1. c. Dead blastomeres (propidium iodide)
Secretome derived from different cell lines in bovine in vitro embryo production
Lange Consiglio A.1, Perrini C.1, Sánchez A.2, Esposti P.1, Compagnoni D.1, Guarneri P.L.2, Cremonesi F.1
1Reproduction Unit, Large Animal Hospital, Università degli Studi di Milano, Italy; 2Embryovet, Practitioner Association, Brescia, Italy
AIMS
Hypotizing that CM could supply a more physiological environment for the embryos by MVs or SN, and knowing that endometrium and amnion have a role in the establishment of pregnancy, aim of this study was to evaluate the effect of endometrial and amniotic CM, SN and MVs on IVP embryos in terms of blastocyst rate and embryo quality.
Co-culture systems are largely used to obtain embryos with a higher number of blastomeres and a better implantation rate [1]. This suggests that paracrine mechanisms of communication exist between “helper” cells and embryos. Conditioned media (CM), composed of soluble factors secreted by cells during their culture, were developed and used in bovine IVP. CM is composed of microvesicles (MVs) and soluble factors that are present in the supernatant (SN) after removal of MVs.
MATERIALS & METHODS
Amniotic and endometrial cells in vitro culture.
CM production and SN and MVs isolation
Oocites collection, in vitro maturation and fertilization.
Embryo culture with SOFaa until day 5.
Culture conditions from day 5:
SOFaa (CTR)
SOFaa + 10% amniotic or endometrial CM
SOFaa + 10% amniotic or endometrial SN
SOFaa + 108/ml amniotic or endometrial MVs
Blastocyst rate and embryo quality evaluation at day 7 (Hoechst 3342 and propidium iodide staining)
RESULTS a b c
Culture Conditions
Blast. rate (%)
Viable blastomeres
SOFaa (CTR)
35,45 ± 2,53
62,36 ± 8,44
SOFaa + amniotic MC
34,17 ± 3,29
81,40 ±4,83
SOFaa + amniotic SN
25,80 ± 2,83
50,33 ± 4,54
SOFaa + amniotic MVs
32,82 ± 3,26
72,42 ± 3,27
SOFaa + endometrial MC
21,69 ± 1,87
37,41 ± 4,63
SOFaa + endometrial SN
27,16 ± 1,92
25,29 ± 2,71
SOFaa + endometrial MVs
13,70 ± 2,05
35,98 ± 2,74 b b a c d d e a d c b
a. Embryo at day 7
b. Blastomeres
(Hoechst 3342)
c. Dead blastomeres (propidium iodide)
d. Merged image
CONCLUSIONS
Embryo culture in SOF supplemented with amniotic CM or MVs on day 5 of IVC results in a blastocyst rate similar to the control medium, but an improvement in embryo quality is detected due to the higher number of viable blastomeres. This suggests that amniotic secretome supplies factors that may have contributed to the achievement of better quality embryos.
REFERENCES:
[1] Rieger et al. Comparison of the effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos. J Reprod Fertil :91-98