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Abstract

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.

Keywords

alkaline phosphatase preattachment embryo preimplantation embryo bovine

The Alkaline Phosphatases are a small family of isozymes expressed in diverse species, including bacteria, mammals, reptiles, amphibians, nematodes, and insects (McComb et al. 1979; Millan 1988,1990). Conservation of alkaline phosphatase expression hints at an important role, but to date there is little biochemical evidence for alkaline phosphatase function except in bone mineralization (Yoon et al. 1989; Beertsen and van den Bos 1992; Fedde et al. 1999) and de-phosphorylation of adenosine nucleotides (Gallo et al. 1997). The alkaline phosphatases have been postulated to be involved in a range of other processes, including cell adhesion (Hui et al. 1993), vitamin B transport (Rindi et al. 1995), metastasis (Manara et al. 2000), and cell signaling (Swarup et al. 1981; Muller et al. 1991). We are interested in their role in the development of mammalian preimplantation embryos.

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 (Garattini et al. 1987; Hsu et al. 1987; Harris 1989; Hahnel et al. 1990; MacGregor et al. 1995; Hoshi et al. 1997; McDougall et al. 1998). Humans with mutations in the TNAP isozyme have defective bone mineralization (infantile hypophosphatasia; Henthorn et al. 1992), and mice engineered to lack TNAP have poor bone mineralization plus a range of defects that result in postnatal death (Narisawa et al. 1997).

The number of tissue-specific alkaline phosphatases expressed depends on the species. There are two tissue-specific isozymes in mice (Manes et al. 1990): intestinal alkaline phosphatase and embryonic alkaline phosphatase (EAP). The EAP isozyme is the major isozyme expressed by 2-cell to blastocyst stage embryos and M-phase spermatogenic cells in the testis (Hahnel et al. 1990; Manes et al. 1990; Narisawa et al. 1992; MacGregor et al. 1995). Mice homozygous null for the mouse EAP gene (EAP knockout) are viable (Narisawa et al. 1997). However, we found that its absence disadvantages preimplantation embryos (Dehghani et al. 2000). Fewer homozygous null embryos survive than wild-type, particularly under even mild stress, such as culture. The results suggest that, in mice, EAP is part of an essential process in blastocyst formation, although its role appears to be duplicated by another molecule. Whether alkaline phosphatase plays a similar role in preimplantation development in other mammals remains to be determined. There are three human tissue-specific alkaline phosphatases: intestinal, germ cell, and placental (Harris 1989). Ectopic expression of alkaline phosphatases is often found in cancer cells (Millan 1988,1992; Millan and Manes 1988). It is not known whether human preimplantation embryos express a tissue-specific alkaline phosphatase. In cattle, several different cDNA sequences have been cloned from intestine (Weissig et al. 1993; Manes et al. 1998), and we have cloned two tissue-specific cDNAs (TSAP2 and TSAP3) from blastocysts (McDougall et al. 1998). Transcription of TSAP2 is first detected at the 4-cell stage in the in vitro-derived bovine embryo and TSAP3 at the 8-cell stage. This is equivalent to the mouse 2-cell stage relative to the initial burst of transcription from the embryonic genome (Telford et al. 1990). Therefore, one or both of these bovine isozymes may be the functional equivalent of the mouse EAP. TSAP2 also is transcribed in testis, spleen, and cumulus cells, and TSAP3 is transcribed by kidney and oviduct epithelial cells (McDougall et al. 1998). A complication in the comparison to mouse is that alkaline phosphatase activity is detected in all stages from oocyte to blastocyst, although transcription of alkaline phosphatase does not begin until the late 4-cell stage (McDougall et al. 1998). It is assumed that the early activity is due to maternal protein from mRNA transcribed during oogenesis. Which isozyme is responsible for the maternal activity is unknown. This research was designed to determine the isozyme(s) responsible for the protein activity encountered in preattachment bovine embryos at the various stages.

