-
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, Unité de Recherches sur les Herbivores, Equipe Tissu
Adipeux et Lipides du Lait,63122 Saint-Genès-Champanelle, France
and INRA, †Laboratoire de Biologie Cellulaire et Moléculaire
and
‡Laboratoire de Génétique biochimique et de Cytogénétique,
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. 80:723–728
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.
-
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
Préalpes 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
3′untranslated region (3′UTR).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 3′untranslated 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.
-
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 (50�L) 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 50°C and 10 min at 95°C. Subsequently,
thermalcycling proceeded with 45 cycles at 95°C for 15 s andat 60°C
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 −35°C 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 inFreund’s complete adjuvant. Three
and six weeks laterthe rabbits were reimmunized with 1 mg of
recombi-nant leptin solubilized in saline buffer and emulsifiedin
Freund’s 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 Duncan’s 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
-
Bonnet et al.726
0.05) than the level assayed at d 80 of pregnancy. More-over, at
d 3 of lactation, the level of leptin mRNA wassignificantly (P <
0.05) lower than those assayed at d15, 80, or 141 of pregnancy.
Immunofluorescent Location of Leptin in OvineMammary Gland
During Pregnancy and Lactation
Immunofluorescence performed with the anti-leptinantiserum
showed a labeling located in adipose, epithe-lial, or myoepithelial
cells depending on the pregnancyor lactation stage. During early
pregnancy (d 15), leptinimmunostaining was located in adipocytes,
mainly intheir cytoplasm (Figure 3A). At the end of pregnancy(d
141), fluorescent labeling was detected on the apicalmembrane of
the epithelial cells (Figure 3B). Just afterparturition (30 min), a
leptin labeling was observed asa continuous fringe surrounding the
acini (Figure 3C)and as a discontinuous fringe until 70 d of
lactation(data not shown). Sections of mammary tissue from oneother
ewe at each pregnancy stage and three other ewesat d 0 of lactation
showed similar location of immuno-stainings. Just after
parturition, the leptin labeling wascolocalized with the F-actin
labeling, indicating thatleptin protein was mainly located in
myoepithelial cells(Figure 3D). At all stages of pregnancy and
lactation,leptin immunostaining was eliminated when anti-lep-tin
antiserum was preadsorbed with leptin (Figures 3E,3F, 3G). No label
was detectable when a non-immunerabbit serum was used instead of
anti-leptin antiserumor when the second antibody alone was used
(datanot shown).
Discussion
We report here the first evidence that ovine mam-mary tissue
expresses leptin mRNA during lactation.This result is in agreement
with the mammary synthe-sis of leptin previously reported in humans
(Smith-Kir-win et al., 1998) and mice (Aoki et al., 1999).
We also report strong variations in ovine mammaryleptin gene
expression depending on the stage of preg-nancy or lactation.
Leptin mRNA was expressedthroughout pregnancy, with a strong
decrease in theexpression at mid-pregnancy. During lactation the
lep-tin mRNA levels were lower than or similar to thoseobserved
during the end of pregnancy. One of the mostinteresting aspects of
our study was the observationthat the cellular location of leptin
changed during thedifferent phases of mammary gland development.
Dur-ing early stages of development, leptin was exclusivelylocated
in mammary adipocytes. In contrast, after fullcell differentiation,
just before parturition, adipose tis-sue had completely regressed
and leptin was presentin mammary epithelial cells, whereas during
lactation,leptin was located in myoepithelial cells. Such a
sequen-tial change in the leptin location between mammary celltypes
suggests a new and complex scheme of mammaryleptin expression.
Indeed, our results suggest a strong
synthesis of leptin by mammary adipocytes at the be-ginning of
pregnancy that decreased to a lower levelduring the second part of
pregnancy when adipocytesdisappeared. At the end of pregnancy,
leptin mRNAlevel increased slightly and leptin protein was
locatedin epithelial cells. A local production is likely becausea
putative transfer of blood and(or) mammary adipocyteleptin would be
reduced, due to the strong decrease inleptin receptor gene
expression by the epithelial cellsat this stage (Laud et al.,
1999). In addition, after partu-rition and throughout lactation,
leptin mRNA was ex-pressed by the mammary tissue and leptin protein
wasfound in myoepithelial cells. It could be hypothesizedthat
leptin is produced exclusively by myoepithelialcells because leptin
receptor is not expressed in thiscell type (Laud et al., 1999).
Further studies, however,are needed to ascertain that leptin is
synthesized byepithelial and myoepithelial cells around
parturition.Nevertheless, in agreement with this hypothesis,
leptinsynthesis was observed in a breast epithelial cell
line(O’Brien et al., 1999) as well as in rat skeletal musclecells
(Wang et al., 1999).
