiogenesis and Spermatogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 927, DOI 10.1007/978-1-62703-038-0_42, © Springer Science+Business Media, LLC 2012

Chapter 42

Testicular Tissue Grafting and Male Germ Cell Transplantation

Jose R. Rodriguez-Sosa, Lin Tang, and Ina Dobrinski

Abstract

Testicular tissue grafting and male germ cell transplantation are techniques that offer unprecedented opportunities to study testicular function and development. While testicular tissue grafting allows recapitu-lation of testis development and spermatogenesis from immature males of different mammalian species in recipient mice, germ cell transplantation results in donor-derived spermatogenesis in recipient testes.

Testicular tissue grafting results in spermatogenesis from a wide variety of large animal donor species and is therefore an attractive way to study testis development and spermatogenesis and preserve fertility of immature males. Germ cell transplantation represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs) in a given donor cell population and serves as a valuable tool to study stem cell biology and spermatogenesis. In this chapter we provide detailed methodology to successfully perform both techniques.

Key words: Testicular tissue grafting, Testis development, Male germ cell transplantation, Spermatogenesis, Spermatogonial stem cells

In the last two decades, important discoveries have provided unprecedented opportunities to explore testicular function and development. In 2002, we first reported that small fragments of testicular tissue from immature males, transplanted under the dorsal skin of immunodeficient mice, were able to survive and undergo full development with the production of sperm (1). Since then, testis tissue xenografting has been shown to be successful in a wide variety of species and emerged as a valuable alternative to study testis devel-opment and spermatogenesis of large animals in mice (2).

1. Introduction

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Testis tissue xenografting is particularly attractive for research as several mice can be transplanted with tissue from a single donor and then assigned to different treatment groups and collection time points. This allows the study of testis tissue from the target species with the advantages of working in a rodent model. For example, this approach reduces the number of large donor animals that are needed for study and the cost associated with it. Moreover, since the resulting sperm are capable of fertilizing and triggering normal embryo development, this approach can also be used to preserve fertility in immature males when sperm collection is not an option (3).

In 1994, Dr. Ralph Brinster and colleagues at the University of Pennsylvania showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipi-ent mice results in donor-derived spermatogenesis and sperm pro-duction by the recipient animal (4, 5). The use of transgenic donors carrying the bacterial b-galactosidase gene easily allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals. Through the tracing of donor cells, it was revealed that donor germ cells, after being transplanted into the lumen of the seminif-erous tubules, were able to move from the luminal compartment to the basement membrane where spermatogonia are located (6). It is generally accepted that only SSCs are able to colonize the stem cell niche in the seminiferous epithelium of recipient mice and initiate spermatogenesis.

Germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to evaluate the stem cell potential of specific populations of germ cells. Moreover, germ cell transplantation can be used to elucidate basic stem cell biology (6–9), to produce transgenic animals through genetic manipula-tion of germ cells prior to transplantation (10, 11), and to study Sertoli cell–germ cell interaction (12, 13).

1. Suture, Ethicon 6-0 silk. 2. Wound clips. 3. #5 Dumont Forceps. 4. Hemostat. 5. Extra Fine Graefe Forceps—0.5 mm Tip, Curved. 6. Extra Thin Iris Scissors.

2. Materials

2.1. Surgical Instruments

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1. Phosphate-buffered saline (PBS). 2. Dulbecco’s modified Eagle’s medium (DMEM). 3. Disposable scalpels, No. 10. 4. Tissue culture dishes (60 × 15 mm).

1. Collagenase, type IV: dissolve 7 mg of collagenase powder in 7 ml of DMEM to make a working solution of 1 mg/ml. Filter-sterilize the solution (see Note 1).

2. Trypsin–EDTA: prepare 4 ml (0.25 % trypsin plus 1 mM EDTA) (see Note 1).

3. DNase I: dissolve 14 mg of DNase I in 2 ml of DMEM to make a working solution of 7 mg/ml. Filter-sterilize the solu-tion (see Note 1).

