ARTICLE |
Correspondence to: Hans-Martin Seyfert, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany. E-mail: seyfert@fbn-dummerstorf.de
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The activity of the enzyme acetyl-CoA-carboxylase (ACC-
) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PIPIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-
-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-
synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution. (J Histochem Cytochem 51:10731081, 2003)
Key Words:
milk fat synthesis, acetyl-CoA carboxylase , in situ hybridization, mammary gland, bovine, ovine, ruminants, promoters, gene expression
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ORGANISMS need fatty acids for a variety of unrelated functions. They serve in all cells to form the cell membrane lipid bilayers and are also used in specialized tissues to store energy in the form of fat. In mammals, the fat secreted into milk nourishes the suckling infant. Milk fat, like most other milk ingredients, is synthesized in the mammary epithelial cells (MECs; (ACC-
; E.C.6.4.1.2) is one of the components crucial to this pathway.
Activity of the ACC- is known to be rate-limiting for the de novo formation of fatty acids (for reviews see
Different tissue-specific restrictions are imposed on the initiation of transcription from these promoters. PI is primarily active in adipose tissue and its activation is nutritionally regulated in liver (10-fold) by lactation in rats (
-encoding gene (
All three promoters are equipped with a unique set of cis-regulatory elements. Given the interest in understanding the hormonal and nutritional controls influencing the formation of milk fat, it is necessary to elucidate the specific role of any one of these promoters in the formation of milk fat. Therefore, we used in situ hybridization (ISH) to examine which of these promoters is used in MECs, the relevant cell type for milk fat formation.
![]() |
Materials And Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of Probes
Probes of bovine origin were used throughout. Isolation and molecular characterization of the entire bovine promoter region have been described (
Probe Labeling
Probes were labeled with 20 U of either T7 or SP6 polymerases (Promega) in 20 µl reaction volumes. They contained the appropriate reaction buffer as provided by the supplier and we added 10 mM DTT, 1 U of RNasin (Promega), 10 mM each of rATP, rGTP, rCTP, and 25 µCi of [35S]-UTP (Amersham; Little Chalfont, UK).
Slide Preparation and General Procedures for ISH
Serial sections (7-µm) were cut from paraformaldehyde (4%)-fixed and wax-embedded udder samples prepared from freshly slaughtered, pregnant, lactating, or involuting sheep and cows. They were mounted adjacently on ATS (SigmaAldrich, St Louis, MO; cat. no. A3648)-coated slides. We followed the general procedures for slide preparation and hybridization with radioactively labeled riboprobes to such sections essentially as previously described (S1-casein control probe used. 35S-labeled riboprobes were generated from the respective subclones. Approximately 40,000 cpm of the radiolabelled sense and antisense probe were added per µl of hybridization mix. Then 30 µl of hybridization mix was applied to each section. Probes were hybridized overnight at 58C. The hybridization mixes contained 0.2M DTT and washing solutions contained 20 mM of ß-mercaptoethanol. The slides were washed in 50% formamide, 2 x SSC, 20 mM ß-mercaptoethanol at 55C (twice for 15 min), then with several changes of 0.2 x SSC. Subsequently, all single-stranded RNA was digested away with RNase A and T1 for 30 min (10 µg/ml and 2.5 µg/ml, respectively; Roche Diagnostics, Basel, Switzerland), then rewashed in 0.2 x SCC. Slides were dehydrated in a graded series of ethanol, all containing 10 mM ß-mercaptoethanol. They were coated with LM-1 nuclear emulsion RPN40 (Amersham), allowed to develop at 4C in sealed desiccant-containing boxes, and developed after an exposure of 2 weeks (
S1-casein) or 2.5 months (all ACC-
probes). They were counterstained with Gill's rapid haematoxylin and eosin.
Quantification of ISH Signal Intensities over Various Cell Types
An Olympus BH2 microscope and PM-10ADS photomicrographic equipment were used to record the relative signal intensities by setting the camera controller to spot metering (1% of the field view) and positioning the measuring area over the cell types under examination. The x40 objective was used for the cow measurements and the x100 objective used for the sheep measurements. Objective measurements were obtained by setting the illumination to full intensity and reading the exposure time displayed by the camera controller. This gave a direct measure of the signal strength, since the length of the exposure time corresponded to the extent to which the deposited silver grains blocked the light and hence gave a measure of their number. Measurements were taken of 10 representative cell types and averaged. Background was corrected by subtracting the average of six measurements from corresponding cell populations from the adjacent control sections.
