Growth factor expression pattern of homologous feederlayer for culturing buffalo embryonic stem cell-like cells

Ruchi SharmaA, Aman GeorgeA, Nitin M. KambleA,Manmohan S. ChauhanA,Suresh SinglaA, Radhey S. ManikA and Prabhat PaltaA,B

AEmbryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute,

Karnal-132001, Haryana, India.BCorresponding author. Email: prabhatpalta@yahoo.com

Abstract. The present study examined the expression profile of buffalo fetal fibroblasts (BFF) used as a feeder layer for

embryonic stem (ES) cell-like cells. The expression of important growth factors was detected in cells at different passages.Mitomycin-C inactivation increased relative expression levels of ACTIVIN-A, TGF-b1, BMP-4 and GREMLIN but notof fibroblast growth factor-2 (FGF-2). The expression level of ACTIVIN-A, transforming growth factor-b1 (TGF-b1),bone morphogenetic protein-4 (BMP-4) and FGF-2 was similar in buffalo fetal fibroblast (BFF) cultured in stem cellmedium (SCM), SCMþ 1000 IUmL�1 leukemia inhibitory factor (LIF), SCMþ 5 ngmL�1 FGF-2 or SCMþLIFþFGF-2 for 24 h whereas GREMLIN expression was higher in FGF-2-supplemented groups. In spent medium, theconcentration of ACTIVIN-A was higher in FGF-2-supplemented groups whereas that of TGF-b1 was similar in SCM

and LIFþFGF-2, which was higher than when either LIF or FGF-2 was used alone. Following culture of ES cell-like cellson a feeder layer for 24 h, the TGF-b1 concentration was higher with LIFþFGF-2 than with LIF or FGF-2 alone which, inturn, was higher than that in SCM. In the LIFþ FGF-2 group, the concentration of TGF-b1 was lower and that of

ACTIVIN-Awas higher in spent medium at 24 h than at 48 h of culture. These results suggest that BFF produce signallingmolecules that may help in self-renewal of buffalo ES cell-like cells.

Additional keywords: ACTIVIN-A, fetal fibroblast, mitomycin-C, pluripotency, TGF-b1.

Received 1 December 2011, accepted 23 March 2012, published online 24 April 2012

Introduction

Embryonic stem (ES) cells, which are derived from the inner cellmass of blastocysts, possess unique properties of self-renewaland pluripotency. Following their isolation in mouse (Evans and

Kaufman 1981;Martin 1981) and human (Thomson et al. 1998),ES cell-like cell lines have been established in other mammalianspecies such as rat, rabbit, hamster, mink and rhesus monkey,

and in farm animal species such as sheep, pig, horse, goat, cattleand buffalo. The support of feeder cells is essential for themaintenance of pluripotency by ES cells. Feeder cells maysupport the attachment of ES cells through expression of

adhesion molecules and production of extracellular matrix(ECM) or support the growth and survival of ES cells by pro-duction of growth factors, or both. Though inactivated mouse

embryonic fibroblasts (MEF) have been the most commonlyused type of feeder cells used for the maintenance of ES cellsacross various species, the use of homologous feeder cells has

also been reported in cattle (Mitalipova et al. 2001), pig (Li et al.2004) and buffalo (Verma et al. 2007).

Very few comparative reports are available on the efficacy of

different types of feeder cells to support the self-renewal of EScells from farm animal species. A comparison of the efficacy of

MEF, bovine embryonic fibroblasts (BEF) and mixed

MEFþBEF feeder cells in supporting the growth and mainte-nance of bovine ES cells indicated that the feeder layers derivedfrom mixed cells were more efficient than those derived from

MEF or BEF alone (Kitiyanant et al. 2000). Gong et al. (2010)reported that the primary colony formation rate of bovine EScell-like cells was similar on MEF and BEF. These results

suggest that for the self-renewal of bovine ES cells, unlike thosefrom human (hES) and murine (mES) species, the use ofhomologous feeder layers offers no additional advantage.However, in a recent study from our laboratory, feeder layers

prepared from buffalo embryonic fibroblasts were found to besuperior to those derived from goat or sheep embryonic fibro-blasts in terms of attachment rate, time required to attach to the

feeder layer, primary colony formation rate, time required toform the primary colonies, maximum number of passages forwhich ES cell-like cells survived and colony morphology,

suggesting that buffalo fetal fibroblasts (BFF) produce factorsthat helped to maintain buffalo ES cell-like cells in an undiffer-entiated state (Kumar et al. 2011).

