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Slow freezing, but not vitrification supports complete spermatogenesis in cryopreserved, neonatal sheep testicular xenografts.

Pukazhenthi BS, Nagashima J, Travis AJ, Costa GM, Escobar EN, França LR, Wildt DE - PLoS ONE (2015)

Bottom Line: The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly.Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue.Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa.

View Article: PubMed Central - PubMed

Affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, Virginia, United States of America.

ABSTRACT
The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly. The purpose of this study was to determine, for the first time, the ability of two substantially different cryopreservation approaches, slow freezing versus vitrification, to preserve testicular tissue of the neonatal sheep and subsequently allow initiation of spermatogenesis post-xenografting. Testis tissue from four lambs (3-5 wk old) was processed and then untreated or subjected to slow freezing or vitrification. Tissue pieces (fresh, n = 214; slow freezing, then thawing, n = 196; vitrification, then warming, n = 139) were placed subcutaneously under the dorsal skin of SCID mice and then grafts recovered and evaluated 17 wk later. Grafts from fresh and slow frozen tissue contained the most advanced stages of spermatogenesis, including normal tubule architecture with elongating spermatids in ~1% (fresh) and ~10% (slow frozen) of tubules. Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue. Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa. Although a first for any ruminant species, findings also illustrate the importance of preemptive studies that examine cryo-sensitivity of testicular tissue before attempting this type of male fertility preservation on a large scale.

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Histological appearance of immature lamb testis tissue exposed to one of three conditions.(A) control (fresh), (B) slow freezing and then thawing or (C) vitrification and then thawing (400x). Inset in each panel depicts the seminiferous cords (SC) at a higher magnification. Slow freezing preserved normal SC integrity similar to the fresh control, whereas vitrification caused disruptions, including evidence of shrinkage around the SC (*). G = gonocyte, S = Sertoli cell, M = peritubular myoid cell. Scale bar = 100 μm.
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pone.0123957.g002: Histological appearance of immature lamb testis tissue exposed to one of three conditions.(A) control (fresh), (B) slow freezing and then thawing or (C) vitrification and then thawing (400x). Inset in each panel depicts the seminiferous cords (SC) at a higher magnification. Slow freezing preserved normal SC integrity similar to the fresh control, whereas vitrification caused disruptions, including evidence of shrinkage around the SC (*). G = gonocyte, S = Sertoli cell, M = peritubular myoid cell. Scale bar = 100 μm.

Mentions: After storage and at the time of grafting, the tissue pieces were comprised of seminiferous cords containing virtually only Sertoli cells, gonocytes and interstitial tissue (Fig 2A). Structural integrity and architecture were comparable between the fresh control and the slow freezing and thawing groups as revealed in Fig 2A and 2B. By contrast, thawed vitrified samples displayed moderate disruptions, including shrinkage of Sertoli cells within the seminiferous cords and an increase in interstitial space indicative of freeze-thaw induced damage (Fig 2C).


Slow freezing, but not vitrification supports complete spermatogenesis in cryopreserved, neonatal sheep testicular xenografts.

Pukazhenthi BS, Nagashima J, Travis AJ, Costa GM, Escobar EN, França LR, Wildt DE - PLoS ONE (2015)

Histological appearance of immature lamb testis tissue exposed to one of three conditions.(A) control (fresh), (B) slow freezing and then thawing or (C) vitrification and then thawing (400x). Inset in each panel depicts the seminiferous cords (SC) at a higher magnification. Slow freezing preserved normal SC integrity similar to the fresh control, whereas vitrification caused disruptions, including evidence of shrinkage around the SC (*). G = gonocyte, S = Sertoli cell, M = peritubular myoid cell. Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414479&req=5

pone.0123957.g002: Histological appearance of immature lamb testis tissue exposed to one of three conditions.(A) control (fresh), (B) slow freezing and then thawing or (C) vitrification and then thawing (400x). Inset in each panel depicts the seminiferous cords (SC) at a higher magnification. Slow freezing preserved normal SC integrity similar to the fresh control, whereas vitrification caused disruptions, including evidence of shrinkage around the SC (*). G = gonocyte, S = Sertoli cell, M = peritubular myoid cell. Scale bar = 100 μm.
Mentions: After storage and at the time of grafting, the tissue pieces were comprised of seminiferous cords containing virtually only Sertoli cells, gonocytes and interstitial tissue (Fig 2A). Structural integrity and architecture were comparable between the fresh control and the slow freezing and thawing groups as revealed in Fig 2A and 2B. By contrast, thawed vitrified samples displayed moderate disruptions, including shrinkage of Sertoli cells within the seminiferous cords and an increase in interstitial space indicative of freeze-thaw induced damage (Fig 2C).

Bottom Line: The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly.Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue.Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa.

View Article: PubMed Central - PubMed

Affiliation: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, Virginia, United States of America.

ABSTRACT
The ability to spur growth of early stage gametic cells recovered from neonates could lead to significant advances in rescuing the genomes of rare genotypes or endangered species that die unexpectedly. The purpose of this study was to determine, for the first time, the ability of two substantially different cryopreservation approaches, slow freezing versus vitrification, to preserve testicular tissue of the neonatal sheep and subsequently allow initiation of spermatogenesis post-xenografting. Testis tissue from four lambs (3-5 wk old) was processed and then untreated or subjected to slow freezing or vitrification. Tissue pieces (fresh, n = 214; slow freezing, then thawing, n = 196; vitrification, then warming, n = 139) were placed subcutaneously under the dorsal skin of SCID mice and then grafts recovered and evaluated 17 wk later. Grafts from fresh and slow frozen tissue contained the most advanced stages of spermatogenesis, including normal tubule architecture with elongating spermatids in ~1% (fresh) and ~10% (slow frozen) of tubules. Fewer than 2% of seminiferous tubules advanced to the primary spermatocyte stage in xenografts derived from vitrified tissue. Results demonstrate that slow freezing of neonatal lamb testes was far superior to vitrification in preserving cellular integrity and function after xenografting, including allowing ~10% of tubules to retain the capacity to resume spermatogenesis and yield mature spermatozoa. Although a first for any ruminant species, findings also illustrate the importance of preemptive studies that examine cryo-sensitivity of testicular tissue before attempting this type of male fertility preservation on a large scale.

Show MeSH
Related in: MedlinePlus