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 (Harris 1989; Lepire and Ziomek 1989; Millan 1990). The tissue-specific isozymes are comparatively insensitive to levamisole and L-homoarginine and sensitive to L-phenylalanine inhibition (Harris 1989; Lepire and Ziomek 1989; Millan 1990). The human and murine TNAPs are inactivated by temperatures less than 60C, whereas the tissue-specific isozymes typically withstand higher temperatures (up to 90C; Harris 1989; Lepire and Ziomek 1989). Therefore, we began this study with examination of the response of bovine tissues transcribing various alkaline phosphatase isozymes to heat and chemical inhibition and subsequently used the information to assay the AP isozymes expressed in preattachment bovine embryos. Because of the difficulty of obtaining sufficient material for biochemical tests of alkaline phosphatase from preattachment bovine embryos, a histochemical assay was used throughout.

Materials and Methods

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 (Xu et al. 1992), with the following modifications. Cumulus-oocyte complexes were aspirated from visible follicles less than 1 cm in diameter using an 18-gauge needle into modified Ham's F-10 medium (Gibco BRL; Burlington, Ontario, Canada) containing 10 mM HEPES (Gibco BRL), 2 U/ml heparin (Hepalean; Organon Teknica, Toronto, Ontario, Canada), and 2% heat-inactivated steer serum. They were matured for 22–24 hr in TCM-199 medium (HEPES buffered medium 199 with Earle's salts; Gibco BRL) containing 55 mg/ml sodium pyruvate and 29 μg/ml L-glutamine (Sigma) and 1.2% heat-inactivated steer serum. After maturation, oocytes were washed twice in sperm-TALP (Greve et al. 1987) buffered with 10 mM HEPES (IVF medium) and placed in this medium for fertilization. Sperm were prepared by thawing two 0.5-ml straws at 38C for 1 hr in IVF medium, subjecting to swim-up for 1 hr in IVF medium, and pelleting by centrifugation. Sperm were added to the matured oocytes at a concentration of 1.0 × 10⁶ sperm per ml, and incubated for 18 hr at 38C in 5% CO₂ in humidified air. Presumptive zygotes were stripped of cumulus by gentle vortexing and co-cultured with oviduct epithelial cells in Medium 199 with Earle's salts to which was added 55 mg/ml pyruvate, 29 μg/ml glutamine, 0.35% bovine serum albumin (fraction V; Sigma) and 1.2% steer serum (IVC medium). Embryos were removed from culture at specific stages of development (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst) to assay for AP activity. They were rinsed three times in PBS+PVP, fixed in 4% paraformaldehyde for 1 hr, washed three times in PBS+PVP, and held in PBS+PVP.

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, 18–24 hr before adding embryos (Xu et al. 1992).

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.

Results

Heat Inactivation

Bovine tissues known to transcribe only tissue-specific alkaline phosphatase mRNA (McDougall et al. 1998) were found to require a higher temperature (90C) to fully eradicate alkaline phosphatase activity than bovine liver (65C), which transcribes only tissue-nonspecific alkaline phosphatase (Table 1). Three trials gave identical results. Figure 1 shows examples of intestinal tissue incubated at room temperature, 80C, and 90C. Bovine spleen, which transcribes mRNA for TSAP2 and TSAP3, also required incubation for 1 hr at 90C to fully eradicate activity (Figures 1I-1J). Kidney, bovine oviduct epithelial cells, and cumulus cells transcribe both TNAP and a tissue-specific isozyme, as determined by RT-PCR (McDougall et al. 1998), and therefore could, in theory, express a combination of alkaline phosphatase proteins. These tissues required a temperature similar to that of liver TNAP for inactivation (Table 1). As shown for kidney in Figures 1E-1G, all positive cells were inactivated at the same temperature (60C); there were no cells with residual thermotolerant activity.