4. DMEM. 5. Trypan blue. 6. Nylon mesh cell strainer, 40 and 70 mm (BD biosciences). 7. Busulfan: dissolve Busulfan powder in DMSO and then add an

equal volume of sterile distilled water to make a final concen-tration of 4 mg/ml. Keep solution warm at 35–40 °C prior to use to avoid precipitation of Busulfan (see Note 2).

8. Thin-wall glass capillary tubes: used as the injection needle. 9. Polyethylene tubing: attach the tubing to a 1 ml syringe to

make a simple injection apparatus. 10. Sigmacote (Sigma). 11. 1 ml Syringe.

1. X-gal: Dissolve X-gal powder in N,N-dimethyl formamide to give a stock solution of 10 mg/ml. Wrap in aluminum foil (light sensitive) and stored at −20 °C.

2. Potassium Ferrocyanide: make 500 mM stock in water. 3. Potassium Ferricyanide: make 500 mM stock in water. 4. Magnesium chloride: make 1 M stock in water. 5. Sodium deoxycholate: make 1 % stock solution in water. 6. N,N-dimethylformamide: used to dissolve X-gal powder. 7. Igepal CA-630 (Sigma): make 10 % stock solution in water. 8. LacZ rinse buffer: 0.2 M sodium phosphate, pH 7.3; 2 mM

magnesium chloride; 0.02 % Igepal CA-630; 0.01 % sodium deoxycholate; make up to 200 ml with PBS.

9. LacZ staining buffer: 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml X-gal, make up to 10 ml with LacZ rinse buffer.

2.2. Testicular Tissue Grafting

2.3. Germ Cell Transplantation

2.4. X-Gal Staining

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From Dobrinski and Rathi (14) and Rodriguez-Sosa et al. (15).

1. Obtain testis tissue by castration or biopsy from a donor male.

2. Place testis in PBS or biopsies into culture medium, maintain-ing sterile conditions.

3. Keep the collected tissue on ice and transport to the laboratory (see Note 3).

4. For preparation of donor tissue, perform in the tissue culture hood to maintain sterility. Wash each testis in ice-cold PBS containing antibiotics two to three times before transferring into a culture dish with PBS. In the case of biopsies, wash testis fragments two to three times with ice-cold culture medium containing antibiotics by centrifugation at 150 × g for 2 min and resuspend in fresh ice-cold culture medium.

5. If intact testes are collected, remove tunica vaginalis by making an incision along the surface and extrude the testis. Remove from testis all annex structures (spermatic cord, epididymis, connective tissue). Wash testes once in cold PBS and transfer into a culture dish with PBS. Carefully remove the tunica albuginea of the testis by using a scalpel blade and a pair of scissors. If the testis is very small, the tunica can be removed by squeezing the testicular tissue out of the tunica through a small incision made on one end while holding the tunica with a pair of small forceps on the other end.

6. Depending on the size of the testis either the whole testis tissue can be cut into small pieces of around 1–2 mm3 in size using a disposable No. 10 scalpel or large pieces of testis tissue can first be removed from the testis and then cut into smaller pieces. All this should be done in ice-cold culture medium and under sterile conditions in a small culture dish (60 × 15 mm).

7. Transfer the prepared tissue fragments to ice-cold culture medium in small culture dishes on ice until grafting.

1. Anesthetize recipient mouse following approved protocols and prepare sterile surgical field by clipping or plucking the hair (not necessary in nude mice), wiping with 70 % ethanol and betadine solution.

2. Make a 0.5–1 cm ventral midline skin incision to expose the abdominal wall (see Note 4).

3. Carefully expose the testis, the testicular artery and epididymis, and detach the tail of the epididymis from the gubernaculum by blunt dissection (Fig. 1a).

3. Methods

3.1. Testicular Tissue Grafting in Mice

3.1.1. Collection of Donor Tissue

3.1.2. Castration of Recipient Mouse

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4. Ligate the testicular artery and the vas deferens together with the blood vessel with silk, and section the ligated structures by cutting between the testis and the ligature (Fig. 1b).

