Lab Anim Res.  2016 Dec;32(4):257-266. 10.5625/lar.2016.32.4.257.

Establishment of a surgically induced cryptorchidism canine recipient model for spermatogonial stem cell transplantation

• Lee WY
• Lee R
• Song H
• Hur TY
• Lee S
• Ahn J
• Jhun H

• KMID: 2362918
• DOI: http://doi.org/10.5625/lar.2016.32.4.257

Abstract

Transplantation of spermatogonial stem cells (SSCs) in experimental animal models has been used to study germ line stem cell biology and to produce transgenic animals. The species-specific recipient model preparation is important for the characterization of SSCs and the production of offspring. Here, we investigated the effects of surgically induced cryptorchidism in dog as a new recipient model for spermatogonial stem cell transplantation. Artificially unilateral or bilateral cryptorchidism was induced in ten mature male dogs by surgically returning the testis and epididymis to the abdominal cavity. The testes and epididymides were collected every week after the induction of artificial cryptorchidism (surgery) for one month. To determine the effect of surgical cryptorchidism, the seminiferous tubule diameter was measured and immunohistochemistry using PGP9.5 and GATA4 antibodies was analyzed. The diameters of the seminiferous tubules of abdominal testes were significantly reduced compared to those of the scrotal testes. Immunohistochemistry results showed that PGP9.5 positive undifferentiated spermatogonia were significantly reduced after surgical cryptorchidism induction, but there were no significant changes in GATA-4 positive sertoli cells. To evaluate the testis function recovery rate, orchiopexy was performed on two dogs after 30 days of bilateral cryptorchidism. In the orchiopexy group, SCP3 positive spermatocytes were detected, and spermatogenesis was recovered 8 weeks after orchiopexy. In this study, we provided optimum experimental conditions and time for surgical preparation of a recipient canine model for SSC transplantation. Additionally, our data will contribute to recipient preparation by using surgically induced cryptorchidism in non-rodent species.

Keyword

Spermatogonial stem cell transplantation; cryptorchidism; recipient preparation; canine; orchiopexy

MeSH Terms

Abdominal Cavity
Animals
Animals, Genetically Modified
Antibodies
Biology
Cryptorchidism*
Dogs
Epididymis
Germ Cells
Humans
Immunohistochemistry
Male
Models, Animal
Orchiopexy
Recovery of Function
Seminiferous Tubules
Sertoli Cells
Spermatocytes
Spermatogenesis
Spermatogonia
Stem Cell Transplantation*
Stem Cells*
Testis
Antibodies

Figure

• 

  Figure 1 Surgically induced cryptorchidism and orchiopexy procedures. (A) The testis was pushed out through the paramedial incision. The arrow indicates the inguinal ring. (B) The testicle was introduced into the abdominal cavity to induce experimental cryptorchidism. (C) The inguinal ring was sutured to prevent the normal descent of the testicle. (D) The cryptorchid testicle was moved into the scrotum and permanently fixed there.

• 

  Figure 2 Morphological changes in scrotal (control) and surgically induced cryptorchidism in dog testes. (A) Hematoxylin and eosin staining results at 7, 14, 21, and 30 days after surgery (D7, D14, D21 and D30). (B) Comparison of seminiferous tubule diameter of dog testes in control and experimental groups. Data are presented as mean±SD. ***P<0.01, compared with the control. The scale bars indicate 100 mm.

• 

  Figure 3 Macroscopic and histological comparison of control dog testes and testes subjected to artificial cryptorchidism for 30 days. (A) Comparison of testis size in cryptorchidism and scrotal testis on day 30 after surgery. Representative section of the cauda epididymis in cryptorchid (B) and scrotal (C) testis. C (Left) is a cryptorchid testis and S (Right) is a scrotal testis. Arrow indicates sperm. The scale bars indicate 100 mm.

• 

  Figure 4 Canine spermatogonia as detected by PGP9.5 immunohistochemisty staining. (A) Positive staining for anti-PGP9.5 antibody 7, 14, 21, and 30 days after surgery (D7, D14, D21, and D30). (B) Comparison of positive cell numbers in control and cryptorchidism groups on different days. Arrows indicate PGP9.5 positive cells. Results are expressed as the mean±SD. P<0.05 was considered statistically significant. The scale bars indicate 100 mm.

• 

  Figure 5 Proportion of canine Sertoli cells as detected by GATA4 immunohistochemical staining. (A) Positive staining of GATA4 antibody 7, 14, 21, and 30 days after surgery (D7, D14, D21, and D30). Arrows indicate GATA4 positive cells. (B) Comparison of positive cell numbers in the control cryptorchidism groups on different days. Results are expressed as the mean±SD. P<0.05 was considered statistically significant. The scale bars indicate 100 mm.

• 

  Figure 6 Histological and immunohistochemical analyses of testis and epididymis tissue from testis after orchiopexy (A, B, C and D). (A) H & E staining of testis and (B) epididymis. (C) PGP9.5 and (D) SCP3 staining of testis 4 weeks after orchiopexy. (E) Ejaculated sperm 8 weeks after orchiopexy. The scale bars indicate 100 mm.

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