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M Bonnet, I Gourdou, C Leroux, Y Chilliard and J Djianeadipose,
epithelial, and myoepithelial cells during pregnancy and
lactation.
Leptin expression in the ovine mammary gland: putative
sequential involvement of
2002, 80:723-728.J ANIM SCI
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Leptin expression in the ovine mammary gland: Putative
sequentialinvolvement of adipose, epithelial, and myoepithelial
cells
during pregnancy and lactation1
M. Bonnet*2, I. Gourdou, C. Leroux, Y. Chilliard*, and J.
Djiane
*INRA, Unite de Recherches sur les Herbivores, Equipe Tissu
Adipeux et Lipides du Lait,63122 Saint-Gene`s-Champanelle, France
and INRA, Laboratoire de Biologie Cellulaire et Moleculaire and
Laboratoire de Genetique biochimique et de Cytogenetique, 78352
Jouy-en-Josas cedex, France
ABSTRACT: We examined the ability of the ovinemammary gland to
synthesize leptin throughout preg-nancy and lactation. Leptin gene
expression was as-sayed by real-time reverse transcription and
polymer-ase chain reaction in mammary gland from ewes at 15,80,
106, 112, or 141 d of pregnancy and at 0 (30 minafter parturition),
3, 48, or 70 d of lactation. LeptinmRNA level was high at the
beginning (the first 80 d)and at the end of pregnancy and was lower
at mid-pregnancy and throughout lactation. Furthermore, dur-ing
these periods of mammary leptin expression, the
Key Words: Lactation, Leptin, Mammary Tissue, Pregnancy,
Sheep
2002 American Society of Animal Science. All rights reserved. J.
Anim. Sci. 2002. 80:723728
Introduction
Leptin is mainly, but not exclusively, produced byadipose tissue
(Zhang et al., 1994; Ahima and Flier,2000) and contributes to the
regulation of energy bal-ance by informing the brain about fat
store levels, thenregulating food intake and energy expenditure in
adultanimals (Houseknetch et al., 1998; Casanueva and Die-guez,
1999). Leptin, via its receptors located in mosttissues, has been
implicated in numerous other roles,including modulation of
reproduction, endocrine sys-tem, tissue metabolism, blood pressure,
hematopoiesis,angiogenesis, brain and bone development, wound
heal-ing, and cell differentiation and proliferation (Ahima
1The authors acknowledge A. Gertler for recombinant leptin,
L.Belair for total RNA extraction and leptin antibody preparation,
S.Taourit for DNA sequencing, B. Vigier, R. Boischard, A.
Aubourg,M. Olivier-Bousquet, and M. Guillomot for advice and help
in immu-nofluorescence analysis, F. Fort for photographic work, and
K. Laudand P. Martin for helpful discussions.
2Correspondence: Centre de Recherches de Clermont-Ferrand/Theix,
(phone: 33-4-73-62-47-01; Fax: 33-4-73-62-45-19;
E-mail:[email protected]).
Received March 1, 2001.Accepted August 21, 2001.
723
location of leptin protein, as determined by
immunohis-tochemical analysis, changed within mammary tissue.It was
located in adipose cells during early stages ofpregnancy, in
epithelial cells after full cell differentia-tion just before
parturition, and in myoepithelial cellsafter parturition. These
data, compared with publisheddata on leptin receptor gene
expression, provide evi-dence that leptin could be produced by
different celltypes of the mammary gland and could act as a
para-crine factor on mammary cell growth and differentia-tion via
adipose-epithelial cells and myoepithelial-epi-thelial cell
interactions.
and Flier, 2000). The identification of leptin in
human(Casabiell et al., 1997; Houseknetch et al., 1997;
Smith-Kirwin et al., 1998), rat (Casabiell et al., 1997),
murine(Aoki et al., 1999), bovine (Rosi et al., 2000), and
porcine(Estienne et al., 2000) milk suggests that this hormonecould
also be involved in the physiology of the neonate.However, the
presence of leptin in milk opens the ques-tion of the mechanisms by
which the epithelial cellstransfer leptin from the blood and(or)
synthesize it. Atransfer of leptin from maternal blood to milk
throughmammary epithelial cells was suggested by the detec-tion of
[125I]-leptin in milk after intraperitoneal injec-tion of
[125I]-leptin into lactating rats (Casabiell et al.,1997) and by
the characterization of leptin receptormRNA in ovine mammary
epithelial cells (Laud et al.,1999). However, the detection of
leptin mRNA and(or)protein in human (Smith-Kirwin et al., 1998) and
mu-rine (Aoki et al., 1999) mammary tissue suggests alsothat leptin
could be produced in the mammary gland.To address the ability of
the ovine mammary gland tosynthesize leptin, we quantified leptin
mRNA levels byreal-time reverse transcription and polymerase
chainreaction (RT-PCR) throughout pregnancy and lacta-tion. In
addition, we used immunofluorescence detec-tion to localize the
leptin protein among mammarycell types.