5. Repeat the procedure for the second testis. 6. Suture the abdominal wall with one or two surgical stitches. 7. Close the skin incision with one or two Michel clips.

Fig. 1. Key methodological steps in recipient preparation and ectopic transplantation of testicular tissue. (a) Photograph illustrating the detachment of the testis (T) from the gubernaculum (G) prior to ligation of the testicular artery and annexed structures. (b) Once the testis has been detached from the gubernaculum, the testicular artery and vas defer-ens are ligated. In this photograph the ligature has already been placed (arrow ) and the sectioning of the ligated structures is about to be performed. (c) Once the mouse has been castrated and its back has been aseptically prepared, 0.5–1 cm incisions are made in the skin to introduce each testis fragment. (d) In order to place each testis fragment under the dorsal skin, the subcutaneous tissue is teased apart with small scissors to produce a small cavity. (e) The testis fragment is placed with fine forceps deep into the subcutane-ous cavity by holding the border of the skin incision with small forceps to expose the cavity. (f) Once the testis fragment has been placed, the incision is closed with a Michel clip. Bars = 1 cm.

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1. Position the mouse in ventral recumbency and prepare a sterile surgical field on its back as above.

2. Depending on how many grafts are to be inserted (generally 4–8/mouse), make ~0.5 cm long skin incisions on each side of the midline of the back of the mouse (see Note 5).

3. Use forceps to hold a border of the skin incision, and make a subcutaneous cavity by teasing apart the connective tissue using scissors.

3.1.3. Ectopic Grafting (Summarized in Fig. 1c–f )

Fig. 2. Germ cell transplantation procedure. Place recipients in dorsal recumbence after anesthesia and make a ~1 cm midline abdominal incision (a). Expose testis by withdrawing the fat pad attached to the epididymis and testis and place a thin sterile drape underneath the fat pad/testis for better visual identification (b). Position the testis and epididymis so that the efferent ducts buried in the fat pad are discernible (c–e). Identity the efferent ducts and gently remove fat tissue around the ducts. A piece of colored paper or plastic can be placed underneath the ducts for better visualization (g). Break or grind the pipette tip according to the size of the ducts and load cell suspension into the pipette (h). Carefully insert the pipette into a duct in the bundle of efferent ducts, gently thread a few millimeters toward the testis (the arrow in H shows the direction of pipette injection and threading). A testis with successful injection into the seminiferous tubules is shown (i). Ts Testis, Ep epididymis.

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4. Using an iris forceps, place a piece of testis tissue deep into the subcutaneous cavity, holding the border of the skin incision with another iris forceps.

5. Close the skin incision with one Michel clip and keep mouse on heating pad until it starts to recover from anesthesia.

6. Transfer the mouse to a cage with additional insulation and cover and monitor until mice are fully recovered (see Note 6).

1. Sacrifice the host mouse according to animal care and use guidelines and make a midline skin incision on the back skin running from the tail to the neck, and open the skin. This exposes the grafts which can be located either on the subcuta-neous tissue or attached to the skin (see Note 7).

2. Carefully remove the grafts using a pair of forceps and a pair of scissors.

3. Record the number of grafts recovered, along with the size and weight of individual grafts.

4. Retrieve the seminal vesicles from the abdomen of the mouse and record their weight as an indication of testosterone pro-duction by the grafted tissue (see Note 8).

5. For histological analysis, suspend xenografts into a sample vial containing Bouin’s solution (or other fixative) in a volume ~10 times that of the xenograft, and label the vial appropriately. Incubate overnight in the refrigerator followed by washing at least three times in 70 % ethanol preferably at intervals of 24 h, and proceed for processing and embedding in paraffin (see Note 9).

