ARTICLE |
Correspondence to: A. Hahnel, Dept. of Biomedical Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada. E-mail: ahahnel@uoguelph.ca
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Summary |
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The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3. (J Histochem Cytochem 50:415422, 2002)
Key Words: alkaline phosphatase, preattachment embryo, preimplantation embryo, bovine
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Introduction |
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The alkaline phosphatases are a small family of isozymes expressed in diverse species, including bacteria, mammals, reptiles, amphibians, nematodes, and insects (
In mammals, the alkaline phosphatase family consists of two groups, tissue-nonspecific alkaline phosphatase and the tissue-specific alkaline phosphatases. The single tissue-nonspecific alkaline phosphatase (TNAP) gene is expressed in many tissues, including liver, bone, and kidney (
The number of tissue-specific alkaline phosphatases expressed depends on the species. There are two tissue-specific isozymes in mice (
Antibodies are currently available for only bovine intestinal alkaline phosphatase. In other species, however, TNAP and the tissue-specific isozymes have different sensitivities to temperature and chemical inhibition by various L-form amino acids and levamisole. Both mouse and human TNAP are particularly sensitive to inhibition with levamisole and L-homoarginine, and are comparatively insensitive to L-phenylalanine (
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Materials and Methods |
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Tissue Preparation
Adult bovine tissues (liver, kidney, intestine, spleen, testis) were recovered at slaughter, flash-frozen in OCT embedding compound (Immucore Canada; Edmonton, Alberta, Canada) in liquid nitrogen, and stored at -80C. Sections (7 µm) were cut using a cryostat, mounted on slides, fixed for 20 min with 4% paraformaldehyde (Sigma; St Louis, MO), washed three times in PBS, and held briefly in PBS before treatment.
Cumulus cells and bovine oviduct epithelial cells were prepared during the culture of embryos. Cumulus cells were recovered during the stripping of presumptive zygotes by gentle pipetting (see below), washed three times in PBS containing 0.1% polyvinyl pyrolidone (PBS+PVP; Fisher Scientific, Unionville, Ontario, Canada), and the cells were smeared on slides. Bovine oviduct epithelial cells were recovered from co-culture preparation (see below), washed three times in PBS+PVP, and smeared on slides. Cells were allowed to dry overnight, then fixed for 20 min in 4% paraformaldehyde and held in PBS until treatment.
Embryo Preparation
Embryos were produced by in vitro maturation, fertilization, and culture as described previously (
Bovine oviduct epithelial cells for co-culture were prepared as follows. Oviducts were trimmed and rinsed and the epithelium stripped by squeezing the oviduct with fine forceps, forcing sheets of epithelial cells from the oviduct into IVC medium. Large sheets of cells were broken by aspiration through a 25-gauge needle and cultured in 1.5 ml IVC medium for 24 hr. In vitro culture medium for embryos (see above) was conditioned for 24 hr by the addition of the oviduct epithelial cells to culture wells on the day of fertilization, 1824 hr before adding embryos (
Heat Inactivation
There were three independent trials of tissues and embryos. Each trial used tissue from a different individual or a different batch of in vitro-produced bovine embryos. Tissue sections were incubated at temperatures ranging from 25C to 100C (in 5C increments) for 1 hr in PBS in an oven (for temperatures less than 80C) or in a water bath (for temperatures over 80C). Sections were removed after 1 hr and rinsed in PBS. Tissue sections were equilibrated for 10 min in 0.1 M Tris, pH 10.0 (Sigma). They were then incubated for 20 min in the same buffer containing NBT/BCIP substrate [200 µl substrate per 10 ml 0.1 Tris, pH 10.0, NBT (nitroblue tetrazolium chloride), BCIP (5-bromo-4-chloro-3-indolyl phosphate; Boehringer Mannheim, Montreal, Quebec, Canada)] in the dark at room temperature. Sections were rinsed three times with PBS, counterstained for 5 sec with methyl green (0.1% w/v solution; Allied Chemical, New York, NY), rinsed another three times in PBS, and coverslips mounted with Aqua-Polymount (Polyscience; Warrington, PA).