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Bonnet et al.724
Materials and Methods
Tissue Samples
Animal care and use procedures were approved bythe French
Ministry of Agriculture in agreement withFrench regulations for
animal experimentation (guide-line 19/04/1988). Primiparous
Prealpes du Sud ewes (n= 26) were allotted in eight groups
according to theirpregnancy or lactation stage: 15, 80, 106, 112,
or 141d of pregnancy and 0 (30 min after parturition), 3, and48 to
70 d of lactation (three to four animals per group).The diet
distributed during the first 3 mo of pregnancyconsisted of 58%
ammonia-treated straw, 19% barley,13% dehydrated alfalfa, and 10%
peas. The diet distrib-uted from the 3rd mo to the end of pregnancy
consistedof 46% ammonia-treated straw, 26% barley, 20% dehy-drated
alfalfa, and 8% peas. The diet distributed tolactating ewes with
one lamb consisted of 23% wheatand barley straw, 23% pea straw, 9%
oats, 18% barley,23% dehydrated alfalfa, and 4% soybean meal. The
dietdistributed to lactating ewes with two lambs consistedof 20%
wheat and barley straw, 20% pea straw, 8%oats, 20% barley, 24%
dehydrated alfalfa, and 8% soy-bean meal. Vitamin-mineral premix
was added to thefeed at 15 or 30 g/d for the pregnancy and
lactationstages, respectively. The body condition score was 2.4 0.4
(on a 0-to-5 scale). The number of fetuses or lambswas one for 16
ewes and two for 10 ewes. Ewes wereslaughtered by exsanguination
and samples of mam-mary tissue were immediately placed either in 2%
para-formaldehyde-PBS buffer for immunohistochemicalanalysis or
frozen in liquid nitrogen pending gene ex-pression analysis.
Quantification of Leptin mRNA Levelby Real-Time Quantitative
RT-PCR Assay
Total RNA was prepared by the guanidium isothiocy-anate/phenol
method as described by Puissant andHoudebine (1990). Quantification
of leptin mRNA levelwas performed by real-time RT-PCR, using the
fluores-cent TaqMan methodology and a 7700 Sequence Detec-tor
System (PE Applied Biosystems, Courtaboeuf,France) according to a
procedure described previously(Bonnet et al., 2000). Sense
(5-TCAGTGGATGGTCCC-TCGA-3) and antisense primers
(5-GGGAAACC-CAAGCCTCCTC-3) as well as TaqMan probe
(5-CAG-GACCAGCCCCCAGGAGCC-3) (PE Applied Biosys-tems) were chosen
(Figure 1) after characterizing 1,076bp of the leptin mRNA
3untranslated region (3UTR).This 1,076-bp cDNA fragment was
reverse-transcribedand amplified by PCR with the forward
(5-CTTTGTTTCTACTGTGACTGACT-3) and the
reverse(5-AGTGCAAGCAGGGTTAGCCTGTG-3) primersand sequenced using an
ABI 377A automated se-quencer (PE Applied Biosystems) as described
pre-viously (Bonnet et al., 2000). The sequence accessionnumber of
this 1,076-bp cDNA fragment is AF310264.
Figure 1. Partial nucleotide sequence of ovine leptincDNA and
position of the primers and TaqMan probeused for the real-time
reverse transcription and polymer-ase chain reaction assay. We
characterized 1,076 bp of theovine leptin cDNA corresponding to a
part of the leptinmRNA 3untranslated region (accession
numberAF310264). This ovine (o) fragment was aligned with itshuman
(h) and pig (p) counterparts (sequence accessionnumbers U43653 and
AF026976, respectively). Gaps (.)have been placed to maximize the
similarity. Dashes ()correspond to nucleotides that are identical
to those ofthe ovine leptin sequence. The primers and TaqMan
probeused for quantitative analysis of leptin mRNA level areshaded.
Alignment was performed with the Clustalw pro-gram (Version 1.81).
This ovine 1,076-bp sequence shows67 and 78% identity with the
human and pig se-quences, respectively.