The efficacy of feeder layers in supporting the self-renewalof ES cells differs considerably depending upon the species and

CSIRO PUBLISHING

Reproduction, Fertility and Development, 2012, 24, 1098–1104

http://dx.doi.org/10.1071/RD11298

Journal compilation � CSIRO 2012 www.publish.csiro.au/journals/rfd

cell type. The primary reason for this is the difference in theprofile of growth factors and signalling molecules secreted by

feeder cells (Eiselleova et al. 2008), which play an importantrole in maintaining self-renewal and pluripotency of ES cells.The feeder layers derived from mouse and human cells, which

have been classified into supportive and non-supportive typesbased on their ability tomaintain hES cells in an undifferentiatedstate, have been shown to differ in their growth factor expression

profile. Genes encoding FGF-2, GREMLIN (a BMP4 inhibitor)and several ECM components were found to be highlyexpressed in supportive fetal skin feeder cells compared withnon-supportive fetal lung feeder cells (Kueh et al. 2006).

The expression pattern of growth factors produced by thefeeder cells is affected by many factors. Mitomycin-C treatmentand g-irradiation have been reported to alter the expression of

growth factors in feeder-layer cells derived from MEF andhuman embryonic fibroblasts (Xie et al. 2004; Eiselleovaet al. 2008). Exogenous supplementation of ES cell culture

media with growth factors has also been shown to modulate theexpression of other related growth factors. For example, FGF-2has been reported to regulate the expression ofTGF-B1,GREM1

and BMP4 in MEF feeder cells (Greber et al. 2007). A study of

growth factors produced by feeder-layer cells may help indeveloping better systems for the culture of ES cells. The presentworkwas thus carried out to study the expression profile of some

growth factors and signalling molecules involved in regulatingthe self-renewal of ES cells in BFF feeder-layer cells culturedunder different conditions.

Materials and methods

Reagents and media

All the chemicals and media were purchased from SigmaChemical Co., St. Louis, MO, USA, unless otherwise indicated.Disposable cell culture Petri dishes, 15- and 50-mL Falcontubes, Unopettes and syringes were purchased from Becton,

Dickinson and Co., Lincoln Park, NJ, USA. The 0.22-mm filterswere from Millipore Corp., Bedford, MA, USA.

Preparation of feeder layers

BFF feeder layers were prepared as described earlier (Vermaet al. 2007). Briefly, buffalo fetuses (around 45–50 days old)obtained from slaughtered animals were separated from uteri

and were washed twice with sterile phosphate-buffered saline.Ear skin biopsies were taken aseptically in sterile Dulbecco’sphosphate-buffered saline (DPBS). After removing the skin, the

remaining tissue was cut into small pieces, which were culturedin 60-mm cell culture dishes containing Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 20% FBS and50 mgmL�1 gentamicin sulfate in a CO2 incubator (5% CO2 in

air) at 378C. The explants were removed after proliferation andestablishment of fibroblasts. Themonolayers of fibroblasts wereallowed to grow till around 70% confluence. Afterwards these

were sub-cultured by partial trypsinisation. For the preparationof the feeder layer, fibroblasts at Passage 3–5 were inactivatedby treatment with 10 mgmL�1 mitomycin-C for 3 h, after which

the monolayer was trypsinised and the cells were washed3–4 times with DPBS containing 10% FBS and 50mgmL�1

gentamicin sulfate and then finallywith DMEMcontaining 10%FBS and 50 mgmL�1 gentamicin sulfate. In all experiments, this

step was the same except for the difference in the medium inwhich BFF were cultured.

Culture and maintenance of ES cells

Buffalo ES cell-like cells were cultured and maintained as

described earlier (Sharma et al. 2011). Briefly colonies of EScell-like cells were cultured on a BFF feeder layer in stem cellmedium (SCM), which comprised Knockout DMEM (Invitro-gen Corporation, Carlsbad, CA, USA) plus 15% Knockout

serum replacement (Invitrogen), 2mML-glutamine, 50mgmL�1

gentamicin sulfate, 1% MEM non-essential amino acids and0.1mM b-mercaptoethanol, which was supplemented with

5 ngmL�1 FGF-2 and 1000 IUmL�1 mLIF. The colonies werepassaged after being split mechanically every 5–6 days with asplit ratio of 1 : 2 or 1 : 3 depending upon the colony size. ES cells

were regularly checked for their pluripotency by studying theexpression of transcription-based markers like OCT4, NANOGand SOX-2 or surface based-markers (SSEA-1, SSEA-3,SSEA-4, TRA-1–60 and TRA-1–81), or both, as described

earlier (Sharma et al. 2011).

