Z-LEHD-FMK | Sphk Signal

Caspase-9 inhibitor Z-LEHD-FMK enhances the yield of in vitro produced buffalo (Bubalus bubalis) pre-implantation embryos and alters cellular stress response
N. Mullani, M.K. Singh, A. Sharma, K. Rameshbabu, R.S. Manik, P. Palta, S.K. Singla, M.S. Chauhan ⁎
Embryo Biotechnology Laboratory, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal 132001, India

a r t i c l e i n f o

Article history:
Received 4 July 2014
Received in revised form 2 November 2015
Accepted 12 November 2015

Keywords: Buffalo Oocyte Embryo Stress Caspase-9
Z-LEHD-FMK
a b s t r a c t

The present investigation was done to study the effect of caspase-9 inhibitor Z-LEHD-FMK, on in vitro produced buffalo embryos. Z-LEHD-FMK is a cell-permeable, competitive and irreversible inhibitor of enzyme caspase-9, which helps in cell survival. Buffalo ovaries were collected from slaughterhouse and the oocytes were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The culture medium was sup- plemented with Z-LEHD-FMK at different concentrations i.e. 0 μM (control), 10 μM, 20 μM, 30 μM and 50 μM dur- ing IVM and IVC respectively. After day-2 post-insemination, the cleavage rate was significantly higher (74.20 ± 5.87% at P b 0.05) in the group treated with 20 μM of Z-LEHD-FMK than at any other concentration. Same trend was observed in the blastocyst production rate which was higher at 20 μM (27.42 ± 2.94% at P b 0.05). The blas- tocysts obtained at day-8 of the culture at different concentrations were subjected to TUNEL assay, to determine the level of apoptosis during the culture medium supplied with 20 μM Z-LEHD-FMK which showed apoptotic index significantly lower (1.88 ± 0.87 at P b 0.05). There was a non-significant increase in total cell number in all Z-LEHD-FMK treated blastocysts. The quantitative gene expression of CHOP and HSP10 genes showed signifi- cant increase (P b 0.05) in the group treated with 50 μM Z-LEHD-FMK, while, HSP40 showed significant increase (P b 0.05) at 30 μM and 50 μM Z-LEHD-FMK concentrations. From the afore mentioned results we conclude that, Z-LEHD-FMK at 20 μM increased the cleavage and blastocyst rate of buffalo pre-implantation embryos also affect- ing the rate of apoptosis and cellular stress at various concentrations.

⦁ Introduction

India possesses the richest source of germplasm and the best breeds of buffalo in the world which contributes over 55% of total milk produc- tion. Only 0.1% of buffaloes have been reported to produce 3500 to 4000 kg of milk in 305-day lactation period (Misra et al., 1990).The assisted reproductive technologies essentially focused on conservation and multiplication of superior germplasm. In vitro production of buffalo embryos is an extraordinary and economical way of producing the em- bryos for further experiments at cellular, molecular and developmental level (Nandi et al., 2006). The raw material for the technique can be ob- tained from slaughterhouse or by obtaining them from elite animals through ultra sound guided ovum pick-up (Manik et al., 2006). Al- though various advances in the culture conditions with different sup- plements in embryo culture media were developed, the problem of low blastocyst yield has still persists.
Besides, caspases or cysteinyl aspartate proteases are involved in initi- ating caspase cascade pathway as well as cell proliferation, adhesion, dif- ferentiation. Caspase-9 is reportedly expressed in a wide variety of fetal

