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a University of Cambridge, Department of Clinical Veterinary Medicine, Equine Fertility Unit, Mertoun Paddocks, Newmarket, CB8 9BH, United Kingdom
b The Babraham Institute, Babraham, Cambridge, CB2 4AT, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blot analysis of tissues and fluids collected from early-pregnant mares demonstrated significant quantities of P19 in the endometrium and uterine secretions and in the embryonic capsule, the chorion, and the yolk sac fluid, showing that the protein is transferred through to the developing embryo. Concentrations of immunoreactive P19 declined during gestation so that, by Day 30, it had virtually disappeared from both maternal and fetal tissues and fluids.
Immunohistochemical staining of endometrial biopsies collected during early pregnancy localized P19 to the glandular and luminal epithelia and to the lumina of the endometrial glands. The capsule and the trophoblast layer of the chorion from early (Days 16–17) horse conceptuses also stained positively with localization of P19 to the apical surface of the trophoblast cells. There was no detectable staining either in or on the embryonic disc. The presence of P19 in both the trophoblast layer and the yolk sac fluid suggests that P19 passes into the yolk sac fluid via trophoblast cells.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P19 is produced in large quantities by the endometrium of the mare during the luteal phase of the estrous cycle and early pregnancy, and its secretion can be induced by the administration of exogenous progesterone ([4, 6]; J. Hyland, personal communication). In addition, it is very likely that the endometrial protein "U1" described by Zavy et al. in 1982 [3], and later shown to be progesterone dependent by Hinrichs and Kenny [7], was P19. However, the protein was first isolated (and N-terminally sequenced) from an equine embryonic capsule [4], suggesting that significant quantities of it are taken up by the developing conceptus. Northern blot analysis failed to detect expression of P19 in the conceptus and showed that expression in the mare was confined to the endometrium [5]. In addition, in situ hybridization studies localized mRNA for P19 to the glandular, and to a lesser extent the luminal, epithelial cells within the endometrium, while Northern blot analysis also confirmed earlier SDS-PAGE analysis of uterine secretions to show that, during the estrous cycle, expression of P19 correlated well with progesterone concentrations in the mare's peripheral blood. During pregnancy, however, expression was reduced to basal levels by Days 20–24 after ovulation, despite the continuation of high progesterone levels. This indicated that expression of P19 is regulated by additional factors. Furthermore, analysis of the predicted amino acid sequence showed P19 to be a novel lipocalin [5].
The lipocalins are a rapidly growing family of mainly extracellular proteins that are characterized by a range of binding properties. For example, they bind small, hydrophobic molecules within a central binding pocket, they bind to specific cell-surface receptors, and they form macromolecular complexes [8]. They are classified on the basis of three conserved sequence motifs and an 8-stranded ß-barrel structure. P19 has most sequence identity with the major urinary proteins, but it has several distinct differences, particularly within one of the three conserved motifs, that identify it as a member of the family [5].
The lipocalins have been identified in a wide range of mammals and also in other organisms including bacteria and yeast [9]. They have been shown to exhibit great functional diversity, with roles in olfaction, invertebrate coloration, modulation of immune responses, and enzymatic synthesis of prostaglandins. However, most of them act as transport molecules, the best known example being retinol-binding protein [10]. There is growing evidence that, as transport proteins, the lipocalins play an important role in cell development and homeostasis. Furthermore, several members of the family, in addition to P19, have been identified as uterine secretory proteins, including PP14, isolated originally from human placental tissues [11], and retinol-binding protein [12–14]. Most appear to transport maternal factors that are important for embryonic sustenance and development, and although P19 has not yet been identified in other species, it may be widespread, particularly in those animals that exhibit an extended preimplantation period.
To study the role of P19 in supporting embryonic development, it was necessary to raise high-titer antisera for localizing the protein in situ and for structure-function studies. However, previous attempts to purify P19 from endometrial secretions all failed due to problems with aggregation. In the present study, antisera were raised against a P19 carboxyl terminal peptide and a recombinant-derived glutathione S-transferase-fusion protein (GST-rP19). These antisera were validated and used to localize P19 in the fetal and maternal tissues and fluids of the pregnant mare by Western blotting and immunohistochemical staining.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fourteen mares aged 3–7 yr were used. Follicular development and ovulation were monitored by daily transrectal ultrasonographic examination of the ovaries. To obtain pregnancies, the mares were inseminated once with freshly collected, extended stallion semen when they showed an ovarian follicle of > 35 mm in diameter. Ovulation was confirmed by measuring a rise in progesterone concentrations in peripheral serum samples recovered daily during and beyond estrus [15], and Day 1 of the cycle or pregnancy was taken as the first day on which the serum progesterone concentration rose to > 1 ng/ml serum.
