Hiroyuki Kaneko, Kazuhiro Kikuchi, Michiko Nakai

Animal Development and Differentiation Research Unit, Transgenic Pig Research Unit

［Abstract］

We successfully produced live piglets by intracytoplasmic sperm injection (ICSI) using sperm from immature testicular tissues after long-term cryopreservation and transplantation into nude mice.

［Keywords］

cryopreservation, immature testis, xenografting, spermatogenesis, ICSI, offspring, pigs

［Background］

Sperm collected from sexually mature males have been utilized for animal production. However animals with genetic modifications usually result in neonatal lethality and could not be used for conventional collection of sperm. Grafting of testicular tissue from a donor into recipient mice (xenografting) is a promising strategy for harvesting mature sperm from spermatogonia present in the testes of young males. In order to develop a new reproductive system using sperm obtained from young males that have not yet reached sexual maturation, we examined whether cryopreserved xenogeneic sperm from immature pig testicular tissue would have the capacity to produce live piglets.

［Results and Discussion］

1. In this study, the testicular tissue from neonatal piglets was cryopreserved after vitrification and xenografted into nude mice. A single sperm obtained from a mouse was injected into an in vitro-matured porcine oocyte. The ICSI oocytes were then transferred to the oviducts of estrus-synchronized gilts to assess the full-term fetal development of the oocytes (Fig. 1).
   
2. A clear structural degradation with distinct spermatogonia was not observed in the tubules of testicular tissue even after long-term cryopreservation of more than 140 days. Sperm appeared in the tubules after 180 days of grafting (day 0 = grafting) (Fig. 2)
   
3. Many porcine sperm were isolated from the testicular grafts after 200 days of grafting (Fig. 3).
   
4. After the ICSI oocytes were transferred to the oviducts of estrus-synchronized gilts, two out of eight gilts gave birth producing 4 male and 3 female piglets (Fig. 4).
   
5. This new system consisting of cryopreservation, xenografting and ICSI allows us to generate offspring from neonatal males when offspring production is desirable.

1. Pigs produced by the new system have undergone a normal maturation process and acquired adequate ability to produce offspring.
   
2. This system is applicable to preservation and multiplication of genetically modified pigs as well as other animal species.
   
3. Cryopreservation of testicular tissue is another option that enables us to conserve genetic information of rare breeds of pigs.

Fig.1. New reproductive system involving cryopreservation, xenografting and intracytoplasmic sperm injection (ICSI).

Fig.2. Images of porcine neonatal testicular tissues before and after cryopreservation, and the cryopreserved tissue after xenografting. Yellow and red arrows indicate spermatogonia and sperm, respectively.

Fig.3. Images of porcine neonatal testicular tissues before and after cryopreservation, and the cryopreserved tissue after xenografting. Yellow and red arrows indicate spermatogonia and sperm, respectively.	Fig.4. Generation of piglets after ICSI using sperm from porcine cryopreserved xenografts.

 

[Reference]

1. Kaneko H, Kikuchi K, Nakai M, Somfai T, Noguchi J, Tanihara F, Ito J, Kashiwazaki N (2013) Generation of live piglets for the first time using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice PLoS ONE 8(7):e70989
   
2. Kaneko H, Kikuchi K, Tanihara F, Noguchi J, Nakai M, Ito J, Kashiwazaki N (2014) Normal reproductive development of pigs produced using sperm retrieved from immature testicular tissue cryopreserved and grafted into nude mice Theriogenology 82:325-331