These sequential changes of leptin cellular location,together
with the synthesis of leptin receptor exclu-sively by epithelial
cells and mainly between 70 and106 d of pregnancy (Laud et al.,
1999), suggest thatleptin could act as a paracrine factor in
mammary glandgrowth, development, and function. Indeed,
mammarygland growth and development during pregnancy arehighly
dependent on steroids and protein hormonesfrom ovaries, placenta,
and pituitary gland (Lyons,1958). However, in vitro studies
indicate that thesehormone effects are largely indirect, being
mediatedby growth factors synthesized by mammary adipocytes(Levine
and Stockdale, 1984; Rudland et al., 1984;Woodward et al., 1998).
Leptin could be one of thesesteroid-inducible proteins synthesized
by mammary fatcells; the strong leptin gene expression that we
observedat 80 d of pregnancy occurred concurrently with thestart of
the increase in plasma estradiol concentration(Martinet and
Houdebine, 1999). Moreover, the mainte-nance of alveolar structures
during lactation is partlycontrolled by growth factors such as
IGF-I, trans-forming growth factor-α and -β, and fibroblast
growthfactor-1, 2, and 7, mainly synthesized by myoepithelialcells
(Gomm et al., 1997; Plath et al., 1998; Martinetand Houdebine,
1999). Hence, the myoepithelial cellleptin observed in our study
could participate in thecontrol of epithelial cell growth and
survival(apoptosis). Finally, leptin gene expression by
sheepmammary gland around parturition could be relatedto leptin
secretion in colostrum and milk; it has beenobserved in women
(Casabiell et al., 1997; Houseknetchet al., 1997; Smith-Kirwin et
al., 1998), rats (Casabiellet al., 1997), cows (Rosi et al., 2000),
pigs (Estienne etal., 2000), and mice, mainly during the first 2 d
of lacta-tion (Aoki et al., 1999). It could be hypothesized
thatleptin, as a colostral protein, may promote immunity
-
Leptin expression by the ovine mammary gland 727
Figure 3. Representative photographs of immunofluorescent
location of leptin in mammary tissue from ewes at d15 or 141 of
pregnancy and 30 min after parturition. Tissues were fixed and
frozen. Sections were stained with theanti-leptin antiserum
(1:100), followed by a staining with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG(1:200). A, B, C: in
sections of mammary tissue from ewes at d 15 or 141 of pregnancy
and 30 min after parturitionincubated with the anti-leptin
antiserum, leptin is located in adipose, epithelial, or
myoepithelial cells, respectively.D: in sections of mammary tissue
from ewes 30 min after parturition, incubated with the anti-leptin
antiserum plusthe tetramethylrhodamine phalloidin, leptin labeling
was colocalized with the F-actin labeling in myoepithelial cells.E,
F, G: in sections of mammary tissue from ewes at d 15 or 141 of
pregnancy and 30 min after parturition incubatedwith the
anti-leptin antiserum fully adsorbed with leptin no label was
observed. 1 cm = 31 �m.
-
Bonnet et al.728
(Lord et al., 1998) and(or) intestinal cell functionality(Morton
et al., 1998) in newborn mammals.
Implications
We show for the first time that leptin is producedby the ovine
mammary gland; its gene expression ismaximal at the beginning of
pregnancy and, to a lesserextent, at the end of pregnancy.
Moreover, we reporthere the presence of leptin protein in adipose,
epithelial,or myoepithelial cells at the beginning or the end
ofpregnancy, or after parturition, respectively. These re-sults
suggest that leptin, besides being a colostral pro-tein, may be a
paracrine factor acting on mammarygland growth, development, and
function, via adipose-epithelial cells and myoepithelial-epithelial
cell inter-actions favored by epithelial cell leptin receptors.
Fur-ther studies are needed to clarify putative implicationsof
leptin in the physiology of newborns as well as inmammogenesis
around puberty and during pregnancy,and in mammary cell apoptosis
during lactation. Thiswould help us to better understand the
mechanisms forthe known effect of nutritional factors and body
fatnesson peripubertal mammogenesis.
Literature Cited
Ahima, R. S., and J. S. Flier. 2000. Adipose tissue as an
endocrineorgan. Trends Endocrinol. Metab. 11:327–332.
Aoki, N., M. Kawamura, and T. Matsuda. 1999.
Lactation-dependentdown regulation of leptin production in mouse
mammary gland.Biochim. Biophys. Acta 1427:298–306.
Bonnet, M., C. Leroux, Y. Faulconnier, J. F. Hocquette, F.
Bocquier,P. Martin, and Y. Chilliard. 2000. Lipoprotein lipase
activityand mRNA are up-regulated by refeeding in adipose tissue
andcardiac muscle of sheep. J. Nutr. 130:749–756.
Casabiell, X., V. Pineiro, M. A. Tome, R. Peino, C. Dieguez, and
F.F. Casanueva. 1997. Presence of leptin in colostrum and/orbreast
milk from lactating mothers: a potential role in the regula-tion of
neonatal food intake. J. Clin. Endocrinol. Metab.82:4270–4273.
Casanueva, F. F., and C. Dieguez. 1999. Neuroendocrine
regulationand actions of leptin. Front. Neuroendocrinol.
20:317–363.