Table 1

Inhibitor concentrations (mM) and temperature (C) required to inactivate alkaline phosphatase (AP) enzyme activity in adult bovine tissues with a 1 hr incubation^a

Tissue	mRNA^b	Temp	PHE	HOM	LEV
Intestine	IAP	90	50	>50	10
Liver	TNAP	65	>50	25	<1
Kidney	TNAP TSAP3	60	50	25	<1
BOEC	TNAP TSAP3	65	>50	>50	1
Cumulus	TNAP TSAP2	60	>50	>50	5
Spleen	TSAP2 TSAP3	90	25	50	5

BOEC, bovine oviduct epithelial cells; PHE, L-phenylalanine; HOM, L-homoarginine; LEV, levamisole; IAP, intestinal AP; TSAP2, tissue-specific AP 2; TSAP3, tissue-specific AP 3; TNAP, tissue-nonspecific AP.

Alkaline phosphatase mRNA present as determined by RT-PCR (McDougall et al. 1998).

Table 2

Inhibitor concentrations (mM) and temperature (C) that inactivate alkaline phosphatase activity in preattachment bovine embryos^a

Stage	mRNA^c	Temp^d	PHE	HOM	LEV	AP(d)^e
Oocyte	—^b	<70	<10	<10	<1	TNAP
2-cell	—^b	<70	25	<10	<1	TNAP
4-cell	TSAP2	<70	25	50	<1	TNAP
8-cell	TSAP2 TSAP3	<70	25	50	<1	TNAP
16-cell	TSAP2 TSAP3	<70	25	>50	5	TNAP TSAP
Morula	TSAP2 TSAP3	>70	>50	>50	5	TSAP
Blastocyst	TSAP2 TSAP3	>70	>50	>50	10	TSAP
Hatched blastocyst	TSAP2 TSAP3 TNAP IAP	>70	>50	>50	>10	TSAP

PHE, L-phenylalanine; HOM, L-homoarginine; LEV, levamisole; IAP, intestinal AP; TSAP2, tissue-specific AP 2; TSAP3, tissue-specific AP 3; TNAP, tissue-nonspecific AP.

No AP mRNA was detected in oocytes and 2-cell embryos by RT-PCR (McDougall et al. 1998).

Messenger RNA found to be present by McDougall et al. (1998).

Embryos were incubated at 70C; <70 represents inhibition of AP activity while >70 denotes activity detected after incubation.

AP isozyme determined to be present based on the data presented.

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. Figures 2A, 2B, 2E, and 2F compare alkaline phosphatase activity of 8-cell embryos and early blastocysts incubated at room temperature and 70C.

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. Figures 1D and 1H show examples of intestine and kidney incubated with 10 mM and 1 mM levamisole, respectively, and Figures 1K and 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 Figures 2C, 2D, 2G, and 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.

Figure 1

Effects of heat and chemical inhibition on alkaline phosphatase enzyme activity. Cryosections of adult bovine intestine (A-D), kidney (E-H), and spleen (I-L) were reacted with NBT/BCIP after treatment with heat or chemical inhibitors for 1 hr. Dark precipitate indicates areas of alkaline phosphatase activity. (A-D) Intestinal epithelium incubated at room temperature (A), 80C (B), 90C (C), and with 10 mM LEV (D). Intestinal crypts (c) are negative; the epithelium of the intestinal villi (v) is positive for AP activity. (E-H) Kidney cortex incubated at 50C (E), 55C (F), 60C (G), and with 1 mM LEV (H). Glomeruli (g) are negative, and proximal convoluted tubules (pt) are positive for AP activity. (I-L) Spleen incubated at 75C (I) and 80C (J), and with 10 mM HOM (K) and 25 mM HOM (L). White pulp (wp) is positive for AP activity. (C,G,H,J,L) Considered negative. Bars: A,I = 200 μm; E = 100 μm.

