Whole ovary cryopreservation is currently under study for preserving female fertility but may result in DNA damage, even in the absence of evident morphological changes. We used immunolabelling and image analysis to evaluate the expression of γH2AX (marker of DNA damage) and of RAD51 (marker of DNA repair) molecules that allow a sensitive assessment of cellular damage, and compare the effect of conventional (CF) and directional freezing (DF) on sheep whole ovary. Fresh controls and thawed samples previously subjected to CF or DF freezing, were cut in 2x2x1 mm pieces and cultured for 7 days. They were then fixed in formalin, embedded in paraffin and sectioned. Slides were incubated over-night with primary antibodies and then with fluorescent secondary antibodies for 45 minutes. Sections were observed under an Eclipse-E600 microscope. Images were acquired with Nis-Elements-Software and analyzed using ImageJ-Software. As expected, no γ-H2AX and RAD51 signals were detected in fresh control tissue. Conversely, γ-H2AX signal was observed in frozen samples, immediately after thawing. Its intensity was significantly stronger in CF than in DF samples. After 7 days of culture, γ-H2AX signal decreased in DF ovaries concomitantly with an increase of RAD51. By contrast, CF ovaries showed no signal for RAD51 leaving the rate of DNA damage unchanged. The results obtained show that cryopreservation induces DNA damage with both methods, however, the ability of DF tissue to repair DNA damage demonstrates that this method allows a better preservation than CF. Supported by AIRC IG 10376, by Carraresi Foundation and by RAS-Legge 7

Assessment of cellular damage in sheep ovaries subjected to different freezing methods / S. Maffei, G. Pennarossa, T. Brevini, F. Gandolfi. ((Intervento presentato al 9. convegno Congresso Nazionale Associazione Italiana dei Morfologi Veterinari tenutosi a Roma nel 2013.

Assessment of cellular damage in sheep ovaries subjected to different freezing methods

S. Maffei;G. Pennarossa;T. Brevini;F. Gandolfi
2013

Abstract

Whole ovary cryopreservation is currently under study for preserving female fertility but may result in DNA damage, even in the absence of evident morphological changes. We used immunolabelling and image analysis to evaluate the expression of γH2AX (marker of DNA damage) and of RAD51 (marker of DNA repair) molecules that allow a sensitive assessment of cellular damage, and compare the effect of conventional (CF) and directional freezing (DF) on sheep whole ovary. Fresh controls and thawed samples previously subjected to CF or DF freezing, were cut in 2x2x1 mm pieces and cultured for 7 days. They were then fixed in formalin, embedded in paraffin and sectioned. Slides were incubated over-night with primary antibodies and then with fluorescent secondary antibodies for 45 minutes. Sections were observed under an Eclipse-E600 microscope. Images were acquired with Nis-Elements-Software and analyzed using ImageJ-Software. As expected, no γ-H2AX and RAD51 signals were detected in fresh control tissue. Conversely, γ-H2AX signal was observed in frozen samples, immediately after thawing. Its intensity was significantly stronger in CF than in DF samples. After 7 days of culture, γ-H2AX signal decreased in DF ovaries concomitantly with an increase of RAD51. By contrast, CF ovaries showed no signal for RAD51 leaving the rate of DNA damage unchanged. The results obtained show that cryopreservation induces DNA damage with both methods, however, the ability of DF tissue to repair DNA damage demonstrates that this method allows a better preservation than CF. Supported by AIRC IG 10376, by Carraresi Foundation and by RAS-Legge 7
Settore VET/01 - Anatomia degli Animali Domestici
Associazione Italiana dei Morfologi Veterinari
AMV
http://www.morfovet.it/Abstract%20Book.pdf
Assessment of cellular damage in sheep ovaries subjected to different freezing methods / S. Maffei, G. Pennarossa, T. Brevini, F. Gandolfi. ((Intervento presentato al 9. convegno Congresso Nazionale Associazione Italiana dei Morfologi Veterinari tenutosi a Roma nel 2013.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/227002
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