Estienne, M. J., A. F. Harpe, C. R. Barb, and M. J. Azain.
2000.Concentrations of leptin in serum and milk from sows that
dif-fered in body condition at farrowing J. Anim. Sci.
78(Suppl.1):204 (Abstr.).
Gomm, J. J., P. J. Browne, R. C. Coope, G. S. Bansal, C.
Yiangou,C. L. Johnston, R. Mason, and R. C. Coombes. 1997. A
paracrinerole for myoepithelial cell-derived FGF2 in the normal
humanbreast. Exp. Cell Res. 234:165–173.
Houseknecht, K. L., C. A. Baile, R. L. Matteri, and M. E.
Spurlock.1998. The biology of leptin: A review. J. Anim. Sci.
76:1405–1420.
Houseknecht, K. L., M. K. McGuire, C. P. Portocarrero, M. A.
McGu-ire, and K. Beerman. 1997. Leptin is present in human milkand
is related to maternal plasma leptin concentration
adiposity.Biochem. Bioph. Res. Commun. 240:742–747.
Laud, K., I. Gourdou, L. Belair, D. H. Keisler, and J. Djiane.
1999.Detection and regulation of leptin receptor mRNA in ovine
mam-mary epithelial cells during pregnancy and lactation. FEBS
Lett.463:194–198.
Levine, J. F., and F. E Stockdale. 1984. Cell-cell interactions
promotemammary epithelial cell differentiation. Exp. Cell
Res.151:112–122.
Lord, G. M., G. Matarese, J. K. Howard, R. J. Baker, S. R.
Bloom,and R. I. Lechler. 1998. Leptin modulates the T-cell
immuneresponse and reverses starvation-induced
immunosuppression.Nature (Lond.) 394:897–901.
Lyons, W. R. 1958. Hormonal synergism in mammary growth. Proc.R.
Soc. Lond. Ser. Biol. Sci. 149:303–325.
Martinet, J., and L. M. Houdebine. 1999. Mammary gland,
mammo-genesis, growth factors, lactogenesis. In: J. Martinet, L.
M.Houdebine, and H. Herbert (ed.) Biology of Lactation. pp
1–27.INRA Publications, Paris, France.
Morton, N. M., V. Emilsson, Y. L. Liu, and M. A. Cawthorne.
1998.Leptin action in intestinal cells. J. Biol. Chem.
273:26194–26201.
O’Brien, S. N., B. H. Welter, and T. M. Price. 1999. Presence of
leptinin breast cell lines and breast tumors. Biochem. Biophys.
Res.Commun. 259:695–698.
Plath, A., R. Einspanier, C. Gabler, F. Peters, F. Sinowatz, D.
Gos-podarowicz, and D. Schams. 1998. Expression and localizationof
members of the fibroblast growth factor family in the bovinemammary
gland. J. Dairy Sci. 81:2604–2613.
Puissant, C., and L. M. Houdebine. 1990. An improvement of
thesingle-step method of RNA isolation by acid guanidinium
thiocy-anate-phenol-chloroform extraction. Biotechniques
8:148–149.
Raver, N., M. Taouis, S. Dridi, M. Derouet, J. Simon, B.
Robinzon,J. Djiane, and A. Gertler. 1998. Large-scale preparation
of biolog-ically active recombinant chicken obese protein (leptin).
ProteinExpr. Purif. 14:403–408.
Rosi, F., V. Bontempo, R. Capalbo, and A. Baldi. 2000. Effects
ofrumen-protected methionine on milk yield, milk leptin and
milknitrogenous compounds during lacatation. In: A. Baldi and
K.Stelwagen (ed.) Livestock Production Science, Special Issue.
p178. Elsevier Science Publishers, Amsterdam, The Netherlands
Rudland, P. S., A. C. Twiston Davies, and S. W. Tsao. 1984.
Ratmammary preadipocytes in culture produce a trophic agent
formammary epithelia-prostaglandin E2. J. Cell.
Physiol.120:364–376.
Smith-Kirwin, S. M., D. M. O’Connor, J. De Johnston, E. D.
Lancey,S. G.Hassink, and V. L. Funanage. 1998. Leptin expression
inhuman mammary epithelial cells and breast milk. J. Clin.
Endo-crinol. Metab. 83:1810–1813.
Wang J., R. Liu, L. Liu, R. Chowdhury, N. Barzilai, J. Tan, and
L.Rossetti. 1999. The effect of leptin on Lep expression is
tissue-specific and nutritionally regulated. Nat. Med.
5:895–899.
Woodward, T. L, J. W. Xie, and S. Z. Haslam. 1998. The role
ofmammary stroma in modulating the proliferative response toovarian
hormones in the normal mammary gland. J. Mamm.Gland Biol. Neoplasia
3:117–131.
Zhang, Y., R. Proenca, M. Maffei, M. Barone, L. Leopold, and J.
M.Friedman. 1994. Positional cloning of the mouse obese gene andits
human homologue. Nature (Lond.) 372:425–432.