Samples for Western Blot Analysis
For the initial Western blot analysis of P19, samples were collected from a mare that was killed on Day 16 of pregnancy. The conceptus was removed intact from the uterus and washed in several changes of PBS before a sample of yolk sac fluid was aspirated using a 21-gauge needle and syringe. The embryonic capsule was removed, and the remainder of the conceptus, consisting of the trophectoderm and embryonic disc (see Fig. 1), was washed in PBS to remove any traces of uterine and yolk sac fluids. The uterus was then flushed with 20 ml PBS, and biopsies of the endometrium and the liver (negative control) were collected. All the fluids and tissues were stored at -70°C until required. Prior to analysis, the samples of endometrium, liver, capsule, and conceptus tissues were weighed and homogenized with approximately 100 µl denaturing buffer (30 mM Tris, pH 6.8, 1.5 M urea, 5% v:v ß-mercaptoethanol, 0.5% w:v SDS) per milligram tissue. They were then centrifuged at 15 000 x g for 1 min, and 50 µl of the supernatant was loaded for Western blot analysis (see below). Since P19 was the major protein in the uterine flush sample (see Fig. 3), the approximate concentration of P19 in this sample was estimated using protein assays and by comparison with a BSA standard. Small aliquots (50 µl) of this sample (containing approximately 1 µg protein) were then stored at -20°C for use as a reference sample on all Western blots. Since the other proteins amounted to around 50% of the total protein content, this suggested a concentration of about 10 (± 3) µg P19/ml uterine flush. The quantity of fluid analyzed on all subsequent Western blots was 50 µl for the uterine flushes and 100 µl for the yolk sac samples.
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To examine the relationship between P19 in the uterine secretions and P19 in the yolk sac fluid during pregnancy, additional samples were collected nonsurgically from three mares on Days 16, 20, and 30 of gestation. The uterine secretions were obtained by infusing 20 ml sterile PBS into the nongravid horn of the uterus via a catheter passed through the working channel of a strobed light videoendoscope as described by Bracher and Allen [16]. The uterus was massaged gently per rectum before as much fluid as possible was recovered by directing the tip of the catheter into the pool of fluid on the uterine floor. A sharpened catheter was then used through the videoendoscope to sample the yolk sac fluid from the older (Days 20 and 30) conceptuses, after which the collapsed conceptuses were removed from the uterus for other studies. The intact Day 16 conceptus was collected by placing an endotracheal tube through the mare's cervix and irrigating the uterus with approximately 500 ml sterile PBS. It was further washed in PBS to rid it of contaminating uterine secretions before the yolk sac fluid was aspirated as described above. The secretions and yolk sac fluids were stored at -20°C until analyzed.
Samples for Immunohistochemistry
For immunolocalization of P19 in the endometrium, biopsies were taken from two mares at estrus (Days 18 and 19 of the cycle), on Days 2, 14, and 16 of the cycle, and on Days 14, 16, 18, 30, and 50 of gestation. The Day 50 biopsy was collected via midline laparotomy performed under general anesthesia, because at this stage the membranes have attached and it is not possible to take a biopsy nonsurgically. The biopsies were fixed in 4% paraformaldehyde in PBS at 4°C for 1–2 h, dehydrated, and embedded in paraffin wax.
For immunolocalization of P19 in the conceptus, a Day 16 and a Day 17 conceptus were flushed from the uterus as described above and fixed in 4% paraformaldehyde at 4°C for 1–2 h. To ensure proper fixation, some of the fixative was injected into the yolk sac cavity. The embryonic disc with some surrounding chorion (Fig. 1) and a piece of the capsule were then dissected, dehydrated, and embedded in paraffin wax.