6. For sperm harvesting, wash xenografts by spinning them down at 300 × g for 1 min and resuspend them in culture medium containing antibiotics. Cut grafts into small pieces and mince carefully with the forceps in a tissue culture dish containing 3–5 ml of culture medium. Finally, filter minced tissue through the 40-mm cell strainer.

1. Recipients should be immunologically tolerant (either genetically matched to donors or immunodeficient) to the donor testis cells.

2. Recipients should be either naturally devoid of spermatogene-sis (e.g., W/Wv mice) or depleted of endogenous germ cells. Germ cell depletion can be achieved by irradiation or chemo-therapeutic drugs such as Busulfan. In this protocol, we describe the method of preparing recipient mice with Busulfan.

3. Treat recipients at 4–6 week of age through intraperitoneal injection of Busulfan (see Note 10). The optimal dose of Busulfan is strain dependent. In commonly used recipient strains, a dose of 40–50 mg/kg is sufficient to deplete endogenous

3.1.4. Collection of Testicular Grafts for Analysis and Sperm Harvesting

3.2. Male Germ Cell Transplantation

3.2.1. Preparation of Recipient Mice with Busulfan

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germ cells (e.g., 44 mg/kg for nude mice, 50 mg/kg for B6/129 F1 recipients).

4. Dissolve Busulfan powder in DMSO and then add an equal volume of sterile distilled water to make a final concentration of 4 mg/ml. Keep solution warm at 35–40 °C before use to avoid precipitation of Busulfan. Discard solution if precipita-tion is observed.

5. After Busulfan treatment, allow at least 1 month before using recipients. Recipients can be used between 1 and 3 months post-Busulfan treatment.

1. Choose borosilicate glass pipettes (capillary tubes) with a 1.0 mm outer diameter, a 0.75 mm inner diameter, and a length of 3 in.

2. Siliconize glass pipettes with Sigmacote, rinse pipettes with methanol and blow dry.

3. Pull the pipettes using a pipette puller (see Note 11). 4. Break the pipette tips under a dissecting microscope prior to

use to achieve a diameter of approximately 50 mm at the tip.

1. Choice of donor strain is dependent on the experimental question studied. If quantification of spermatogenesis is used as the endpoint, use donors with an easily identifiable genetic marker such as Lac-Z (e.g., B6.129 S7-Gt(ROSA)26Sor/J from Jackson Laboratory) or GFP (e.g., C57BL/6-Tg(CAG-EGFP)1Osb/J from Jackson Laboratory).

2. Prepare enzyme solutions (see Note 1). 3. Collect testes aseptically and remove the tunica albuginea.

Spread seminiferous tubules gently with fine forceps to facili-tate enzymatic digestion.

4. Transfer tubules into collagenase solution, incubate at 37 °C for 5–10 min, and agitate frequently.

5. Wash tubules twice by spinning (200–300 × g for 3 min) and resuspend in PBS w/o Ca2+.

6. Resuspend tubules in trypsin–EDTA and shake until they become sticky and cloudy.

7. Monitor the digestion of tubules at 37 °C as it should occur within 1–2 min.

8. Add DNase solution and shake well. Incubate for 1–2 min. 9. Add 3 ml of FBS to stop the action of enzymes. 10. Filter the cell suspension using a nylon mesh with 40–70 mm

pore size to remove cell/tissue chunks. 11. Collect cells at 600 × g for 5 min and resuspend cells in a small

amount of DMEM (<100 ml).

3.2.2. Preparation of Microinjection Pipettes

3.2.3. Preparation of Donor Cells for Transplantation

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12. Count cells and adjust the volume to a desired final concentration (usually 100 × 106). Keep cell suspension on ice prior to use.

13. Each Busulfan-treated mouse testis will be filled with only 10–15 ml of cell suspension. Due to potential waste and leak-age, aim to prepare 30–50 ml cell suspension per testis.

1. Anesthetize recipients. Place in dorsal recumbency and surgi-cally prepare the abdominal area. Make a ~1 cm midline abdominal incision to expose the abdominal wall.