⁎ Corresponding author.
E-mail address: [email protected] (M.S. Chauhan).
and adult human tissues (Kuida, 2000). Caspase-9 alongside caspase-2 are present in the mitochondria as a zymogen, which get activated and redistributed in the cytosol after the disruption of the outer mitochondrial membrane (Susin et al., 1999) as a part of apoptosis pathway.
Further, varieties of synthetic caspase inhibitors act as pseudo sub- strates and are competitive inhibitors. The linking of appropriate pep- tides like LEHD/IETD to ﬂuoro- or chloro-methyl ketone (−FMK,
−CMK,) groups gives irreversible, competitive inhibitors (Ekert et al.,
1999). Z-Leu-Glu (O-Me)-His-Asp (O-Me) ﬂuoromethyl ketone triﬂuoroacetate salt hydrate (Z-LEHD-FMK) is a cell permeable, compet- itive and irreversible inhibitor of caspase. Ozoren et al. (2000) reported Z-LEHD-FMK when supplemented along with tumor necrosis factor- related apoptosis-inducing ligand (TRAIL), inhibited TRAIL-induced ap- optosis in normal cells whereas the cancer cells were affected with the same. Loureiro et al. (2007) reported that using Z-LEHD-FMK on heat stressed bovine embryos, decreased the number of TUNEL positive cells in both heat stressed and TNF-α induced apoptotic cells.
A wide range of external and internal stressors including environ- mental and hormonal changes incites a stress response within a cell to counteract stress known generally as cellular stress response. Stress re- sponse has been widely studied in organelles like endoplasmic reticu- lum and recent studies show that mitochondria also are equipped

http://dx.doi.org/10.1016/j.rvsc.2015.11.008

with cellular stress response machinery (Zhao et al., 2002). Factors like, mutation in the mtDNA, heat, ethidium bromide treatment, electron transport chain (ETC) protein mutation or uncoupling of OXPHOS and physiological stimuli-induced accumulation of reactive oxygen species (ROS) can either disrupt membrane potential or increase the load of misfolded proteins interfering with mitochondrial homeostasis and function. This activates mitochondrial unfolded protein response (UPRmt) where certain genes and enzymes play a functional role leading to apoptosis (Zhao et al., 2002).
C/EBPβ Homologous Protein (CHOP) codes for a transcription factor C/EBPβ. Elevated levels of CHOP are during ER and mitochondrial stress responses (Zhao et al., 2002; Schroder, 2008). CHOP is also expressed during early embryogenesis (Fawcett et al., 1996; Horndasch et al., 2006).
HSP10, HSP60, HSP40 (chaperone DnaJ) and CLPP (an ATP-dependent protease) are mitochondrial stress target proteins, that get upregulated with the induction of mitochondrial stress (Zhao et al., 2002). Neuer et al. (2000) reported the expression of HSP60 at different developmen- tal stages in mice. DNAJ is expressed in varied organisms ranging from bacteria to humans (Caplan et al., 1993; Qiu et al., 2006).
Caspase-3 is an enzyme which is involved in apoptosis and interacts with caspase-8 and caspase 9. An increase in the expression of CASP-3 was observed when buffalo preimplantation embryos were subjected to oxidative and heat stress, (Elamaran et al., 2012; Yadav et al., 2013). By examining the above information we have observed that there are a few reports available on the effect of caspase-9 inhibitor Z- LEHD-FMK in in vitro embryo production and there was no attainable report on how supplementation of Z-LEHD-FMK possibly affects cellular
stress response in buffalo pre implantation embryos.

⦁ Materials and methods

⦁ Reagents and media

The chemicals and media that were used during the present study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). TCM- 199 used in the experiments was supplemented with HEPES. In vitro culture medium RVCL was obtained from Research Vitro Cleave, Cook, Brisbane, Australia. The disposable 35 mm × 10 cell culture Petri dishes were procured from Nunc (Roskilde, Denmark). The 15 ml and 50 ml Falcon tubes, 100 mm × 100 mm square Petri dishes were purchased from Becton, Dickinson and Co., Lincoln Park, NJ, USA. Syringe filters of the pore size 0.22 μm were purchased from Millipore Corp., Bedford, MA, USA while the syringes were from Henke Saas Wolf GmBH, Tuttingen, Germany.