Using bovine tissue sections, incubation at 70C was found to distinguish TNAP from tissue-specific alkaline phosphatase activities, so embryos were incubated for 1 hr at 70C in PBS+PVP in capped 200 µl Eppendorf microcentrifuge tubes (to prevent loss of volume due to evaporation). Embryos were recovered from tubes, equilibrated for 10 min in 0.1 M Tris, pH 10.0, and subjected to the AP protein activity assay as previously described, except that embryos were not counterstained with methyl green. Embryos were mounted in PBS under coverslips sealed with petroleum jelly to prevent evaporation and were scored immediately.
Chemical Inactivation
As for heat inactivation, tissues from different individuals or different batches of embryos were used in each of three trials. Inhibitors were prepared as 500 mM stock solutions in PBS, then further diluted to working solutions in 0.1 M Tris, pH 10.0, directly before use. Working inhibitor concentrations were 50 mM, 25 mM, 10 mM, 5 mM, and 1 mM for levamisole (Sigma), L-phenylalanine (Sigma), and L-homoarginine (ICN Biomedicals; Costa Mesa, CA). Tissue sections on slides were incubated for 1 hr in 0.1 M Tris, pH 10.0, containing an inhibitor. Alkaline phosphatase enzyme activity was then determined by incubation for 20 min in the same inhibitor as the previous incubation, containing NBT/BCIP substrate as above, at room temperature in the dark. Sections were rinsed three times in PBS and coverslips were mounted with Aqua-Polymount and observed under a light microscope.
Results from adult bovine tissues showed that incubation in 50, 25, or 10 mM L-phenylalanine and L-homoarginine, and 10, 5, or 1 mM levamisole, in 0.1 M Tris, pH 10.0, for 1 hr would provide a characteristic pattern of alkaline phosphatase inactivation. Embryos were incubated in inhibitor in 0.1 M Tris, pH 10.0, for 1 hr, then for 20 min in 0.1 M Tris, pH 10.0, plus inhibitor containing NBT/BCIP substrate as described above. Embryos were washed three times in PBS+PVP, mounted, and scored as above.
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Results |
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Heat Inactivation
Bovine tissues known to transcribe only tissue-specific alkaline phosphatase mRNA (
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Embryos were incubated at 70C for 1 hr, because we found that this temperature distinguished TNAP, or a combination of isozymes containing TNAP, from the tissue-specific isozymes in adult tissues. Oocytes and embryos, up to and including the 16-cell stage, were negative for alkaline phosphatase activity when incubated for 1 hr at 70C. Morulae, blastocysts, and hatched blastocysts still were positive after 1 hr of incubation (Table 2), showing a change from TNAP to TSAP2 or 3-expression after the 16-cell stage. Fig 2A, Fig 2B, Fig 2E, and Fig 2F compare alkaline phosphatase activity of 8-cell embryos and early blastocysts incubated at room temperature and 70C.
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Chemical Inactivation
Table 1 summarizes the concentration of inhibitor that eliminated bovine alkaline phosphatase activity in adult tissues. Again, this occurred at the same incremental step for each tissue in all three trials. Fig 1D and Fig 1H show examples of intestine and kidney incubated with 10 mM and 1 mM levamisole, respectively, and Fig 1K and Fig 1L compare spleen incubated in 10 mM and 25 mM L-homoarginine. Levamisole is a potent bovine TNAP inhibitor, effectively blocking all alkaline phosphatase activity in liver at <1 mM, whereas levamisole did not block the intestinal activity until 10 mM and spleen activity (TSAP2 or -3) until 5 mM. Kidney and bovine oviduct epithelial cells, which transcribe a combination of TNAP and TSAP3, require 1 mM LEV, similar to liver TNAP, but cumulus cells, which contain message for TNAP + TSAP2, required 5 mM levamisole for inactivation, like spleen. Bovine liver TNAP was inactivated by 25 mM L-homoarginine, bovine intestinal alkaline phosphatase by >50 mM, and bovine spleen by 50 mM. But kidney again behaved as TNAP, while bovine oviduct epithelial cells and cumulus cells required concentrations like those of the intestinal isozyme (>50 mM L-homoarginine). Liver required >50 mM L-phenylalanine for inactivation, while intestinal tissue required 50 mM and spleen 25 mM. Again, kidney and oviduct epithelial cells did not match, although containing mRNA for the same isozymes, with kidney requiring 50 mM and the oviduct cells needing >50 mM. Cumulus cells also required >50 mM L-phenylalanine for inactivation.