RNA isolation, RT-PCR and quantitative real-time PCR

The expression of growth factors that regulate pluripotency

were first examined by reverse transcriptase (RT)-PCR. TotalRNAwas isolated from buffalo fetal fibroblast cells both beforeand after the mitomycin-C treatment using Tri Reagent. The

quality and integrity of RNA was checked by Nanoquant(Teccan, Salzburg, Austria) and agarose gel electrophoresis.The RNA was reverse transcribed using Revertaid First-Strand

cDNA Synthesis Kit (Fermentas Life Sciences, Hanover, MD,USA), as per the manufacturer’s protocol. The annealing tem-perature and PCR conditions of the target genes are given inTable S1 available as Supplementary Material to this paper. For

examination of relative mRNA abundance, quantitative real-time PCR (qRT-PCR), was performed on a CFX 96 I Cycler(Biorad, Hercules, CA,USA) in a 10-mL reaction volume con-

taining 5mL SYBR Green master-mix (Maxima SYBR GreenMastermix; Fermentas), 0.5mM of each primer and 5� dilutedc-DNA. Thermal cycling conditions consisted of initial dena-

turation at 958C for 5min followed by 40 cycles of 10 s at 958C,15 s at the appropriate annealing temperature (Table S1) and 15 sat 728C, followed by 958C for 10 s and the melting curve. All

primer pairs used were confirmed for their PCR efficiency, andspecific products were checked by melting curve analysis andfor the appropriateness of size by agarose gel electrophoresis.Primer sequences are provided in the supplementary data

(Table S1). The expression data were normalised to the expres-sion ofGAPDHandwere analysed usingCFXManager software(BioRad). RelativemRNA levels were presented as a percentage

of the calibrator used in each experiment. In all experiments threetrials were carried out, each with three replicates.

Determination of protein concentrations by ELISA

The concentrations of FGF-2, TGF-b1, ACTIVIN-A andBMP-4 were measured in the conditioned media with the

Growth factors expressed by buffalo feeder cells Reproduction, Fertility and Development 1099

following commercially available enzyme-linked immunosor-bent assay (ELISA) kits (R and D Systems, Minneapolis, MN,

USA) according to the manufacturer’s instructions: QuantikineHS human FGF basic, Quantikine human TGF-b1, DuoSethuman ACTIVIN-A and Quantikine human BMP-4. All the

experiments were repeated twice, with three replicates in eachexperiment.

Experimental design and statistical analyses

In Experiment 1, BFF feeder-layer cells were cultured inDMEMþ 10%FBS up to Passage 7. At Passage 3, 5 and 7, thesewere trypsinised and processed for RNA isolation for qualitativeRT-PCR studies.

In Experiment 2, BFF that had been cultured in SCM up toPassage 3–5 were either subjected to mitomycin-C treatment for3 h or remained untreated (Controls), following which these

were trypsinised and processed for RNA isolation for qRT-PCRstudies. In another set, spent medium collected from both thegroups 24 and 48 h aftermitomycin-C treatment was centrifuged

at 200g for 10min at 48C and was stored in aliquots at �808Cuntil analysis by ELISA.

In Experiment 3, mitomycin-C-treated feeder layers at

Passage 3–5 were divided into four groups and were culturedwith different culture media for 24 h, after which the spentmedium and the feeder-layer cells were processed as explainedunder Experiment 2. The four groups were as follows: SCM,

SCM alone; LIF, SCMþ 1000 IUmL�1 LIF; FGF-2: SCMþ5 ngmL�1 FGF-2 and LIFþFGF-2, SCMþ 1000 IUmL�1

LIFþ 5 ngmL�1 FGF-2.