⦁ In vitro embryo production

⦁ Collection and in vitro maturation of oocytes
Buffalo ovaries were collected from slaughterhouse and carried in 0.9% saline solution antibiotic fortified (400 I.U/ml penicillin and 500 μg/ml streptomycin), and were transported to the laboratory within of 6 h of the slaughter. Oocytes were collected by aspirating the surface follicles (2–8 mm diameter) with an 18 gauge needle attached to a 10 ml syringe containing the aspiration medium (TCM-199 + 0.3% BSA + 0.68 mM L-glutamine + 50 μg/ml gentamycin sulfate). The oo- cytes were searched under a zoom stereomicroscope at around 20 × magnification. The oocytes were then transferred to 35 mm Petri dishes containing the washing medium (TCM-199 + 10% fetal bovine serum (FBS) + 0.81 mM sodium pyruvate + 0.68 mM L-glutamine + 50 μg/ ml gentamicin sulfate). Oocytes with N 2 layers of cumulus cells and ho- mogenous cytoplasm were considered to be of usable quality and used for in vitro maturation (IVM). The oocytes were washed six times in washing medium (TCM-199 + 10% FBS + 0.81 mM sodium pyruvate + 50 μg/ml gentamicin sulfate), then twice with the IVM me- dium (TCM-199 + 10% FBS + 5% follicular ﬂuid + 1 μg/ml estradiol-
17β + 5 μg/ml pFSH + 0.81 mM sodium pyruvate + 0.68 mM glutamine + 50 μg/ml gentamicin sulfate). For IVM, groups of 18–20 cu- mulus–oocyte complex (COCs) were placed in 100 μl droplets of the IVM medium, overlaid with sterile mineral oil in 35 mm Petri dishes and cultured for 24 h in a humidified CO2 incubator (5% CO2 in air) at
38.5 °C.

⦁ In vitro fertilization and culture
Frozen semen from the same bull of proven fertility in IVF trials was used throughout the study. The spermatozoa were prepared for fertili- zation as described by Chauhan et al. (1998). Brieﬂy, two straws of frozen-thawed buffalo semen were washed twice with the washing Brackett and Oliphant (BO) medium (BO medium containing 10 μg/ml heparin, 137.0 μg/ml sodium pyruvate and 1.942 mg/ml caffeine sodium benzoate). The pellet was re-suspended in around 0.5 ml of the wash- ing BO medium. The in vitro matured oocytes were washed thrice with the fertilization BO medium and transferred to 50 μl droplets (18–20 oocytes/droplet) of the capacitation and fertilization BO me- dium (washing BO medium containing 10 mg/ml fatty acid-free BSA). The spermatozoa in 50 μl of the capacitation and fertilization BO medium (2–4 million spermatozoa/ml) were then added to the droplets containing the oocytes, covered with sterile mineral oil and placed in a CO2 incubator (5% CO2 in air) at 38.5 °C for 16–18 h for IVF. At the end of sperm–oocyte co-incubation, the cumulus cells were removed from the oocytes by gentle pipetting. The oocytes were then washed several times with modified Charles Rosenkrans medium supplemented with amino acids (mCR2aa) containing 0.6% BSA and the presumed zygotes were cultured in the IVC medi- um (RVCL + 1% fatty acid free BSA) alone or in presence of different concentrations of Z-LEHD-FMK on monolayer beds of granulosa cells for up to 8 days post insemination in a humidified CO2 incubator (5% CO2 in air) at 38.5 °C to obtain different embryonic stage i.e. 2-cell (cleaved), morale and blastocyst.

⦁ Assessment of apoptosis by TUNEL assay

For examining the health of the embryos, total cell number and the level of apoptosis in Day 8 blastocysts was determined by TUNEL stain- ing as described previously (Elamaran et al., 2012). Brieﬂy, the embryos were washed three times with PBS containing 0.3% polyvinyl alcohol (PVA) in 4-well dishes and were fixed in 4% paraformaldehyde for 1 h at room temperature. Then embryos were washed three times with PBS + 0.3% PVA to remove any traces of paraformaldehyde. The embry- os were permeabilized by incubation in 0.5% Triton X-100 in PBS for 1 h after which these were incubated with ﬂuorescein isothiocyanate (FITC)-conjugated dUTP and terminal deoxy nucleotidyl transferase (TdT) enzyme for 1 h at 37 °C in the dark. The embryos were then treated with 50 μg/ml RNase at room temperature for 1 h, and were counterstained with 10 μg/ml propidium iodide for 20 min at 37 °C. Along with the treatment groups, positive and negative controls were also processed. For the positive controls, the blastocysts were incubated in DNase solution (100 U/ml) for 20 min at 37 °C prior to incubation with FITC-conjugated dUTP and TdT. The stained blasto- cysts were washed with DPBS then mounted on glass slides in 3 μl droplets of anti-fade solution and were ﬂattened with a cover slip. The images were captured at both red and green filters for examining the nuclei and the site of apoptosis, respectively. The merged images generated from red and green filters showing yellow bodies on the exact site of green nuclei were considered as apoptotic cells. Cell counting was performed from digital images obtained on inverted Nikon ﬂuorescence microscope. The total apoptotic indices were calculated for each embryo as follows: Apoptotic index of blasto- cyst = (number of TUNEL-positive nuclei / total number of nuclei in blastocyst) × 100.