C-Terminal Peptide Antiserum
A peptide consisting of the carboxyl-terminal 15 residues of P19 (Ile Val Asp Leu Thr Gln Thr Asp Arg Cys Leu His Ala Arg His; see [5] and GenBank accession number X98459 for the full sequence) was synthesized on a Perceptive Biosystems (Foxholes Ind. Est., Hertford, U.K.) synthesizer, conjugated to a purified protein derivative of tuberculosis toxin, and used to raise a polyclonal antiserum in a Dutch rabbit. The rabbit was immunized three times with approximately 1 mg conjugated peptide on the first occasion and 0.5 mg on the second and third booster immunizations, each after an interval of 3 wk. The specificity and titer of the antiserum were assessed on Western blots of endometrial secretions containing P19. Although this antiserum detected P19 very well on Western blots, it did not bind to the protein in tissue sections. It was therefore necessary to make recombinant-derived P19.
Recombinant-Derived Protein and Antiserum
The full-length cDNA clone for P19 was isolated originally in a lambda Uni-Zap vector (Stratagene Ltd., Cambridge, UK) and then excised into a pBluescript SK phagemid [5]. To construct the glutathione S-transferase (GST)-fusion protein vector (pGEX-P19), two oligonucleotide primers (5' CCC GGA TCC TGC ACA TGG GCC CTG GGG AC 3' and 5' CGA ATT CGG AGA CGC CCC TGG ACC CTT GG 3') were used to amplify (using the polymerase chain reaction [PCR]) the coding region for the mature form of P19. The 5' overhangs were removed using Pfu polymerase (Stratagene); the PCR product was blunt-end ligated into pBluescript, sequenced using the T7 and T3 primers, and then subcloned into the pGEX-3X expression vector. Positive colonies were selected by PCR. To check for expression, 5-ml aliquots of Luria Broth were inoculated with the positive clones and grown overnight at 37°C, and 20 µl of each culture was then analyzed by Western blotting using the antiserum raised against the P19 C-terminal peptide.
The Escherichia coli strain BL21(DE3)pLysS was used for expression of the construct, but initial trials (see Results) showed that it was necessary to incubate the transformed cells at 30°C (rather than 37°C) and without the inducer, isopropyl-ß-D-thiogalactopyranoside (IPTG). After incubating overnight (16–20 h), the bacteria were isolated by centrifugation, resuspended in 1/20th volume sterile PBS, and sonicated. Triton X-100 (0.1% v:v) was added, the mixture was incubated on ice for 50 min, and insoluble debris was removed by centrifugation. The supernatant was incubated at room temperature for 30 min in the presence of DNase I (10 ng/ml), filtered through a 0.45-µm filter, and passed through a glutathione Sepharose 4B column (Pharmacia, St. Albans, UK) that had been equilibrated with PBS/Triton X-100. The column was washed with 10 bed volumes of PBS and then with 3 bed volumes of elution buffer (5 mM glutathione in 50 mM Tris HCl, pH 8.0). Samples were collected at all stages for analysis by SDS-PAGE and Western blotting. The SDS-PAGE gels were stained with Coomassie blue and destained using the method of Hervieu [17]. The majority of the eluted fusion protein (GST-rP19) was stored at 4°C prior to use. However, approximately 50 µg was incubated with 1 mg of factor Xa (New England Biolabs, Hitchin, UK) at room temperature for 16 h and applied to a glutathione Sepharose 4B column, and the cleaved recombinant-derived P19 (rP19) was collected in the flow-through. The GST and undigested fusion protein (GST-rP19) were eluted with 3 bed volumes of elution buffer, and samples were analyzed by SDS-PAGE, using the Day 16 uterine flush sample as reference. The remainder of the GST-rP19 was immobilized on 100 µl of 50% (v:v) glutathione agarose slurry (Sigma, Poole, UK) and analyzed by SDS-PAGE to estimate the amount of incorporated GST-rP19. An antiserum was then raised in a Dutch rabbit by injecting the equivalent of 500 µg GST-rP19 s.c. and intramuscularly. Two subsequent boosts, each of approximately 250 µg protein, were given s.c. at intervals of 3 wk. Serum samples were collected 10 days after each boost, stored at -70°C, and assessed by Western blot analysis using the Day 16 uterine flush sample as reference.
