in vitro culture in the presence of amorphous calcium ...cme-utilities.com/mailshotcme/material for...

www.oc2017.cme-congresses.com

In vitro culture in the presence of amorphous calcium

carbonate improves mice embryos compaction and

hatching rates

Amir Arav DVM PhD

Consulting to Amorphical LTD

Examples of CaCO3 occurring in nature: mollusk shells

10µm

calcite vaterite

aragonite

Amorphous Calcium Carbonate

ACC seeds

0.1µm

Polymorphism CaCO3

Calcium carbonate nano-particles have a large number of

applications due to their beneficial properties such as high

porosity, high surface area to volume ratio, non-toxicity and

biocompatibility

Amorphous Calcium Carbonate (ACC)

CaCO3 2HCl Ca CO2 2H2O + + +

CaCO3 will only increase pH in acidic environments

Buffering capacity

ACC Control

Compaction D3 (60h)

Control ACC

Dose dependence of ACC

ACC 17µl/ml ACC 34µl/ml

D5 Hatching blastocysts (110h) - 10-20% more hatching

ACC Control

Cleavage medium (Quinn-SAGE) with ACC or CCC

Arav ESHRE 2017

Arav ESHRE 2017

100% hatching blastocysts in the presence of ACC

Testing SAGE one-step medium

Mouse

#

No. of Embryos % Compaction (60h) % Blastocysts (86h) % F. Hatching

without

ACC With ACC

without

ACC With ACC

without

ACC With ACC

without

ACC With ACC

1 13 13 75 80 75 80 50 60

2 19 19 30 67 30 45 13 100

3 18 16 40 89 80 67 70 78

4 13 14 46 86 30 57 61 100

5 14 11 36 90 18 90 27 90

6 10 20 27 70 55 85 0 35

7 14 22 75 100 37 100 37 100

8 20 10 40 67 20 80 20 80

9 9 11 33 100 25 100 25 100

10 10 15 70 90 80 90 80 100

11 15 19 67 78 67 56 78 100

12 19 18 40 60 20 60 30 70

Testing IRVINE CMC medium

Mouse #

No of Embryos % Compaction (60h) % Blastocysts (86h) % F. Hatching

without

ACC With ACC

without

ACC With ACC

without

ACC With ACC

without

ACC With ACC

1 13 11 69 78 54 55 46 60

2 14 14 57 72 43 72 55 72

3 18 20 69 70 54 71 46 100

4 14 14 0 22 21 22 29 100

5 23 29 73 74 20 28 52 62

6 13 15 20 62 31 87 23 80

7 8 11 12 55 0 34 0 37

8 13 11 69 78 54 55 46 57

9 14 13 36 62 43 38 43 70

10 14 13 36 38 43 48 43 92

11 11 15 55 60 36 27 27 60

12 11 14 55 64 36 36 27 68

Arav ESHRE 2017

Cathepsin- Lysosomal Proteases (A,B,D, K,V…) Most of the members become activated at low pH

Richard Willstätter 1872 –1942 Nobel price 1915

Willstätter & Bamann (1929) Hoppe-Seylers Z. Physiol. Chemie 180, 127-143

Intracellular Cathepsin B Activity Is Inversely Correlated With

the Quality and Developmental Competence of Bovine

Pre-implantation Embryos

A.Z. Balboula, K. Yamanaka, M. Sakatani, A.O. Hegab, S.M.

Zaabel AND M. Takahashi

E-Cadherins (named for "calcium-dependent adhesion") are a class of type-1 transmembrane proteins. They play important roles in cell adhesion, forming adherent junctions to bind cells within tissues together. They are dependent on calcium (Ca2+) ions to function.

E-cadherin as a target substrate of cathepsins B, L, S but not C and S

Amorphous CaCO3

Proposed mechanism

Low pH

Ca++ CO3--

Increase pH Reduce Cathepsin activity

Support embryo compaction

E-cadherin

Arav et al., 2017

Low Glucose and low cathepsin

High Glucose and high cathepsin Oxidative Glycolysis

pH 7.6

pH 6.9

Amorphous calcium carbonate (ACC) at a concentration

of 1.7mM acts as a buffer increasing the compaction,

blastocyst and hatching rates of mice embryos.

Conclusion

Yosi ben

Orit Ecker Cohen

Pliensky Ylena

Yehudit Natan

Otto Warburg 1883-1970 Nobel price 1931

Warburg effect

Science, New Series, Vol. 123, No. 3191. (Feb. 24, 1956), pp. 309-314

Proliferation Migration Invasion

Angiogenesis

Aerobic Glycolysis

H --- pHe (6.5-6.9)

Lysosome Cathepsin

Embryo implantation metabolism

E-Cadherine

Metaloproteinase Glycolysis

Oxidative

Arav et al., 2017

H --- pHe (6.5-6.9)

VEGF

+

Lysosome Cathepsin

Metalloproteinase (MMPS)

Cancer cell

Angiogenesis

Acid mediates invasion

Immune response

APOPTOSIS AND NECROSIS

Normal Tissue

Arav et al., 2017

oncogenic

mutations

P53

Out of 8 transfer with >80 embryos none has produced pregnancies

Blastocyst transfer with ACC