Table 1
Primer sequence and conditions for real time PCR.

Gene Primer sequence Prod size (bp) Annealing temp. (°C) Accession no.
DDIT3 F-TGAAAGCAGAGCCTGATCCA 176 58 NM_001078163.1

HSP60 R-TTGATTCTTCCTCCTTGTTTCCAG
F-ACTGGCTCCTCATCTCACTC
146
56
NM_001166610.1
R-TGTTCAATAATCACTGTCCTTCCC
HSP10 F-GAGTATTAGTTGAAAGAAGTGCGG 198 58 NM_174346.2

DnaJ R-ACTTTGGTGCCTCCATATTCTG
F-CACCATCCAGATCGACACAG
151
58
NM_001205976.1

ClpP R-ATGCCCTCTTCATCAAGGTC
F-AGCTCTACAGCATCTACGCC
124
58
NM_001046414.2

CASP-3 R-CAGAACCTTGTCCAAGATGCC
F-GACCATAGCAAAAGGAGCAG
220
60
NM_001077840.1
R-CCTCAGCACCACTGTCTGTC
GAPDH F- TCAAGAAGGTGGTGAAGC R- CCCAGCATCGAAGGTAGA 122 58 GU324291.1

⦁ Quantitative expression of genes

⦁ RNA isolation and cDNA synthesis
RNA was isolated from embryos by using the RNAqueous Micro Kit (Ambion, TX, USA) according to the manufacturer’s instructions with slight modifications. Brieﬂy, the 10–12 blastocysts from different groups were lysed with the lysis buffer after which RNA was eluted with col- umn after several washings with the wash solutions. The genomic DNA contamination was removed by DNase treatment at 37 °C for 20 min. Before cDNA synthesis, the concentration of RNA was measured and set at 20 ng/μl for embryos.
The cDNA was synthesized using Superscript III first strand synthesis system for RT-PCR (Invitrogen, CA, USA) according to the manufacturer’s instructions. For cDNA synthesis 1 μl of total RNA (20 ng), 1 μl dNTP mix (10 mM), 1 μl oligodT (50 μM), and DEPC treated water was added to make volume up to 10 μl. Then the mixture was heated at 65 °C for 5 min and the reaction was quenched by placing the tube immediately on ice for 1 min. Then 2 μl 10 × RT buffer, 4 μl 25 mM MgCl2,2 μl 0.1 mM DTT, 1 μl RNaseOUT and 1 μl superscript RT enzyme were added. The reaction was incubated at 50 °C for 50 min followed by 85 °C for 5 min. The reaction was chilled on ice and then 1 μl RNase H was added and incubated for 20 min at 37 °C. The cDNA prepared was stored at −20 °C until use in Real-Time PCR. For confir- mation of cDNA synthesis, GAPDH was amplified at each stage as the house keeping marker gene. The PCR cycle included denaturation (at 94 °C for 30 s) followed by repeated cycles of denaturation (at 94 °C for 30 s), annealing (at the temperature as indicated in Table 1 for 30 s), and extension (at 72 °C for 45 s) followed by a final extension (at 72 °C for 10 min). The synthesized cDNA was validated by agarose gel electrophoresis (AGE).
⦁ Real time quantiﬁcation
The relative quantification of mRNA of various genes with their cor- responding primers from Sigma Chemical Co. (St. Louis, MO, USA), (Table 1) was done, by using CFX96 Real time system (BIO-RAD, CA, USA). The cDNA from control and the entire treatment groups was used for gene expression study and GAPDH was used as reference gene for all experiments. The qRT-PCR reactions were performed using the SYBER green (double stranded DNA-specific ﬂuorescent dye) master mix (BIORAD, CA, USA). Each run was performed in dupli- cate in a 10 μl reaction volume which contained 5 μl ﬂuorescence dye,
t
0.6 μl of gene specific primers (forward and reverse) from 10 μM stock and 1 μl template. The final volume was made up with nuclease-free water. The PCR condition used for all genes was as follows: initial dena- turation at 95 °C for 3 min, 40 cycles (denaturation 95 °C for 10 s, an- nealing (Table 1) for 10 s, and extension at 72 °C for 10 s), melting cycle starting from 65 °C up to 95 °C with a 0.5 °C/s transition rate. The qRT-PCR specificity was confirmed by the analysis of the melting curve shown by machine by CFX Manager Software. During data analy- sis, the Ct value of housekeeping gene was subtracted from the Ct value of target gene to obtain change in Ct (ΔCt). The Δ Ct value of target gene sample was subtracted from the calibrator (control) to get ΔΔ Ct values. Difference in the transcript abundance for the target genes was calculat- ed using the equation 2−ΔΔC .