Figure 2

Effects of heat and chemical inhibition on alkaline phosphatase enzyme activity in preattachment bovine embryos. Whole-mount preparations of 8-cell embryos (A-D) and early blastocysts (E-H) were reacted with NBT/BCIP after treatment with heat or chemical inhibitors for 1 hr. Dark precipitate indicates alkaline phosphatase activity. (A-D) Eight-cell embryos incubated at room temperature (A) and 70C (B), and with 1 mM LEV (C) and 25 mM PHE (D). E-H Early blastocysts incubated at room temperature (E) and 70C (F), and with 5 mM LEV (G) and 50 mM PHE (H). In the early blastocysts (E-H), the dark precipitate obscures the fluid-filled cavity (blastocoel) in the embryonic ball of cells in all but G. Bar = 50 μM.

Discussion

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 Lepire and Ziomek (1989) and with data from humans reported by Harris (1989).

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 (Narisawa et al. 1992). However, formation of functional heterodimers has been found in human cell lines (Wray and Harris 1982; Kodama et al. 1994) and in neutrophils of individuals with Down's syndrome (Vergnes et al. 1999). In bovine preattachment embryos there is simultaneous transcription of TSAP2 and TSAP3 by what appears to be a homogeneous population of embryonic cells. Therefore, it is interesting to compare the bovine tissues transcribing a combination of isozymes to tissues transcribing a single class of alkaline phosphatase. The results suggest that TNAP/TSAP2 heterodimers may be formed in cumulus cells. If there were two distinct homodimer populations in cumulus cells, it would be expected that TNAP would be inactivated before TSAP2 with heat and levamisole. This would result in a loss of activity from some cells or a partial loss of activity in all cells at approximately 60C and <1 mM levamisole, with a further loss at 90C and ≥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 (Hoylaerts et al. 1997). In contrast, bovine kidney and oviduct epithelial cells transcribe TNAP and TSAP3. Neither tissue had an inhibition profile that was clearly distinct from liver with TNAP alone. It is possible that TSAP3 is not translated or that it behaves differently from TSAP2 in combination with TNAP. Unfortunately, bovine TSAP2 and TSAP3 messages were not detected alone in a tissue, and therefore their individual temperature and levamisole sensitivities could not be determined.

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 (Lepire and Ziomek 1989) and human (Fish-man 1974; Harris 1989; Millan 1990). However, the differences between the classes were smaller in our histochemical assay and were not helpful in further distinguishing the different 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 (McDougall et al. 1998). However, maternally derived activity is found in the oocyte and is presumably the predominant activity present on at least 1-cell to 4-cell embryos. The inhibition profile of alkaline phosphatase activity on oocytes through 8-cell embryos was distinctly that of TNAP, with inactivation at low temperature and high levamisole. Therefore, TNAP is the maternally-inherited isozyme and the predominate alkaline phosphatase during cleavage. By the 16-cell stage the inhibition was similar to that of cumulus cells, suggesting residual maternal TNAP plus a newly transcribed tissue-specific isozyme (either TSAP2 or TSAP3). After the 16-cell stage the profile was that of a tissue-specific alkaline phosphatase, indicating full replacement of maternal TNAP with embryonic TSAP2 or −3. In hatched blastocysts, TNAP message is again detected but the activity profile still is that of a tissue-specific isozyme, indicating that little or no functional TNAP is synthesized before implantation. Whether both TSAP2 and TSAP3 are translated in morulae and blastocysts could not be determined, but this study shows that TNAP is expressed during early cleavage and is supplanted by TSAP2 or TSAP3 by the morula stage.

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 (Telford et al. 1990), and an outer epithelium (the trophectoderm) differentiates over the 16- to 32-cell stage (morula). During the equivalent stages in mice, there is evidence for chromatin remodeling and post-translational modifications in the activities of a variety of proteins commonly associated with epithelia (reviewed in Pratt 1989; Collins and Fleming 1995; Thompson 1996). Although there is strong evidence that phosphorylation is important in these events, there has been little research on the enzymes that dephosphorylate proteins in preimplantation embryos. We found that a homozygous null mutation of EAP in preimplantation mouse embryos is associated with loss of 8-cell stage embryos, when epithelialization of the outer cells begins (Dehghani et al. 2000). In the bovine, TSAP2 or TSAP3 protein activity is first detected in 16-cell stage embryos and morulae, as an epithelium begins to form in this species. This lends support to a hypothesis that a tissue-specific alkaline phosphatase is generally expressed by mammalian preimplantation embryos, and that it plays a role in forming the trophectoderm.

