This study investigated the effects of vitrification on morphology and mRNA expression of apoptotic genes in sheep immature cumulus oocyte complexes. Immature oocytes were collected from sheep ovaries and divided into groups for cryopreservation using different cryoprotectants. The oocytes were evaluated for morphology before and after vitrification and thawing. Real-time PCR was used to analyze expression of apoptotic genes BAD, BAK, BAX, BID, BOK, BCL2A1, BCL2, MCL1 in fresh and vitrified oocytes. Results showed varying recovery and damage rates among oocytes cryopreserved with different cryoprotectants. Morphological changes included cracked or split zona pell

1. CRYO2020: Rosemont ,Illinois U.S.A
EFFECTS OF VITRIFICATION ONMORPHOLOGY AND mRNA EXPRESSION IN
APOPTOTIC GENES IN OVINE IMMATURE CUMULUS OOCYTE COMPLEXES
Satish Kumar, Associate Professor
Maharishi Markandeshwar (deemed to be) University, Mullana, Ambala Haryana ,India

2. Introduction
 Cryobiology is the study of the effects of low temperatures on living organisms.
 In the future, it may be possible to cryopreserve human and animal cells, whole
organs, such as kidneys, hearts and livers for subsequent transplantation,
preserve corneas and other delicate tissues with minimal damage long enough to
allow them to be shared all over the world Oocyte is female gametocyte or germ
cell involved in reproduction.
 Oocyte cryopreservation make possibility of salvaging genetic material from
prepubertal, infertile, pregnant or even from dead animal. Creating oocyte bank
and revival of extinct and endangered species in animals.

3.  Apoptosis is a programmed cell death (PCD) as compared to accidental death of infected transformed or
damaged cells by activation of a specific biochemical pathway.
 In vertebrates, apoptosis can be induced by a number of distinct death inducing stimuli, including the
lack of extracellular survival factors, steroid hormones, activation of membrane bound death receptors,
viral infection, heat shock, oxidative stress, excitotoxicity, ionizing radiation and various other cellular
insults.
 Sheep are placid animals and, because of their size, can easily be handled. Sheep are quite similar in
body weight to humans, and sufficiently large to allow serial sampling and multiple experimental
procedures (Hubrecht and Kirkwood, 2010).
 Researchers have successfully studied fetal circulatory system function, anesthesia, catheterization
techniques, and steroid radio immunoassays with sheep models. Subsequent to this, pregnant ewes
became common subjects in noninvasive studies by ultrasonography, by which the monitoring of fetal
physiology could be performed easily and safely.
Oocyte Cryopreservation

4. Objectives
1. Retrieval of immature cumulus oocyte complexes (COC) from sheep ovaries.
2. Cryopreservation of immature cumulus oocyte complexes (COC) by
vitrification.
3. Comparative morphological studies of cumulus oocyte complexes (COC)
before and after cryopreservation by Zoom stereo microscope
4. Apoptotic gene BAD, BAK,BAX,BID,BOK,BCL2A1,BCL2,MCL1 expression
study in non-vitrified and vitrified thawed cumulus oocyte complexes by Real
Time-PCR using GAPDH as reference genes

5. Solutions for oocyte collection
Normal saline containing antibiotics
Composition - Volume (1000ml)
Sodium chloride - 9.0 gm
Penicillin G - 0.12 gm
Distilled water - 1000 ml
Aspiration medium (for about 250-300 ovaries)
Composition -Volume (100 ml)
TCM-199 (Hepes modified) - 50 ml
DPBS - 50 ml
BSA - 0.3 gm
Gentamycin - 50g/ml
L-glutamine - As recommended by
the manufacturer

6. The aspirated oocytes were graded according to the following criteria already in use in the laboratory:
 Grade-A: Oocytes with homogeneous cytoplasmic granulation, multilayerd unexpanded cumulus cell
layers associated with the oocyte.
 Grade-B: Oocytes with homogeneous cytoplasmic granulation, 3-4 layers of unexpanded cumulus cell
layers associated with the oocyte.
 Grade-C: Oocytes with homogeneous cytoplasmic granulation, with a few or irregularly arranged
unexpanded cumulus cell layers associated with the oocyte. Some of the oocytes with expanded
cumulus cells were also graded as C grade oocytes.
 Grade-D: deformed or irregular ooplasm without no cumulus cells associated. C and D grade oocytes
were not used, though the number was recorded.
 Oocytes of grades A and B were used for experimentation.

7. Experiment
 The immature oocytes were divided into 3 groups after
morphological evaluation for observing effects of
different cryoprotectants and subjected to vitrification.
 Oocytes were thawed at least after three weeks of
cryopreservation for morphological evaluation
 The percentage of recovered oocytes recorded. This
experiment was replicated 6 times.