⦁ Experimental design

Three experiments were performed to research the objectives of the present study. For each experiment, the immature oocytes were divided into groups based upon the different concentrations of Z- LEHD-FMK.

Table 2
Effect of Z-LEHD-FMK supplementation on developmental competence of buffalo oocytes.

Z-LEHD-FMK No. of oocytes (n) % cleavage rate (n) % Morula (n) % blastocyst (n)
(μM)
0 (control) 249 46.26 ± 5.11a 27.06 ± 5.15a 19.72 ± 2.18a

10
250 (112)
65.73 ± 7.86a,b (26)
33.67 ± 4.11a (21)
21.46 ± 1.79a,b

20
276 (167)
74.20 ± 5.87b (59)
37.52 ± 2.33a (37)
27.42 ± 2.94b

30
208 (205)
45.53 ± 4.22a (75)
35.96 ± 6.11a (58)
17.47 ± 0.99a

50
215 (87)
52.65 ± 9.29a (30)
23.74 ± 4.39a (15)
15.68 ± 2.45a
(107) (26) (17)
All values are mean ± S.D. (n = 4).
Values with different superscripts within the same column differ significantly (P b 0.05).

In Experiment 1, in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were carried out without the addition of Z-LEHD-FMK in the maturation and IVC media (Control), while treat-
Table 3
Effect of Z-LEHD-FMK supplementation on total cell number and level of apoptosis in embryos.

ment were given at 10 μM, 20 μM, 30 μM and 50 μM concentrations re- spectively during IVM and IVC. In all the groups, IVF was carried out without the addition of Z-LEHD-FMK.
In Experiment 2, the apoptotic index and total cell number of blasto- cysts of all the groups from experiment 1 was determined by TUNEL assay.
In Experiment 3, the relative abundance of mRNA of cellular stress
Concentration of Z-LEHD-FMK
Total number of cells in blastocyst
Apoptotic index

0 μM (control) 138.75 ± 12.74 3.65 ± 0.79a,b
10 μM 160 ± 10.00 5.93 ± 1.28b,c
20 μM 160 ± 30.77 1.88 ± 0.20a
30 μM 158.75 ± 16.37 2.80 ± 1.55a
50 μM 158.33 ± 13.64 7.75 ± 2.30c

response genes (CHOP, CLPP, HSP10, HSP40 and HSP60) and CASPASE-3 was compared among the blastocysts in different experimental groups by real time PCR.

⦁ Statistical analysis

The percentage data was normalized using arcsine transformation. With the help of one way ANOVA significant differences in treatment level was determined at (P b 0.05). Once the treatment effect was found significant Duncan’s Multiple Range Test was done to know the differences among treatment means. The data was analyzed using SPSS16.0 (SPSS Inc. Chicago, IL, USA).

⦁ Results

⦁ Effect of Z-LEHD-FMK supplementation on in vitro production of blastocyst

On day 2, at concentration 20 μM Z-LEHD-FMK, the cleavage rate post insemination was significantly higher (P b 0.05) as compared to control and 10 μM, 30 μM and 50 μM concentrations of Z-LEHD-FMK (Table 2). Rate of morula formation was high at a concentration of 20 μM and increased up to 30 μM of Z-LEHD-FMK. While at 50 μM Z- LEHD-FMK, a decreased rate of morula formation was observed when compared to control. An increase in blastocyst formation rate was ob- served at 10 μM of Z-LEHD-FMK and it was found to be significant (P b 0.05) at 20 μM Z-LEHD-FMK. On the other hand decreased rate of blastocyst formation was observed at a concentration of 30 μM and 50 μM Z-LEHD-FMK compared to control (Table 2).

