Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage.
The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae. Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), interleukin-6 (1 ng/ml), transforming growth factor (TGF) alpha (2 ng/ml), epidermal growth factor (EGF) (4 ng/ml), platelet-derived growth factor (1 ng/ml), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGFbeta (2 ng/ml). At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis. The following parameters were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining. When contrasted to the medium alone control, co-culture consistently accelerated the development of frozen-thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapidly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achieved with co-culture, all of the growth factors impacted positively on at least one of the morphological parameters studied. Cell proliferation was significantly stimulated by just 48 h exposure to growth factors, either through co-culture or by direct media supplementation. Co-culture again yielded the best results with a mean cell count of 217 +/- 76 cells per blastocyst as compared with 131 +/- 36 in control medium alone. Amongst the factors tested, IGF-I, IGF-II and EGF had the greatest impact, with mean cell counts of 172 +/- 50, 168 +/- 50 and 179 +/- 55 respectively. Whereas only 5% of CT embryos developed to blastocysts with > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25-32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematically describes the effect of culture regimen and growth factor additives on the post-thaw development of cryopreserved embryos.
Desai
N
N
Department of Reproductive Biology, University MacDonald Women's Hospital, Case Western Reserve University, Cleveland, OH 44106, USA.
Lawson
J
J
Goldfarb
J
J
D000818:Animals; D001755:Blastocyst / Q000187:drug effects*; D018920:Coculture Techniques; D015925:Cryopreservation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D006133:Growth Substances / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D051379:Mice; D009028:Morula / Q000187:drug effects*; D013997:Time Factors; D015044:Zona Pellucida / Q000187:drug effects / Q000502:physiology
D006133:Growth Substances; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II