RT- and Real-Time Quantification PCR
RNA for RT- and real-time PCR experiments was extracted using TRIZOL (Life Technologies; Karlsruhe, Germany) as prescribed by the manufacturer. The cDNA for these assays was primed in reverse with the oligonucleotide primer Acex8_9r (5'-CAGCCAGCCCAAACTGCTTGTTGCAC) bridging exons 8 and 9. This cDNA was used to amplify the ovine exon 1 with the primers bovine/ovine Ac1cx1f: 5'-GTCTGTCCATCTGTGAAGTATC; reverse Acex5r: 5'-CTCATGTGTAAGGCCA(G/A) ACCAT (G for bovine, A for ovine amplifications, respectively). The abundance of total and PI-derived transcripts in mammary glands from sheep was assayed with the Light Cycler device and assay kits (Roche Diagnostics), basically as described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clear association of the signals from all of our ACC- specific antisense probes with MECs is the most prominent feature seen in our hybridizations. The sense control probes, on the other hand, did not give rise to any specific accumulation of silver grains above any specific cell type or suborgan region. A set of serial sections from a mammary gland of the sheep at 1 week of lactation is shown in Fig 1. The low-magnification overviews (Fig 1A, Fig 1C, Fig 1E, and Fig 1G) show that all alveoli expressing the
S1-casein-encoding gene are also expressing the ACC-
encoding gene, as transcribed from all three promoters. The higher magnifications (Fig 1B, Fig 1D, Fig 1F, and Fig 1H) clearly show that the MECs are the site of ACC-
expression. Although in this example the lower left bundle of alveoli is devoid of
S1-casein messages, ACC-
-derived transcripts are clearly evident here when hybridized with the exon 5-derived probe detecting messages stemming from both promoters, PI and PII. This shows that
S1-casein and ACC-
gene expression are not strictly coupled, at least in the stage of early lactation.
|
Cells of the connective tissue and those lining the blood vessel (Fig 1B, Fig 1D, and Fig 1F) are clearly labeled with the probe of combined specificity for PI and PII, while the signals of the two other ACC- specific probes appear either to be reduced (blood vessel) or absent (connective tissue).
MECs express transcripts initiated from all ACC--encoding promoters also at full lactation (Fig 2). The example shown was taken from bovine sections and is also representative of the images seen from sheep tissue at full lactation. There appears to be considerable variation in the intensity of ACC-
expression among even closely adjacent alveoli. In addition to the heterogeneity of milk protein gene expression associated with histologically obvious changes in alveolar morphology, hybridization for
S1-casein and
-lactalbumin reveals such substantial variation in the status of alveolar activity for these mRNAs also in cows and sheep alveoli that appear otherwise histologically similar (unpublished observations; and
(e.g., Fig 2, running from upper left to bottom). However, ACC-
expression of such cells may be readily seen in other ducts (not shown).
|
ACC- expression may be uncoupled from
S1-casein gene expression also in stages of mammary gland differentiation (Fig 3). This can be clearly seen in sections taken from either pregnant or involution animals. The examples shown in Fig 3 were taken from cows but, again, are representative for sheep as well. It appears from the sections of the pregnant animal that some groups of alveoli are not yet prepared for
S1-casein gene expression (Fig 3D, arrow), while already well engaged in ACC-
gene expression (Fig 3A3C). Likewise, we observe that, during involution ACC-
gene expression may eventually persist longer in residual alveolar regions than does the expression of the
S1-casein-encoding gene (Fig 3E3H).
|
The images from the pregnant cow (Fig 3A3D) show similar findings to those from the sheep at 1 week of lactation (cf. Fig 1). Specifically, as seen in sheep, it appears that adipocytes are most prominently labeled by the exon 5-derived probe, detecting messages initiated from either PI or PII (Fig 3C, inset). Neither the PI- nor the PIII-specific probe seems to give rise to specific signals in this area. The qualitative evaluation of the hybridization signals therefore augments the conclusion drawn already from sheep (Fig 1) that PII might be the relevant promoter for fatty acid synthesis in the adipocytes of the mammary gland. Likewise, the exon 5-specific probe gives rise to quite abundant silver grains over areas of connective tissue containing stromal cells during involution. Neither one of the other two promoter-specific probes reveals substantial mRNAs there.