⦁ Effect of Z-LEHD-FMK supplementation on total cell number and apo- ptosis level in embryos

Total cell number in the blastocyst was increased at all the concen- trations of Z-LEHD-FMK when compared to control. The apoptotic index was significantly high at 50 μM of Z-LEHD-FMK and minimum at 20 μM Z-LEHD-FMK, while the number of apoptotic nuclei did not show any significant difference at the concentrations of 10 μM and 30 μM Z-LEHD-FMK, compared to control (Table 3, Fig. 1).

⦁ Effect of Z-LEHD-FMK supplementation on the relative expression of mitochondrial speciﬁc stress response genes

Embryos treated with different concentrations of Z-LEHD-FMK showed differences in the level of gene expression of important mito- chondrial specific stress response genes.
CHOP/DDIT3 was upregulated by 6.64 folds at a concentration of
50 μM and slightly increased at 30 μM and 10 μM of Z-LEHD-FMK when compared to control. Whereas no significant difference in expres- sion of CLPP gene at concentrations 10 μM, 20 μM and 30 μM was ob- served, however it increased at a higher concentration of 50 μM (Fig. 2). At the concentration of 20 μM and 30 μM of Z-LEHD-FMK, HSP10 was upregulated, in the treated embryos, while at 50 μM of Z-LEHD-FMK the expression showed a significant difference (P b 0.05) as compared to control group. The gene HSP40 showed a significant upregulation at 30 μM and50 μM of Z-LEHD-FMK, whereas at lower concentrations of
All values are mean ± S.D. (n = 5).
Values with different superscripts within the same column differ significantly (P b 0.05).

Fig. 1. Detection of apoptotic and total nuclei in blastocyst stage embryos by TUNEL (FITC- conjugated dUTP; green channel) and propidium iodide (red channel), respectively when cultured in medium supplemented with Z-LEHD-FMK at concentration 0 μM (control) (A), 10 μM (B), 20 μM (C), 30 μM (D) and 50 μM (E). A’, B’, C’, D’ and E’ show the respective total cell number.

10 μM and 20 μM, there was no significant difference compared to the gene expression of control group (Fig. 2). During the gene expression of HSP60 gene no significant difference was observed at any concentra- tions as compared to control. However, at a higher concentration of 50 μM of Z-LEHD-FMK, a slight increase in the gene expression was ob- served (Fig. 2).
CASPASE-3 was found to be upregulated in the group of embryos treated with 10 μM, 30 μM and 50 μM Z-LEHD-FMK and a slight decrease at 20 μM concentration of Z-LEHD-FMK was observed when compared to control (Fig. 2).