8. Gene expression study

9. cDNA Synthesis and Primers
 Total RNA was isolated from fresh and cryopreserved sheep oocytes (n=30 each)
using the RNAqueous- Micro Kit (Ambion Inc. )
 cDNA was synthesized by First-strand cDNAs Superscript III, cDNA synthesis
kit (Invitrogen) according to the manufacturer’s instructions.
 Primers used in the present study were designed from highly conserved region
from sheep, goat, bovine sequences available in NCBI databases using Primer 3
software . The primers were synthesized from the Sigma Company, Bangaluru.
 The primer sequences, annealing temperature and sizes of amplified products
are shown in Table 2.

10. Reaction mix components and their
concentrations for cDNA synthesis
S.No. Component Sock Conc. Working conc. Per reaction
volume per reation
(20µl)
1 DNase treated RNA - - 5 μl
2 Random primer 50ng/μl 75ng/μl 1.5μl
3 dNTP’s 10mM 500μM 1μl
4 RNase OUT 40U/μl 40U 1μl
5 Reaction buffer 10X 1X 2μl
6 Mgcl2 25mM 2.5mM 2μl
7 DTT 100mM 2.5mM 0.5μl
8 Reverse Transcriptase 200U/μl 200U 0.5μl
9 DEPC Treated Water - - 6.5μl

11. Accession number of genes
Gene Sheep Goat Bovine
BAD
XM_004019650 XM_005699970 NM_001035459
BAK
XM_004018758 ---- NM_001098868
BAX
XM_004015363 XM_005692695 NM_173894
BID
GAAI0100248 XM_005681052 NM_001075446
BOK
XM_004015786 ---- NM_001098868
BCL2A1
XM_004015786 XM_005698059 NM_001037100
BCL2
XM_004020687 XM_005697325 NM_001166486
MCL1
XM_004002457 XM_005677664 NM_001099206
P53
X81705 XM_005677664 X81704
GAPDH
NM_001190390 XM_005680968 XM_001034034

12. List of primers used in the present study
GENE Sequence Annealing Tem./ Cycles Size
BAD F- CCAGAGCATGTTCCAGATCC
R- GTTAGCCAGTGCTTGCTGAG
60/40 125bp
BAK F- GCTACGACACAGAGTTCCAG
R- TTGATGCCGCTCTCAAACAG
60/40 108bp
BAX F- TGAATTGGACAGTAACATGGAG
R- GAAGGAAGTCCAATGTCCAG
60/40 229bp
BID F- CCTTCATCAACCAGAACCTCC
R- GTCTCAAGCCGTCTTCTCTG
60/40 182bp
BOK F- TTGTGTCTCTGTACTCGGTG
R- CAACAGCATGAAGAAGGCAG
60/40 166bp
BCL2A1 F- ACTGCCAGAACAATATTCAACC
R- GTAAGAATACCTTCAAAGGCGA
60/40 103bp
BCL2 F- CCTTCTTTGAGTTCGGAGGG
R-GTTCAGGTACTCGGTCATCC
60/40 100bp
MCL1 F- ACGATGTCAAGTCTTTGTCTC
R-CTGTGATGCTTTCTGCTAGTG
60/40 164bp
P53* F-GGAAGAATCGCAGGCAGAACT
R-GGAGAGCTCGGAGGACAGAA
60/40 109bp
GAPDH F-CCATACTCAGCCATCAAGGA
R-CTGTAGCCATATTCATTGTCGT
60/40 202bp
* Ebrahimi et.al. 2010

13. Real-time quantitative PCR (qPCR)
 The quantification of mRNA was carried out by using CFX96 real time
system (Bio-Rad, Hercules, CA, USA). For qPCR, the relative
quantification method was used. For this, cDNA of test samples was
randomly diluted in 1:3 ratios.
 Genes were quantified using the CFX 96 thermocycler (BioRad) and
detected using Maxima_ SYBR Green/ROX qPCR Master Mix (2X)
(Fermentas).
 All reactions were run in triplicate, and three biological replicates were
carried out. Relative levels of expression were determined using 2-DDCt

14. Results

15. Objective 1
Retrieval of immature cumulus oocyte complexes
(COC) from sheep ovaries.

16. Oocyte aspiration from sheep ovary

17. Objective 2
Cryopreservation of immature cumulus oocyte
complexes (COC) by vitrification.

18. 0
20
40
60
80
100
%
of
oocytes
Cryoprotectants
Oocytes recovered
Morphologically normal
oocytes n (%)
Damaged oocytes n (%)
Effect of different cryoprotectants on
sheep oocytes

19. Objective 3
Comparative morphological studies of cumulus oocyte
complexes (COC) before and after cryopreservation by Zoom
stereo microscope

20. Types of damages after vitrification of immature oocytes in
different cryoprotectants
0
10
20
30
40
50
60
Cracked ZP
Split In two halves
Leaked contents
Shrunken cytoplasm
Change in Shape
Partly/ Fully denuded

21. Different types of damages after
cryopreservation

22. Excessively damaged zona

23. Shrinkage of ooplasm

24. Splitting of oocyte in two halves

26. THANK YOU