Western Blot Analysis
The tissue homogenates, secretions, yolk sac fluid samples, and bacterial cultures were boiled in loading buffer (30 mM Tris, pH 6.8, 1.5 M urea, 5% v:v ß-mercaptoethanol, 7.5% v:v glycerol, 0.5% w:v SDS, 0.05% bromophenol blue) for 5 min and loaded in duplicate onto two 12% SDS-PAGE gels. After electrophoresis, one gel was stained with Coomassie blue [17], and the other was blotted onto a polyvinylidene fluoride membrane (Sartorius, Epsom, UK) by wet blotting (Trans-Blot Cell; BioRad, Hemel Hempstead, UK) for 3 h at 200 mA. The membrane was then blocked with 5% (w:v) milk powder in PBS-Tween 20 (0.1% v:v) for at least 1 h. The primary antiserum (diluted in blocking solution) was allowed to bind for 1 h at room temperature and, after washing, the second antiserum (horseradish peroxidase-conjugated goat anti-rabbit; Sigma) at a dilution of 1:5000 in PBS-Tween-20 was allowed to bind for 1 h. This antiserum was detected using ECL (enhanced chemiluminescence) reagents (Amersham Int., Little Chalfont, UK) and exposure to X-OMAT AR film (Kodak IBI Ltd., Cambridge, UK) for 1–10 min. The antisera to P19 were titrated using the uterine flush from the Day 16 pregnant mare as a reference, and the optimum dilutions were 1:5000 for the anti-peptide serum and 1:10 000 for the anti-GST-rP19 sera. The preimmune sera were tested at a dilution of 1:1000.
Immunohistochemistry
Sections of approximately 6-µm thickness were placed onto microscope slides (Superfrost Plus; BDH Merk, Lutterworth, UK) and stored at room temperature until required. Prior to use, the sections were dewaxed, rehydrated in an ethanol series, washed in TBS (50 mM Tris HCl, pH 7.5, 275 mM NaCl, 5 mM KCl), and blocked with a 1:200 dilution of normal goat serum or 3% w:v BSA in TBS for 30 min. The primary antisera were diluted in 3% w:v BSA in TBS, allowed to bind for 1 h in a humidified chamber at room temperature, and, after thorough washing, were detected using the VectorStain Elite ABC (avidin-biotin-peroxidase) anti-rabbit kit and diaminobenzidine reagents (Vector Laboratories, Peterborough, UK). Finally, the sections were counterstained with Harris hematoxylin and eosin before being mounted. The primary antisera were tested at dilutions of 1:1000 to 1:50 000, and the preimmune sera were tested at a dilution of 1:1000.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of the pGEX-rP19 construct in BL21(DE3)pLysS produced a large amount of recombinant protein, but when the transformants were grown at 37°C in the presence of IPTG, the majority of the GST-rP19 was in the insoluble debris after cell lysis, and therefore in the form of inclusion bodies. However, the proportion of recombinant protein in the inclusion bodies was greatly reduced when the bacteria were grown at 30°C without IPTG, and these conditions were therefore used for all subsequent studies. A summary of the purification of the GST-rP19 fusion protein from the bacterial cell lysates and its cleavage to produce rP19 is shown in Figure 2. The majority of the bacterial proteins passed through the glutathione Sepharose 4B column without binding, and the few that did bind were removed by washing. The GST-rP19 was then successfully eluted from the column (lane 3, Fig. 2), and the yield was estimated as 1.2 mg protein per liter of bacterial culture. This was the material that was used to raise the polyclonal antiserum. Some of the fusion protein was digested with factor Xa and again passed through the affinity column to yield pure rP19 in the flow-through (lane 4, Fig. 2). The GST and uncut GST-rP19 that had bound to the column were then eluted (lane 5, Fig. 2).
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Antisera
The titers of the anti-P19 peptide serum and the anti-recombinant-derived P19 serum increased during immunization, and the rabbits were anesthetized and exsanguinated 10 days after the final booster immunization. At a dilution of 1:10 000, the antiserum raised against the C-terminal peptide detected approximately 10 ng P19 on Western blots, whereas the preimmune serum (at 1:1000) did not show any specific binding. This antiserum was therefore very useful for detecting expression of the recombinant-derived protein and for analysis of tissues and fluids. However, it did not bind to P19 in histological sections of endometrium from diestrous and pregnant mares. The antiserum raised against the recombinant-derived protein was able to detect P19 at dilution of 1:20 000, and when used at 1:10 000 it was able to detect as little as 6 ng P19 on Western blots. It also gave positive and specific staining when used as the primary antiserum in the immunohistochemical analysis of tissue samples (see below).