In Experiment 4, ES cell colonies at Passage 47 were dividedinto four groups as explained under Experiment 3 and werecultured on mitomycin-C-treated feeder layers at Passage 3–5

with the different culture media for 24 h. In the LIFþ FGF-2group, the culture was continued up to 48 h for comparison of theconcentrations of FGF-2, BMP-4, TGF-b1 and ACTIVIN-A inthe spent medium. The feeder-layer cells and the spent medium

were processed as explained under Experiment 2.Wherever required, statistical analysis was carried out using

ANOVA, and the level of significance was kept at P# 0.05.

Results

In Experiment 1, the expression of LIF, FGF-2, BMP-4,GREMLIN, NOGGIN, TGF-b1 and ACTIVIN-A was detectedat Passage 3, 5 and 7whereas that ofWNT3Awas not detected at

any of the passages examined (Fig. 1). In Experiment 2, therelative abundance ofmRNA forACTIVIN-A, TGF-b1, BMP-4and GREMLIN was found to be significantly increased(P, 0.05) by mitomycin-C treatment of BFF cells, whereas

expression of FGF-2 was not affected (Fig. 2a). Followingmitomycin-C treatment, the protein concentrations in the spentmedium were found to be similar between the treated and con-

trol groups for FGF-2, TGF-b1 and ACTIVIN-A at 24 h,whereas at 48 h theACTIVIN-A concentrationwas significantlyhigher (P, 0.05) in the mitomycin-C-treated group than in the

control group (Fig. 2b). BMP-4 protein could not be detected at24 or 48 h following mitomycin-C treatment.

In Experiment 3, following culture of mitomycin-C-treatedfeeder layers with different media supplements, no significant

difference was observed in the expression level of ACTIVIN-A,TGF-b1, BMP-4 or FGF-2 in the feeder-layer cells of the SCM,LIF, FGF-2 or LIFþFGF-2 groups (Fig. 3a). GREMLIN

expression was, however, significantly higher (P, 0.05) inthe FGF-2-supplemented groups compared with those thatlacked it. The concentration of ACTIVIN-A was higher

(P, 0.05) in the FGF-2-supplemented groups than in those thatdid not contain FGF-2. The concentration of TGF-b1, whichwas similar in the SCM and LIFþFGF-2 groups, was higher

(P, 0.05) than that when SCM was supplemented with LIFalone (Fig. 3b) indicating that treatment with LIF or FGF2 alonelowered the expression of TGF-b1, while the two factorstogether merely recovered TGF-b1 to the untreated levels.

In Experiment 4, when ES cells were cultured for 24 h onmitomycin-C-treated feeder layers, the concentration ofTGF-b1 in the spent medium was found be significantly higher

(P, 0.05) in the presence of both LIF and FGF-2 than whenonly one of them was present which, in turn, was higher(P, 0.05) than that in the non-supplemented control group

(Fig. 4a). The concentration of ACTIVIN-A was, however,significantly higher (P, 0.05) in the control and FGF-2groups than that in the LIF and LIFþFGF-2 groups. In theLIFþ FGF-2 group, the ACTIVIN-A concentration was

P3 P5 P7

LIF

FGF-2

BMP-4

GREMLIN

NOGGIN

TGF-β1

ACTIVIN-A

WNT-3A

GAPDH

Fig. 1. Expression pattern of various signalling factors examined by

reverse transcriptase-PCR at Passage (P) 3, 5 and 7 in buffalo fetal fibroblast

feeder layers.

1100 Reproduction, Fertility and Development R. Sharma et al.

significantly lower (P, 0.01) and the TGF-b1 concentrationwas significantly higher (P, 0.01) in the spent medium at 24 hcompared with that at 48 h of culture (Fig. 4b).

Discussion

To our knowledge, this is the first report on the expression

profile of growth factors produced by feeder cells from any farmanimal species. The present study examined the profile ofgrowth factors expressed by BFF, generally used as feeder cells

for supporting the self-renewal of buffalo ES cell-like cells.Transcripts of several growth factors and signalling moleculessuch as LIF, FGF-2, BMP-4, GREMLIN, NOGGIN, TGF-b1and ACTIVIN-A were detected in BFF feeder-layer cells atPassage 3, 5 and 7 by RT-PCR. Among these, Passage 5appeared to be the best for the culture of buffalo ES cells sincethe transcripts of all the factors mentioned above were detected

quite consistently at this passage. The transcripts of many ofthese signalling molecules have been previously shown to bepresent in mouse and human feeder cells (Eiselleova et al.