Footnotes

Acknowledgements

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.

References

Beertsen

van den Bos

(1992) Alkaline phosphatase induces the mineralization of sheets of collagen implanted subcutaneously in the rat. J Clin Invest 89: 1874–1980

Collins

Fleming

(1995) Epithelial differentiation in the mouse preimplantation embryo: making adhesive cell contacts for the first time. Trends Biol Sci 20: 307–312

Dehghani

Narisawa

Millan

J-L

Hahnel

(2000) Effects of disruption of the embryonic alkaline phosphatase gene on preimplantation development of the mouse. Dev Dyn 217: 440–448

Fedde

Blair

Silverstein

Coburn

Ryan

Weinstein

Waymire

Narisawa

Millan

MacGregor

Whyte

(1999) Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 14: 2015–2026

Fishman

(1974) Perspectives on alkaline phosphatase isoenzymes. Am J Med 56: 617–650

Gallo

Dorschner

Takashima

Klagsgrun

Eriksson

Bernfield

(1997) Endothelial cell surface alkaline phosphatase activity is induced by IL-6 released during wound repair. J Invest Dermatol 109: 597–603

Garattini

Hua

Udenfriend

(1987) Cloning and sequencing of bovine kidney alkaline phosphatase cDNA. Gene 59: 41–46

Greve

Callesen

Hyttel

(1987) In vivo development of in vitro fertilized bovine oocytes matured in vivo versus in vitro. J In Vitro Fertil Embryo Transf 4: 281–285

Hahnel

Rappolee

Millan

Manes

Ziomek

Theodosiou

Werb

Pedersen

Schultz

(1990) Two alkaline phosphatase genes are expressed during early development in the mouse embryo. Development 110: 555–564

10.

Harris

(1989) The human alkaline phosphatases: what we know and what we don't know. Clin Chim Acta 186: 133–150

11.

Henthorn

Raducha

Fedde

Lafferty

Whyte

(1992) Different missense mutations at the tissue non-specific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia. Proc Natl Acad Sci USA 89: 9924–9928

12.

Hoshi

Amizuka

Oda

Ikehara

Ozawa

(1997) Immunolocalization of tissue non-specific alkaline phosphatase in mice. Histochem Cell Biol 107: 183–191

13.

Hoylaerts

Manes

Millan

(1997) Mammlian alkaline phosphatases are allosteric enzymes. J Biol Chem 272: 22781–22787

14.

Hsu

HHT

Rouse

Hamilton

Anderson

(1987) Purification and partial amino acid sequencing of bovine kidney alkaline phosphatase. Int J Biochem 19: 413–417

15.

Hui

Tenenbaum

(1993) Changes in cell adhesion and cell proliferation are associated with expression of tissue non-specific alkaline phosphatase. Cell Tissue Res 274: 429–437

16.

Kodama

Asai

Adachi

Mori

Hayashi

Hirano

Stigbrand

(1994) Expression of a heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells. Biochim Biophys Acta 1218: 163–172

17.

Lepire

Ziomek

(1989) Preimplantation mouse embryos express a heat-stable alkaline phosphatase. Biol Reprod 41: 464–473

18.

MacGregor

Zambrowicz

Soriano

(1995) Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells. Development 121: 1487–1496

19.

Manara

Baldini

Serra

Lollini

De Giovanni

Vaccari

Argnani

Benini

Maurici

Picci

Scotlandi

(2000) Reversal of malignant phenotype in human osteosarcoma cells transduced with the alkaline phosphatase gene. Bone 26: 215–220

20.