2. Lift abdominal wall by using a small forceps at the point of the white line to avoid accidentally injuring abdominal organs, and proceed to make a ~0.5 cm incision at the midline of the abdominal wall to expose the peritoneal cavity.

3. Use one iris forceps to hold the abdominal wall and use another pair of iris forceps to search for the fat pads attached to the epididymis and testis in the peritoneal cavity.

4. Gently pull the fat pad out until the testis is exteriorized and the testicular artery and epididymis are clearly visible. Work on one testis at a time.

5. Place a thin sterile drape made from autoclaved index cards underneath the fat pad/testis for better visual identification (optional). The drape also works to absorb fluid.

6. Add a drop of Trypan blue dye into the cell suspension and carefully load the cell suspension into the polyethylene tubing connected to a 1 ml syringe. Attach the pulled pipette into the tubing and gently force the cell suspension into the pipette by applying pressure to the syringe (see Note 12).

7. Identify the efferent ducts (that connect the testis to the epididymis) and gently remove fat tissue around the ducts. Work carefully as the ducts and the membrane around them are translucent.

8. Carefully insert the pipette into a duct in the bundle of efferent ducts; gently thread a few millimeter toward the testis.

9. Hold the injection pipette in place with one hand, and use the other hand to reach for the syringe plunger. Gently depress the plunger to ensure that the suspension flows into the rete testis and the seminiferous tubules begin to fill.

10. The injection rate and flow of cell suspension is regulated by thumb pressure. Avoid sudden increase in pressure; monitor the movement of suspension in tubules. Stop the injection when almost all surface tubules have been filled before the tes-tis starts to become ischemic (see Note 13).

11. Return the testis to the abdominal cavity. Repeat procedure on the contralateral testis. Suture the abdominal wall with 6-0 silk suture and close the skin with metal wound clips. Monitor mice on a warming pad until full recovery.

3.2.4. Transplantation Procedure (Summarized in Fig. 2)

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1. Allow 2–4 months after transplantation before analysis. 2. Sacrifice the recipient mouse according to animal care and use

guidelines and collect the testis and epididymis into PBS. When a Lac-Z transgenic donor strain has been used, the epididymis can be used as a positive staining control as it has endogenous beta-galactosidase activity.

3. For detection of donor cells by Lac-Z staining, fix the testis for 1–2 h at 4 °C in 4 % paraformaldehyde (PFA) in PBS. Rinse 3 × 30 min at room temperature in lacZ rinse buffer.

4. Incubate overnight at 37 °C in lacZ staining solution. Testis can be stained as a whole or after dispersing the tubules.

5. Fix and store in 10 % neutral-buffered formalin. If sections are analyzed use neutral fast red as counterstain.

1. The amount described is for digesting two donor mouse testes. Scale the solution accordingly if digestion of more or fewer testes is intended.

2. Handle Busulfan with caution. Measure and dissolve Busulfan power in a chemical hood. Allow Busulfan to complete dis-solve in DMSO before adding water. Add water very slowing to the Busulfan/DMSO solution. Keep Busulfan solution warm in a preheated block during intraperitoneal injection and discard solution if precipitation is observed.

3. Testes can be stored overnight at 4 °C prior to preparation of pieces for transplantation without affecting the ability of testis to survive and develop after grafting.

4. Castration can also be performed through scrotal incisions. 5. Spermatogenesis and development is variable between xeno-

grafts. Generally, xenografts located closest to the limbs show the best grafting outcome.

6. In some cases gonadotropin supplementation to recipient mice may be required to improve development and germ cell dif-ferentiation of xenografts. Horse and monkey xenografts show higher development and germ cell differentiation when hCG is applied to recipient mice (16, 17).

7. Individual grafts can be removed surgically by anesthetizing the mouse and removing the graft through a skin incision. However, this may affect the endocrine balance similar to the situation in hemi-castration.

