Follicular development in cryopreserved Common Wombat ovarian tissue

Follicular development in cryopreserved Common Wombat ovarian tissue xenografted to immunodeficiency rats (Wolvekamp. M et al, 2001) Australia

Wombat: • 3 species: – Northern Hairy-nosed Wombat (Lasiorhinus krefftii): highly endangered species – Southern Hairy-nosed Wombat (Lasiorhinus latifrons) – Common Wombat (Vombatus ursinus) Northern Hairy-nosed Wombat Common Wombat

Northern Hairy-nosed Wombat • Queensland wombat • (highly endangered species)

Aims and applications of the study: • Preserving the female germline of the endangered species. • Solving the problem of egg maturity. • In species that only ovulate one egg at a time (as in northern. Hairy-nosed Wombat). • And since breeding up mature eggs using in vitro techniques has a very low success rate, the researchers turned to xenografting.

Materials and Methods: • 3 dead wombats Wombat Hours after death at the time of collection (hr) Wombat 1 18 Wombat 2 3. 5 Wombat 3 <1 • immunodeficiency mice.

Method: • Collection of ovarian tissue • Cryopreservation and thawing of ovarian tissue • Surgery • Graft recovery • Histology

Step 1: Ovarian tissue collected from the cortex of wombat ovary cortex medulla

Step 2: Cryopreservation and thawing of ovarian tissue Transplantation time • Pieces of 1 mm 3 • Cryopreservation • Thawing (at transplantation time) Cryopreservation process

Step 3: Surgery • Dorsal incision • 3 pecies of cryopreserved wombate ovarian inserted uner kidney capsule of immunodeficiency mouce. • Skin closed Immunodeficient recipient mouse after subcapsular injection of an ovarian cortex tissue strip. The xenograft can be seen underneath the kidney capsule.

after 10 wombats were injected with human chorionic gonadotrophin (h. CG) Step 4: Graft recovery -abdomen was opened -grafts located -disected free with fine forceps using a stereomicroscope Recovered grafts were immediately placed in 10% neutral buffered formalin for histological examination.

Step 5: Histology using knowing method for preparing slide (fixation, embedding, sectioning, and staining).

Results: • Ovarian tissue histology before transplantation: – Wombat 1: showed some signs of cell degeneration. (why? ) – Wombat 2 & Wombat 3: no such signs were present in the tissues

Results: Ovarian tissue histology after transplantation Wk# Histological observations on grafts WK 2 Revascularization, numbers of atretic follicles were found in these grafts. WK 4 Good revascularization, primordial follicles. WK 10 Wombat 1 -Ovarian grafts were all collagen and fibrous-like in appearance. -Only few primordial follicles. -No signs of follicular development were seen. Wombat 2 -Grafts had many primordial follicles present. -Revascularization. -Follicular development. -Atretic follicles. Wombat 3 -Grafts had many primordial follicles present. -Atretic follicles were also found. -Follicular development.

Conclusion: • Advances in ovarian tissue cryopreservation have made it possible for the female germline to be successfully stored. • While performing xenografting, revascularization of the transplanted tissue is another important aspect to create a successful model. • This study also shows that the time between death of the Wombat and actual collection of the ovarian tissue and subsequent cryopreservation is of uttermost importance.