Manes

Glade

Ziomek

Millan

(1990) Genomic structure and comparison of mouse tissue-specific alkaline phosphatase genes. Genomics 8: 541–554

21.

Manes

Hoylaerts

Muller

Lottspeich

Holke

Millan

(1998) Genetic complexity, structure, and characterization of highly active bovine intestinal alkaline phosphatases. J Biol Chem 273: 23353–23360

22.

McComb

Bowers

Posen

(1979) Alkaline Phosphatase. New York, Plenum Press

23.

McDougall

Beecroft

Wasnidge

King

Hahnel

(1998) Sequences and expression patterns of alkaline phosphatase isozymes in preattachment bovine embryos and the adult bovine. Mol Reprod Dev 50: 7–17

24.

Millan

(1988) Oncodevelopmental expression and structure of alkaline phosphatase genes. Anticancer Res 8: 995–1004

25.

Millan

(1990) Oncodevelopmental alkaline phosphatases: in search of function. In Ogita

Z-I

Markert

eds. Isozymes: Structure, Function, and Use in Biology and Medicine. New York: Wiley-Liss, 453–475

26.

Millan

(1992) Alkaline phosphatase as a reporter of cancerous transformation. Clin Chim Acta 209: 123–129

27.

Millan

Manes

(1988) Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Natl Acad Sci USA 85: 3024–3028

28.

Muller

Schellenberger

Borneleit

Treide

(1991) The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism. Biochim Biophys Acta 1076: 308–313

29.

Narisawa

Fröhlander

Millan

(1997) Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn 208: 432–446

30.

Narisawa

Hofmann

M-C

Ziomek

Millan

(1992) Embryonic alkaline phosphatase is expressed at M-phase in the spermatogenic lineage of the mouse. Development 116: 159–165

31.

Pratt

HPM

(1989) Marking time and making space: chronology and topography in the early mouse embryo. Int Rev Cytol 117: 99–130

32.

Rindi

Ricci

Gastaldi

Patrini

(1995) Intestinal alkaline phosphatase can transphosphorylate thiamin to thiamin monophosphate during intestinal transport in the rat. Arch Physiol Biochem 103: 33–38

33.

Swarup

Cohen

Garbers

(1981) Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatase. J Biol Chem 256: 8197–8201

34.

Telford

Watson

Schultz

(1990) Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 26: 90–100

35.

Thompson

(1996) Chromatin structure and gene expression in the preimplantation mammalian embryo. Reprod Nutr Dev 36: 619–635

36.

Vergnes

Grozdea

Denier

Bourrouillou

Calvas

(1999) Expression of a liver/bone-intestinal hybrid of alkaline phosphatase in neutrophils of Down's syndrome patients. Clin Chim Acta 279: 167–173

37.

Weissig

Schildge

Hoylaerts

Iqbal

Millan

(1993) Cloning and expression of the bovine intestinal alkaline phosphatase gene: biochemical characterization of the recombinant enzyme. Biochem J 290: 503–508

38.

Wray

Harris

(1982) Demonstration using monoclonal antibodies of inter-locus heteromeric isozymes of human alkaline phosphatase. J Immunol Methods 55: 13–18

39.

Yadav

Rorie

Plante

Betteridge

King

(1992) Development and viability of bovine embryos derived from oocytes matured and fertilized in vitro and co-cultured with bovine oviductal epithelial cells. J Reprod Fertil 94: 33–43

40.

Yoon

Golub

Rodan

(1989) Alkaline phosphatase cDNA transfected cells promote calcium and phosphate deposition. Connect Tissue Res 22: 17–25

Inhibitor Profiles of Alkaline Phosphatases in Bovine Preattachment Embryos and Adult Tissues

Abstract

Keywords

Materials and Methods

Tissue Preparation

Embryo Preparation

Heat Inactivation

Chemical Inactivation

Results

Heat Inactivation

Chemical Inactivation

Discussion

Footnotes

Acknowledgements

References