2008). These growth factors have been established to play keyroles in the maintenance of pluripotency in mES and hES cells.

LIF, which is responsible for the activation of the JAK/STATpathway, is capable of feeder-independent support of pluripo-tency in mES cells in the presence of serum (Matsuda et al.

1999). Members of the bone morphogenic protein (BMP)

family, especially BMP-4, synergise with LIF to maintain theself-renewal of mES cells (Ying et al. 2003). However, LIFcannot maintain the pluripotency in hES cells since the JAK/

STAT3 pathway is not activated under conditions that areessential for keeping hES cells in an undifferentiated state(Thomson et al. 1998). hES cells have been maintained in an

undifferentiated state on feeder layers derived from MEF(Thomson et al. 1998) or human feeders (Richards et al. 2002,2003) or on Matrigel with MEF-conditioned medium (Xu et al.

2001). In the absence of feeder-cell support, STAT3 activationis not sufficient for stopping the differentiation of hES cells(Daheron et al. 2004). In contrast to mES cells, the self-renewalof hES cells may be controlled by three different pathways –

FGF,Wnt and TGFb – as indicated by transcriptome analysis ofhES cells (Brandenberger et al. 2004; Wei et al. 2005). Severalstudies have indicated that activation of the FGF signalling

pathway by bFGF is critical for maintenance of hES cells in anundifferentiated state (Amit et al. 2000). TGF-b1/activin/nodal

*

*

**

*

16Control

Mitomycin-C

Control

Mitomycin-C

12

8

Rel

ativ

e ex

pres

sion

fold

4

0

0

20

40

60

80

100

Activin-A

ACTIVIN-A

TGFβ-1

TGF-β1

BMP-4FGF-2

FGF-2

GREMLIN

(a)

(b)

Con

cent

ratio

n (p

g m

L�1 )

Fig. 2. Effect of mitomycin-C treatment for 3 h on various signalling factors in buffalo fetal

fibroblast feeder layers. (a) Relative mRNA expression levels in cells measured by qRT-PCR and

(b) protein concentrations in the spentmediumat 48 h. Barsmarkedwith an asterisk differ significantly

(P, 0.05) from the control.

Growth factors expressed by buffalo feeder cells Reproduction, Fertility and Development 1101

signalling has also been shown to be operative in hES cells

(Vallier et al. 2005).Not much information is available on the signalling path-

ways that need to be activated for the self-renewal of buffalo EScell-like cells. Whereas several earlier studies have indicated

the requirement of LIF for the maintenance of buffalo ES cell-like cells in an undifferentiated state, the presence of LIF aloneis not sufficient for their long-term culture (Verma et al. 2007;

Anand et al. 2011; Kumar et al. 2011). We have recently shownthat a combination of LIF and bFGF is capable of supportinglong-term self-renewal of buffalo ES cell-like cells on BFF

feeder-layer support (Sharma et al. 2011), indicating that boththese signalling pathways are operative in buffalo ES cell-likecells. The expression of mRNA for LIF and the presence ofbFGF in the spent medium of BFF cells, in combination with

the results of our previous studies, in which it was found thatbuffalo ES cells cannot survive under feeder-free conditions(Sharma et al., unpubl. data), suggest that at least LIF and bFGF,

which are critically required by the buffalo ES cells for theirself-renewal (Sharma et al. 2011), may be supplied by the BFFfeeder layer.

Supplementation with LIFþFGF-2 was found to increasethe expression of GREMLIN in the feeder cells and the con-centrations ofACTIVIN-A in the spentmedium. Treatmentwith

LIF or bFGF alone lowered the concentration of TGF-b1, whilesupplementation with both recovered TGF-b1 to the untreatedcontrol levels. This is important since the maintenance of EScells in an undifferentiated state may require the coordinatedactivation of more than one signalling pathway. For example,

with high concentrations of FGF-2, hES cells can be maintainedin feeder-free conditions in combinationwithACTIVIN-A or bysuppression of BMP signalling byNOGGIN due to co-operation

between the FGF and activin/nodal/TGFb pathways (Vallieret al. 2005;Wang et al. 2005; Xu et al. 2005; Xiao et al. 2006). Itis possible that the actions of exogenous LIF and FGF-2 on

buffalo ES cell-like cells leading to the maintenance of apluripotent state (Sharma et al. 2011) could be partly mediatedby altering the production of ACTIVIN-A and TGF-b1 by BFFfeeder cells. FGF-2 was shown to upregulate TGF-b1 and

Grem1 and downregulate Bmp4 in MEF, thus switching themto an overall hES-supportive mode through the concertedderegulation of these factors (Greber et al. 2007). In another

study, a global expression profile analysis using microarraysindicated that 17 secreted factors were downregulated in theabsence of FGF-2 treatment of MEF (Diecke et al. 2008).