The three different ACC--specific probes gave rise to hybridization signals of quite different intensities. Specifically, the signals from the exon 5-derived probe with the combined specificity for transcripts derived from PI and PII appeared to be much stronger than those from the other two probes. This makes it difficult to evaluate the relative abundances of transcripts across hybridizations. Therefore, we measured the density of silver grains at 10 different locations over selected cell types on any given slide to obtain an unbiased estimate of the relative abundance of the respective transcripts in different cell types. The analysis shows (Fig 4) that highly casein-expressing alveoli contain the highest concentrations of transcripts complementary to all three ACC-
-specific probes. Moreover, it becomes very clear that the expression of PIII is not restricted to MECs, but is also quite prominent in other cell types, particularly in the lactating udder of the cow (Fig 4A). Expression of PI, on the other hand, is clearly not restricted to adipose tissue, but is also quite prominent in blood vessels or cells lining the ducts. Finally, we note that the relative abundance of silver grains caused by the exon 5-specific probe above adipose tissue is not stronger than that caused by the other probes (Fig 4C and Fig 4D, filled columns). The mere visual inspection of the hybridizations had provoked a different interpretation (cf. insets in Fig 3A3C).
|
Recently, a lack of PI activity in the mammary gland of the sheep was reported (
|
We used real-time PCR to compare more precisely the abundance of PI-derived transcripts in lactating and pregnant udders of the sheep (Table 1). We assayed RNA preparations from lactating or pregnant sheep (three each) for the abundances of transcripts derived from PI and PIII. Moreover, we measured the relative abundance of all ACC--encoding transcripts from lactating ewes to approximately evaluate the contribution of PI-derived transcripts to all ACC-
-encoding messages in the lactating mammary gland of the ewe. We included a set of four samples from lactating cows to compare this experiment with previous reports (
-encoding transcripts, unlike in cattle (30%). Although these RT-PCR quantifications are not exactly comparable across different transcripts, due to different primer efficiencies, it is clear that the activity of PI contributes much more to the total amount of Acc-
-derived transcripts in the cow than in lactating sheep. Together, these data show that PI is indeed expressed in the lactating mammary gland of the ewe, but at lower levels than in cattle.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The significant result from our study is that all three promoters of the ACC--encoding gene are expressed in MECs at any developmental stage of the gland. This holds for both species examined, sheep and cattle. The data imply that none of these promoters must be neglected in analyzes aiming to disclose regulatory mechanisms governing the extent of milk fat synthesis, particularly in cattle. This conclusion could not have been drawn from previous data, which all used whole gland extracts to analyse ACC-
-derived gene expression. Previous work from other groups as well as our own suggests that milk fat synthesis might be regulated primarily either by PII in murine species (
An evaluation of the relative contribution of any one of the ACC--expressing promoters to control the total abundance of ACC-
-encoding transcripts in the mammary gland at lactation leads to different conclusions for different species. In rat, the activity of PII only was found to increase about 10-fold with lactation (
30% of all ACC-
-encoding transcripts (
-encoding messages. The same applies for PII-derived transcripts (
Our data show that basically all promoters appear to be expressed in all cell types of the mammary gland. The visual inspection of the hybridizations had always revealed very strong signals where the exon 5 probe was applied, having specificity for transcripts from both promoters, PI and PII. Although this could have provoked the view that PII would be more widely expressed than the other two promoters, the photometrical determination of the signal intensities showed indeed that this is not the case (Fig 4). These measurements rather indicate that the ACC- mRNA abundance, as measured from whole gland extracts, cannot be attributed to a specific cell type as single source or to the predominant activity of a specific promoter within a distinct cell type.
The ACC--encoding gene may be expressed in such alveoli and MECs that are not expressing the
S1-casein-encoding gene. This is evident in the sections taken at early lactation (Fig 1) but also from pregnant (Fig 3) or involuting cows or sheep (latter not shown). These qualitative data suggest that the temporal control of expression during lactational development of the gland may be tighter for the
S1-casein-encoding gene than for that of the ACC-
. Perhaps it would be more interesting to know if such an uncoupling of casein and fatty acid synthesis occurs within any one MEC at full lactation. This would have significant implications regarding the molecular control mechanisms coordinating casein and fatty acid synthesis within the MEC. However, this would require electron-microscopic analysis of ultrathin sections and cannot be resolved using our 7-µm sections as analyzed with 35S-labelled probes.
In conclusion, we have shown that all three ACC- promoters are active in MECs of the mammary glands from cow and sheep and that ACC-
expression is not necessarily linked to that of major milk proteins. Our data together also reveal a striking difference between cattle and sheep regarding the relevance of PI for milk fat synthesis. Whereas the contribution of PI may be very limited in sheep, it contributes significantly to establishment of the final concentration of the ACC-
-encoding transcripts in the mammary gland of the lactating cow. This is a remarkable difference in the mammary gland-specific use of this promoter, which is evolutionarily truly conserved between cattle and sheep.
![]() |
Footnotes |
---|
1 Present address: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX.
![]() |
Acknowledgments |
---|
Supported by the Deutsche Forschungsgemeinschaft (DFG grant Se 326/10-2), ISTAD grant No 00-FRG-21-WHEE), and the New Zealand Foundation for Research Science and Technology.