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Leptin expression by the ovine mammary gland 725
The reverse transcription reaction (20 L) of the real-time
RT-PCR assay was performed using 4 g of totalRNA, with 100 U of
SuperScript reverse transcriptase(Gibco BRL, Life Technologies,
Cergy Pontoise, France)and 10 pmol of oligo(dT)18. Amplification
reactions (50L) contained diluted (1:500 in water) cDNA sample(10
L), 10 PCR Master Mix (27.5 L, PE AppliedBiosystem), 40 pmol of
each primer, and 10 pmol ofTaqMan probe. The cycling conditions
included 2 minat 50C and 10 min at 95C. Subsequently,
thermalcycling proceeded with 45 cycles at 95C for 15 s andat 60C
for 2 min. Each assay was performed in tripli-cate. The
concentration of leptin mRNA was deter-mined from a calibration
curve prepared by amplifying59,250, 29,625, 7,406, 1,851, and 592
copies of a recom-binant plasmid containing the 1,076-bp fragment
de-scribed in Figure 1.
The leptin mRNA copy number was normalized bythe mRNA copy
number of the constitutively expressedcyclophilin gene, quantified
by real-time RT-PCR asdescribed previously (Bonnet et al.,
2000).
Leptin Location by Indirect Immunofluorescence
Mammary fragments obtained after dissection werefixed with 2%
paraformaldehyde in PBS buffer, pH 7.2,for 24 h. Fixed tissues were
incubated overnight in 40%sucrose in PBS, frozen in liquid nitrogen
vapors, andcut in 3-m sections at 35C with a Reichert
Cryocut(Leica, Reuil-Malmaison, France).
Leptin antiserum was produced in rabbits by injec-tion of 1 mg
of recombinant chicken leptin (Raver etal., 1998) solubilized in
saline buffer and emulsified inFreunds complete adjuvant. Three and
six weeks laterthe rabbits were reimmunized with 1 mg of
recombi-nant leptin solubilized in saline buffer and emulsifiedin
Freunds incomplete adjuvant. From the 6th wk afterinitial
immunization, antiserum was collected weekly.
Serum and antibody dilutions were made in PBS con-taining 0.2%
of fish gelatin.
For labeling of leptin protein, tissue sections (n =2 or 4 for
tissues from pregnant and lactating ewes,respectively) were
successively incubated with PBS-50mM NH4Cl (20 min), PBS (three
times, 15 min eachtime), goat serum (1:10, 1 h) and rabbit
anti-chickenleptin antiserum (1:100, 2 h); washed in PBS-0.2%
fishgelatin; and then incubated with fluorescein isothiocya-nate
(FITC)-conjugated goat anti-rabbit IgG (1:200 for2 h; Sanofi
Diagnostics Pasteur, Marnes-La-Coquette,France). Sections were
mounted on a drop of Vectas-hield (Vector Laboratories, Burlingame,
CA) and ob-served with a Polyvar Reichert microscope (Leica).
Con-trol sections were treated similarly with nonimmunerabbit serum
or with omission of anti-chicken leptinantiserum. To check the
specificity of the staining, sec-tions were incubated with
anti-chicken leptin antise-rum fully adsorbed with chicken leptin.
This adsorbedantiserum was prepared by incubating, for 2 h,
chicken
Figure 2. Expression of the leptin gene in mammarytissue
throughout pregnancy and lactation determinedby real-time reverse
transcription and polymerase chainreaction assay. Leptin and
cyclophilin mRNA levels weremeasured from mammary gland tissue
collected fromthree or four ewes at 15, 80, 106, 112, or 141 d of
pregnancyand at 0, 3, 48, or 70 d of lactation.
Leptin/cyclophilinmRNA ratios were calculated. a,b,c,dMeans ( SEM)
withdifferent superscripts differ significantly (P < 0.05).
leptin (Raver et al., 1998) with anti-chicken leptin anti-serum
(1:100) to make a final concentration of 1 g/L.
Double-labeling of both leptin protein and F-actinstructures was
performed according to the same proce-dure, with the addition of
0.17 mol of tetramethylrho-damine phalloidin (Molecular Probes,
Eugene, OR) tothe incubation step with FITC-conjugated goat
anti-rabbit IgG.
Statistical Analysis
Data were normalized by log transformation andwere submitted to
an analysis of variance by the GLMprocedure of SAS (SAS Inst. Inc.,
Cary, NC). Since asignificant (P < 0.01) effect of the
physiological statewas shown, the differences between two
physiologicalstages were tested using Duncans test with a
probabil-ity of 0.05.
Results
Temporal Expression of Leptin mRNA in OvineMammary Gland During
Pregnancy and Lactation
Leptin mRNA level, normalized by the level ofcyclophilin mRNA,
varied significantly (P < 0.01) de-pending on the pregnancy or
lactation stage (Figure 2).During pregnancy, the leptin mRNA level
decreasedstrongly between d 80 and d 106 or 112 (P <
0.05),before increasing slightly at d 141 (P < 0.05 for d 141vs
d 106 of pregnancy) to levels similar to those assayedat d 15 and
80. Throughout lactation, leptin mRNAlevels did not vary
significantly but were lower (P