Whether the TGFb/activin/nodal pathway plays any role inthe self-renewal of buffalo ES cell-like cells is, therefore,currently being investigated by our laboratory.

5

2

3

4

bc

c

(a)

(b)

0

1

2

Rel

ativ

e ex

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sion

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abab

3000b

b

ACTIVIN-A TGF-β1 BMP-4 FGF-2 GREMLIN

2000

a a

a a

0

1000

TGF-β1 Activin-A

Con

cent

ratio

n (p

g m

L�1 )

bab

SCM LIF

FGF-2 LIF�FGF-2

SCM LIF

FGF-2 LIF�FGF-2

Fig. 3. (a) Relative mRNA expression levels in cells and (b) protein concentrations in spent medium

of various signalling factors in buffalo fetal fibroblast feeder-layer cells after 24 h of culture in stem

cell medium (SCM), SCMþ 1000 IUmL�1 LIF, SCMþ 5 ngmL�1 FGF-2 or SCM þ1000 IUmL�1

LIFþ 5 ngmL�1 FGF-2. Bars marked with different superscripts differ significantly (P, 0.05).

1102 Reproduction, Fertility and Development R. Sharma et al.

Feeder cells differ in their ability to support the undifferenti-ated state of ES cells depending upon the nature of growth

factors and signalling molecules that they secrete. A compara-tive study of mouse and human feeder cells indicated that bothsecreted comparable levels of TGF-b1. The former secretedlarger quantities of ACTIVIN-A, whereas FGF-2 could not be

detected in the spent medium of mouse cells (Eiselleova et al.2008). In addition to the growth factors produced by the feedercells, the ES cells themselves produce many growth factors that

may affect them either directly in an autocrine manner (Dvoraket al. 2005) or indirectly through their effects on the productionof growth factors and signalling molecules by the feeder cells.

A combination of all these factors and their interactions pro-duces the in vitro stem-cell niche required for the maintenanceof ES cells in an undifferentiated state.

The feeder-layer cells need to be inactivated before their use

in co-culture with ES cells. Among the methods employed forinactivation, mitomycin-C treatment and g-irradiation are mostcommonly used. It was found in the present study that the

relative mRNAabundance ofACTIVIN-A andBMP-4 increasedsignificantly (P, 0.05), that of TGF-b1 and GREMLIN expres-sion increased many-fold (P, 0.01), whereas the expression

level of FGF-2 was not affected by mitomycin-C treatment ofBFF cells. However, protein concentrations of FGF-2, TGF-b1and ACTIVIN-A in the spent medium was similar between the

mitomycin-C-treated and control groups at 24 h, whereas at48 h, the ACTIVIN-A concentration, but not that of others, was

higher (P, 0.05) in the treated than in the control group.WNT-3a expression could not be detected either before or after

mitomycin-C inactivation, unlike MEF, in which expressionof WNT-3A has been reported following inactivation byg-irradiation (Xie et al. 2004). Also, we were not able to detectexpression of BMP-4 in the spent medium of BFF feeder cells at

24 or 48 h following mitomycin-C treatment, which agrees wellwith previous reports, in which BMP-4 was found to beundetectable or barely detectable in mouse and human feeders

(Pera et al. 2004; Eiselleova et al. 2008).In conclusion, these results suggest that buffalo fetal fibro-

blast feeder cells produce many growth factors and signalling

molecules that may be involved in maintaining buffalo ES cell-like cells in an undifferentiated state, and that LIF and FGF-2affect the production of these factors.

Acknowledgements

The present work was funded by a National Agriculture Innovative Project

(NAIP) grant to M. S. C. (C-2067 and 075) and S. K. S. (C 2–1-(5)/2007).

A. G. and R. S. are supported by CSIR senior research fellowships.

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SCM LIFa

ba

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a ab bc

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1104 Reproduction, Fertility and Development R. Sharma et al.