Western Blot Analysis
A Western blot of the tissues and fluids from the mare killed on Day 16 of pregnancy is shown in Figure 3. This blot was probed with the anti-GST-rP19 serum at a dilution of 1:10 000; very similar results were obtained with the C-terminal peptide antiserum used at a dilution of 1:5000. The endometrial biopsy and the uterine flush gave a signal at 19 kDa whereas the liver was negative. The embryonic capsule, the conceptus itself, and the yolk sac fluid sample all gave strong signals, thereby demonstrating that P19 passes through the capsule and chorion and into the yolk sac cavity. The signal at around 40 kDa in the yolk sac fluid probably represents a dimer of P19 [4].
Figure 4 shows a Western blot of the secretions and yolk sac fluids recovered on Days 16, 20, and 30 of gestation using the anti-GST-rP19 serum at a dilution of 1:10 000. The immunoreactivity was very strong in the secretions on Day 16 and Day 20 but was barely detectable on Day 30. This is in keeping with previous observations showing that the secretion of P19 is reduced to basal levels during pregnancy, despite the maintenance of maternal progesterone levels [4, 5]. The 40-kDa putative dimer was also evident in the Day 16 secretion sample. The signal in the yolk sac fluid was also strong on Days 16 and 20 but undetectable on Day 30, thereby showing a positive correlation with levels of P19 in the uterine secretions.
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Immunohistochemistry
The results of the immunohistochemical localization of P19 are summarized in Figure 5. There was no detectable staining in any of the tissues using the preimmune control serum at a dilution of 1:1000 (endometrial biopsy from the Day 2 pregnant mare shown in Fig. 5a) or in the endometrial biopsies taken at estrus (Days 18 and 19 of the cycle) or at Days 30 and 50 of pregnancy (not shown). The earliest detection of P19 was in the endometrial biopsy taken 2 days after ovulation in which staining was evident in the glandular epithelial cells (Fig. 5b). The signal was much stronger on Days 14 and 16 after ovulation in both pregnant and nonpregnant mares (Day 16 shown in Fig. 5, c and d), and by this stage, the secretory material in the lumina of the glands and the luminal epithelial cells had also become positive (Fig. 5c). Figure 5d shows a high-power view of the section in Figure 5c demonstrating dense staining in the cytoplasm of the glandular epithelial cells and within the lumina of the glands. The signal in the endometrial biopsy from the Day 18 pregnant mare (not shown) was just as strong as that on Day 16 (pregnant and nonpregnant), contrasting markedly with the complete lack of staining in the two nonpregnant mares sampled at Days 18 and 19 of the cycle (i.e., during estrus).
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Figure 5e shows a cross section of the area of the embryonic disc of the Day 16 conceptus. Although the embryo itself was negative, there was intense staining in the trophoblast layer of the chorion, which in this area consists of three layers, ectoderm (trophoblast), mesoderm, and endoderm (see diagram in Fig. 1). Furthermore, the capsule, which had been in close contact with the trophoblast layer and outer surface of the embryonic disc in vivo, also stained positively (see insets in Fig. 5). The Day 17 conceptus gave the same picture, with positive staining of the capsule and trophoblast layer of the chorion, but nothing in the embryo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Western blot analysis of tissues and uterine secretions confirmed and extended our earlier studies showing that P19 is secreted by the endometrium when progesterone levels rise during diestrus or early pregnancy but that it was reduced to undetectable levels at the next estrus in the cycling mare or after Day 20 in the pregnant mare, despite the maintenance of high blood progesterone levels in the latter. The mechanisms that control this reduction in P19 synthesis and secretion during pregnancy clearly need further study.