Received for publication October 11, 2002; accepted March 19, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albuquerque LG, Dimov G, Keown JF, Van Vleck LD (1995) Estimates using an animal model of (co)variances for yields of milk, fat, and protein for the first lactation of Holstein cows in California and New York. J Dairy Sci 78:1591-1596
Barber MC, Travers MT (1998) Eludication of a promoter activity that directs the expression of acetyl-CoA carboxylase a with an alternative N-terminus in a tissue-restricted fashion. Biochem J 333:17-25[Medline]
Dematawewa CM, Berger PJ (1998) Genetic and phenotypic parameters for 305-day yield, fertility, and survival in Holsteins. J Dairy Sci 81:2700-2709
Gallego MI, Binart N, Robinson GW, Okagaki R, Coschigano KT, Perry J, Kopchick JJ et al. (2001) Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects. Dev Biol 229:163-175[Medline]
Hardie DG (1989) Regulation of fatty acid synthesis via phosphorylation of acetyl-CoA carboxylase. Prog Lipid Res 28:117-146[Medline]
Hayes JF, Ng KHK, Moxley JE (1984) Heritability of milk casein and genetic and phenotypic correlations with production traits. J Dairy Sci 67:841-846[Medline]
Iritani N (1992) Nutritional and hormonal regulation of lipogenic-enzyme expression in rat liver. Eur J Biochem 205:433-442[Medline]
Kim K-H (1997) Regulation of mammalian acetyl-coenzyme A carboxylase. Annu Rev Nutr 17:77-99[Medline]
Kim K-H, LopezCasillas F, Bai DH, Luo X, Pape ME (1989) Role of reversible phosphorylation of acetyl-CoA carboxylase in long chain fatty acid synthesis. FASEB J 3:2250-2256
LopezCasillas F, PonceCastaneda MV, Kim KH (1991) In vivo regulation of the activity of the two promoters of the rat acetyl coenzyme-A carboxylase gene. Endocrinology 129:1049-1058[Abstract]
Luo X, Kim K-H (1990) An enhancer element in the house-keeping promoter for acetyl-CoA carboxylase gene. Nucleic Acids Res 18:3249-3254[Abstract]
Mao J, Marcos S, Davis SK, Burzlaff J, Seyfert H-M (2001) Genomic distribution of three promoters of the bovine gene encoding acetyl-CoA carboxylase alpha and evidence that the nutritionally regulated promoter I contains a repressive element different from that in rat. Biochem J 358:127-135[Medline]
Mao J, Molenaar AJ, Wheeler TT, Seyfert H-M (2002) Stat5 binding contributes to lactational stimulation of promoter III expressing the bovine acetyl-CoA-carboxylase -encoding gene in the mammary gland. J Mol Endocrinol 29:73-88
Mao J, Seyfert H-M (2002) Promoter II of the bovine acetyl-coenzyme A carboxylase-alpha-encoding gene is widely expressed and strongly active in different cells. Biochim Biophys Acta 1576:324-329[Medline]
Molenaar AJ, Davis SR, Jack LJW, Wilkins RJ (1995) Expression of the butyrophilin gene, a milk fat globule membrane protein, is associated with the expression of the S1 casein gene. Histochem J 27:388-394[Medline]
Molenaar AJ, Davis SR, Wilkins RJ (1992) Expression of -lactalbumin,
-S1-casein, and lactoferrin genes is heterogenous in sheep and cattle mammary tissue. J Histochem Cytochem 40:611-618
Neville MC, Picciano MF (1997) Regulation of milk lipid secretion and composition. Annu Rev Nutr 17:159-183[Medline]
PonceCastaneda MV, LópezCasillas F, Kim K-H (1991) Acetyl-coenzyme A carboxylase messenger ribonucleic acid metabolism in liver, adipose tissues, and mammary glands during pregnancy and lactation. J Dairy Sci 74:4013-4021
Rohlfs EM, Louie DS, Zeisel SH (1993) Lipid synthesis and secretion by primary cultures of rat mammary epithelial cells. J Cell Physiol 157:469-480[Medline]
Travers MT, Vallance AJ, Gourlay HT, Gill CA, Klein I, Bottema CB, Barber MC (2001) Promoter I of the ovine acetyl-CoA carboxylase-alpha gene: an E-box motif at -114 in the proximal promoter binds upstream stimulatory factor (USF)-1 and USF-2 and acts as an insulin-response sequence in differentiating adipocytes. Biochem J 359:273-284[Medline]
Wakil SJ, Stoops JK, Joshi VC (1983) Fatty acid synthesis and its regulation. Annu Rev Biochem 52:537-579[Medline]
Witkiewicz H, Bolander ME, Edwards DR (1993) Improved design of riboprobes from pBluescript and related vectors for in situ hybridization. Biotechniques 14:458-463[Medline]