In preattachment embryos, the inactivation of alkaline phosphatase by chemical inhibitors changed with each stage examined (Table 2). Examples of early blastocysts and 8-cell embryos incubated in 5 mM levamisole and 50 mM L-phenylalanine are shown in Fig 2C, Fig 2D, Fig 2G, and Fig 2H. Oocytes, 2-, 4-, and 8-cell embryos required <1 mM levamisole to inhibit alkaline phosphatase activity. Sixteen-cell through hatched blastocysts required >1 mM levamisole, 5 mM for 16-cell and morula, 10 mM for blastocysts, and >10 mM for hatched blastocysts. Oocytes and 2-cell embryos required <10 mM L-homoarginine, while 4- and 8-cell embryos required 50 mM and 16-cell embryos required >50 mM for inactivation of alkaline phosphatase activity. Oocyte activity was inhibited by <10 mM L-phenylalanine, 2-cell through 16-cell embryos by 25 mM, morula and all blastocysts by >50 mM. Again, there was no evidence of two-step inactivation, even in embryos known to have mRNA for two isozymes, and the activity on all cells seemed equally sensitive within an embryo.
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Discussion |
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Inhibition of alkaline phosphatase activity by temperature and levamisole provided the clearest distinctions between bovine isozymes in this histochemical analysis. Adult bovine tissues transcribing only a tissue-specific or combination of tissue-specific mRNA require higher temperatures and higher levamisole concentrations to inhibit alkaline phosphatase activity than tissues expressing TNAP alone (see intestine and spleen vs liver in Table 1). This corresponds with data from mice reported by
The alkaline phosphatases usually are described as homodimers. In mouse and humans there is expression of different isozymes (homodimers) by different cell types, e.g., in the testis (5 mM levamisole. This was not observed. If only one isozyme formed functional protein in cumulus cells (although two are transcribed), then the inhibition profile would probably resemble that of either TSAP2 or TNAP alone. Heat inactivation of cumulus cells was like TNAP, and the levamisole inhibition like that of a tissue-specific alkaline phosphatase. One explanation for the response to the different inhibitors is suggested by the report that functions of human placental alkaline phosphatase subunits are only partly independent of each other (
Inhibition of bovine intestinal alkaline phosphatase and TNAP with L-homoarginine and L-phenylalanine was similar to what has been described for the mouse isozymes (
The inhibition profiles of the bovine alkaline phosphatases in adult tissues proved to be useful for analyzing expression in preattachment bovine embryos. Bovine embryos transcribe mRNA for TSAP2 and TSAP3 starting at the 4-cell and 8-cell stages, respectively (
Transition from a cleavage-stage embryo to a blastocyst is required for further development and is associated with a number of processes. The first burst of transcription from the embryonic genome in cows occurs at the 8-cell stage (
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Footnotes |
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1 Current address: The John P. Robarts Research Inst., Neurodegeneration Group, London, Ontario, Canada.
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Acknowledgments |
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Supported by grants from NSERC Canada to AH.
We wish to thank Gencor, Inc. (Guelph, Ontario) for donating the bovine sperm used in IVF, the Animal Biotechnology and Embryo Laboratory for providing bovine ovaries, and Kevin O'Reilly for assistance in preparing the figures. KM was supported by an Ontario Government Scholarship.
Received for publication November 11, 2000; accepted October 5, 2001.
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