The Western blots also confirmed our earlier observations that P19 is sequestered by the capsule [4] and, in addition, showed that it is present in the conceptus membranes and the yolk sac fluid up to Day 20 of gestation. It is not possible at this stage to quantify the actual amount of protein that is transferred, but, as with the secretions, P19 was clearly visible in the yolk sac fluid on Days 16 and 20 of gestation on the Coomassie blue-stained gels, as well as giving a strong signal on Western blots. In comparing the relative amounts of protein in the two fluids, it should be noted that the endometrial secretions were diluted with 20 ml PBS as they were flushed from the mare's uterus, whereas the yolk sac fluid samples were recovered neat. Also, the amount analyzed was different (50 µl for uterine flushes and 100 µl for yolk sac fluids), but because the flushes probably represented more than a 5-fold dilution of the uterine secretions, the overall result was that the amount of uterine secretion analyzed on each gel was considerably less than the amount of yolk sac fluid analyzed. This indicates that the concentration of P19 in the undiluted secretion was at least 10 times greater than that in the yolk sac fluid at the same stage of gestation.
Since the equine embryonic capsule has been shown to be quite permeable, allowing diffusion of molecules up to 200 kDa in size [18], P19 could presumably diffuse through it and therefore explain the presence of P19 on and in the conceptus membranes. Furthermore, the complete lack of detectable P19 in the yolk sac fluid by Day 30, coincidentally with the fall in the amount of protein in the uterine secretions, illustrates a more or less direct relationship between the concentrations in the uterine secretions and the yolk sac fluids. This, in turn, indicates that P19 is not stored in the yolk sac compartment for any length of time.
The immunostaining results confirmed that P19 is synthesized in the epithelia of the endometrial glands, and they also showed that the protein can be detected in these cells as early as 2 days after ovulation. This indicates a very rapid response to progesterone, which shows a detectable rise in the peripheral circulation of the mare by about 24 h after ovulation [15]. The staining on Day 2 was confined to the secretory (basal) regions of the endometrial glands, but, by Day 16, additional intense staining was present in the lumina of the glands and in the luminal epithelium. This indicates a time-dependent switching on of expression in these cells, which also agrees with our previous in situ hybridization studies [5]. However, some staining in the luminal epithelium at the later stages could also be due to accumulation of the protein on the surface of the endometrium.
The immunolocalization of P19 in the young (Days 16 and 17) equine conceptuses provides additional evidence for the uptake of P19. Intense staining was visible in the trophoblast layer but not within or on the embryonic disc, which at this stage is devoid of its covering of "polar" trophoblast cells [19]. This suggests that horse trophoblast cells may possess receptors for P19, analogous to retinol-binding protein receptors [20], or that they simply absorb P19 by pinocytosis. This question will be addressed using electron microscopy and by labeling P19 with a suitable probe to investigate its interaction with horse trophoblast cells.
The results of this study, combined with our previous studies on the structure of P19 [5], provide good evidence that P19 is a transport protein, carrying what may be a small, hydrophobic ligand from the mother to the developing embryo. An example of such a ligand is retinol, the principal serum form of vitamin A. Retinoids are well-known morphogens that are involved in pattern formation during embryogenesis, as well as in hematopoiesis and tissue differentiation. McDowell et al. [14] cloned and sequenced the carrier protein for retinol in the horse, and since the deduced amino acid sequence showed only 20% identity with P19, it is unlikely that P19 is another retinol-binding protein. However, the ligand could well be a steroid or eicosanoid. Several members of these two families of molecules have been shown to be lipocalin ligands and are believed to be important in embryonic development [13].
The striking temporal correlation between secretion of P19 into the mare's uterus during pregnancy and the presence of the capsule on the conceptus suggests that they are functionally correlated. The capsule, which consists of highly sialylated mucin oligosaccharides, is negatively charged [2], whereas P19 has a strong positive charge (pI = 9.1; unpublished results), thereby providing a possible explanation for the interaction between P19 and the capsule. The negative charge of the capsule is thought to play a role in the unusual mobility of the equine conceptus in the mare's uterus between Days 6 and 17 after ovulation [2, 21], which has been shown to be necessary for maintenance of pregnancy by the mare [22]. However, the capsule could impede the absorption of small, hydrophobic molecules from the mother, and it is therefore possible that P19 may assist in the uptake of such molecules by acting as a carrier. Studies are now under way to establish whether this is the case and to determine the precise role of P19 in the pregnant mare.
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FOOTNOTES |
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2 Correspondence.
FAX: 44 1223 837912;francesca.stewart{at}bbsrc.ac.uk
Accepted: April 10, 1998.
Received: January 22, 1998.
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