⦁ Discussion

Z-LEHD-FMK is a cell permeable, competitive and irreversible inhib- itor of caspase-9. In the present study, different concentrations of Z- LEHD-FMK during in vitro maturation of oocytes and subsequently cul- tured the buffalo embryos were used. At 20 μM concentration of Z- LEHD-FMK there was a significant increase in the cleavage and blasto- cyst production rate as compared to control. At the lower concentration also cleavage and blastocyst production rate was increased but at higher concentration rate of production of all the developmental stages was re- duced. Further, addition of the Z-LEHD-FMK to the media reduces the cellular stress and rate of apoptosis which enhanced the embryonic de- velopment. This study showed that lower concentration of Z-LEHD-FMK is better than the higher concentration. Reduced apoptosis leads to the increase in rate of early development and better survival of embryonic cells (Loureiro et al., 2007). Chen et al. (2010) used caspase-9 (Z- LEHD-FMK) and caspase-3 (Z-DEVD-FMK) in curcumin treated mouse blastocysts and found more survival of blastomere cells.
The Z-LEHD-FMK treated embryos showed a higher cell number at all the concentrations when compared to control, while the apoptotic index was minimum at 20 μM, and high (P b 0.05) at 50 μM indicating that addition of Z-LEHD-FMK was favorable at lower concentration for cell survival whereas at higher concentration the embryonic cells were more prone to apoptosis. These results are in agreement with the results of Loureiro et al. (2007) and Chen et al. (2010) which showed that the use of Z-LEHD-FMK in bovine pre implantation embry- os decreased the number of apoptotic cells. In human Z-LEHD-FMK de- creased the TRAIL induced apoptosis in the non-cancerous cells (Ozoren et al., 2000). Uchiyama et al. (2007) used Z-VAD-FMK, a broad- spectrum caspase inhibitor of caspase-8, caspase-3/7, and caspase-9 and reported reduced DNA fragmentation and apoptosis in cells. Inhibi- tion of caspase-9 activity by Z-LEHD-FMK prevented most of the cells from apoptosis of G418 resistant mammalian cells (Taghiyev et al., 2005; Jin et al., 2004).
Mitochondrial specific stress response is caused due to the presence of unfolded proteins in the matrix of the mitochondria. Mitochondrial specific stress response occurs in the mammalian cells which causes the upregulation of specific genes like CHOP, CLPP, mtHSP60, mtHSP70 and DNAJ (Zhao et al., 2002).In this present study it was found that lower concentration of Z-LEHD-FMK, did not affect the CHOP gene ex- pression in blastocyst concord with higher blastocyst rate and lower the apoptotic index, but was upregulated when 50 μM of Z-LEHD-FMK was used. The upregulation of CHOP gene leads to DNA damage, growth arrest, and the induction of apoptosis (Zinszner et al., 1998; Ubeda and Habener, 2003).
Haynes et al. (2007) have shown that CLPP, an ATP dependent prote- ase which is essential for the induction of molecular chaperone genes in response to stress. It was observed that, Z-LEHD-FMK treated groups of embryos at different concentrations showed a slight upregulation of CLPP gene, but did not show any significant difference. Zhao et al. (2002) reported an upregulation of CLPP gene during mitochondrial specific stress response in mammalian cells.
HSP10, HSP40 (DnaJ) and HSP60 are chaperonin proteins that assist
in the protein folding and sorting of the mitochondrial target proteins.HSP10 acts along with the chaperonin HSP60 and helps in the proper folding of the misfolded protein (Jia et al., 2011). HSP40, expressed in varied organisms ranging from bacteria to humans (Caplan et al., 1993; Qiu et al., 2006). HSP60 is localized in the mitochon- dria and it is expressed after ovulation during the implantation phase and fertilization. We observed that at lower concentration of Z-LEHD- FMK the relative abundance of HSP10, HSP40 mRNA was decreased which supports the better health and development of embryos; where- as at 50 μM of Z-LEHD-FMK, expression of HSP10, HSP40 was significant- ly high (P b 0.05) possibly causing the retardation of development, which lead to the lower blastocyst rate and higher apoptotic index. HSP60 has been found to be important in the synthesis and transporta- tion of essential mitochondrial proteins from the cytoplasm into the mi- tochondrial matrix during stress response (Sterrenberg et al., 2011).
Supplementation of 20 μM Z-LEHD-FMK in culture medium reduced the expression of CASPASE-3 while it is slightly upregulated at other concentrations compared to control. Aforesaid results support the bet- ter embryonic health and production rate of embryos in the presence of 20 μM Z-LEHD-FMK.
To the best of the author’s knowledge no work has been done on ef- fect of Z-LEHD-FMK on embryo production in any species. The concen- trations during the designed experiment were decided according to previous studies made majorly on different cancerous cell lines. Results of the present study demonstrated that Z-LEHD-FMK used during in vitro embryo production, decreased the rate of apoptosis which is an incurring problem during in vitro culture of embryos and possibly

Fig. 2. Relative mRNA abundance of CHOP, ClpP, HSP10, HSP40, HSP60 and CASPASE-3 in blastocysts at different concentration of Z-LEHD-FMK. Bars with asterisks differ significantly (P b
0.05).

reduced cellular stress which affects early embryonic development of buffalo embryos. From the observations made in this study, we suggest the use of Z-LEHD-FMK, as media supplement for in vitro embryo devel- opment, so that, the embryos may be obtained in favorable numbers for any subsequent analyses, including real time PCR and staining.

Acknowledgments

The present work was partly funded by the National Initiative on Cli- mate Resilient Agriculture project and National Agriculture Innovative Project, grant Numbers (C 2-1-(5)/2007) and (C-2067 & 075).

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