3.2.5. Analysis of Recipient Testes

4. Notes

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8. The seminal vesicles are androgen-dependent accessory sex glands. Their weight is an indicator of the levels of bioactive testosterone in the host mouse. In intact male mice, seminal vesicles weigh 100–300 mg, while in castrated male mice the weight is <10 mg.

9. If the grafts are large (weighing over 100 mg), they should be cut into small pieces to assure sufficient penetration of the fixative.

10. For transplantation to work efficiently, choice and treatment of recipient animals is important. Recipients should be immuno-logically tolerant (either genetically matched to donors or immunodeficient) to the donor testis cells. Recipients should be depleted of endogenous germ cells either by irradiation or by chemotherapeutic drugs such as Busulfan. Alternatively, recipients that are naturally devoid of spermatogenesis (e.g., W/W v mice) can be used without germ cell depletion.

11. Different pipette puller machines have different settings, there-fore, test a few settings. Generally, a setting similar to that used for making enucleation pipettes will work.

12. Break the pulled pipette tip under a dissecting microscope before attaching it to the tubing. Make sure the diameter of the broken tip does not exceed the diameter of the ducts. After loading the pipette, test and adjust the flow of cell suspension on a piece of sterile absorbent paper to ensure that there is no solution leakage without pressure applied. Also, make sure that the pipette is not blocked by cell aggregates.

13. Watch the speed of tubule filling closely under a dissecting microscope. Depending on the total cell number injected and the number of stem cells present in the injected cell suspen-sion, injection volume can be customized. With good practice, a greater than 70 % filling of the tubules can be easily achieved.

References

1. Honaramooz A et al (2002) Sperm from neo-natal testes grafted in mice. Nature 418:778–781

2. Rodriguez-Sosa JR, Dobrinski I (2009) Recent developments in testis tissue xenografting Reproduction 138:187–194

3. Schlatt S, Rodriguez-Sosa JR, Dobrinski I (2011) Testicular xenografting. In: Orwig K, Hermann B (eds) Male germline stem cells: developmental and regenerative potential. Springer/Humana Press, New York, pp 205–225, Chapter 10

4. Brinster RL, Avarbock MR (1994) Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci USA 91:11303–11307

5. Brinster RL, Zimmermann JW (1994) Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 91:11298–11302

6. Nagano M, Avarbock MR, Brinster RL (1999) Pattern and kinetics of mouse donor sper-matogonial stem cell colonization in recipient testes. Biol Reprod 60:1429–1436

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7. Nagano MC (2003) Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice. Biol Reprod 69:701–707

8. Kubota H, Avarbock MR, Brinster RL (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA 101:16489–16494

9. Oatley JM et al (2006) Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci USA 103:9524–9529

10. Nagano M et al (2001) Transgenic mice pro-duced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci USA 98:13090–13095

11. Ryu BY et al (2007) Efficient generation of trans-genic rats through the male germline using lenti-viral transduction and transplantation of spermatogonial stem cells. J Androl 28:353–360

12. Hess RA et al (2006) Mechanistic insights into the regulation of the spermatogonial stem cell niche. Cell Cycle 5:1164–1170

13. Oatley MJ, Racicot KE, Oatley JM (2011) Sertoli cells dictate spermatogonial stem cell niches in the mouse testis. Biol Reprod 84:639–645

14. Dobrinski I, Rathi R (2008) Ectopic grafting of mammalian testis tissue into mouse hosts. In: Xou SX, Singh SR (eds) Methods in molecular biology, germ line stem stem cells. Humana Press, New Jersey, pp 139–148, Chapter 10

15. Rodriguez-Sosa JR, Schlatt S, Dobrinski I (2011) Testicular tissue transplantation for fer-tility preservation. In: Seli E, Agarwal A (eds) Fertility preservation: emerging technologies and clinical applications. Springer, New York, Chapter 25

16. Rathi R et al (2006) Germ cell development in equine testis tissue xenografted into mice. Reproduction 131:1091–1098

17. Rathi R et al (2008) Maturation of testicular tissue from infant monkeys after xenografting into mice. Endocrinology 149:5288–5296

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