Department of Anatomy and Reproductive Biology, Medical School, RWTH University of Aachen, Wendlingweg 2, 52057 Aachen, Germany
1 To whom correspondence should be addressed. Email: aherrler{at}ukaachen.de
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Abstract |
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Key words: amino acid sequence/embryo/haptoglobin/oviduct/uterus
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Introduction |
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The active protein is formed by two - and two
-subunits, resulting in a tetramer. Furthermore, in humans two
-alleles are known (Dobryszycka, 1997
). Haptoglobin mRNA is expressed in mice liver already during fetal development, and in the adult organism in lung, spleen, kidney and skin (D'Armiento et al., 1997
). The protein is present in most body fluids (e.g. serum, urine, saliva, ascites) (Dobryszycka, 1997
). Furthermore, the haptoglobin
-subunit is detectable in human uterine secretions (Beier and Beier-Hellwig, 1998
), as well as the haptoglobin
-subunit (Berkova et al., 2001
) and haptoglobin in the endometrium (Sharpe-Timms et al., 2000
). As Sharpe-Timms et al. (2000)
detected haptoglobin expression in the endometrial stroma by in-situ hybridization, at least some of the haptoglobin detected in the secretions seems to be of local origin. Hoffman et al. (1996)
identified the rabbit 42 kDa glycoprotein (GP42) to be
-haptoglobin. GP42 has been shown to be secreted by the rabbit endometrium shortly before implantation (Thie et al., 1984
; Anderson et al., 1986
). Although haptoglobin expression in the late preimplantation uterus is well established, nothing is known about haptoglobin mRNA expression and protein content in the oviduct, the early preimplantation uterus or in the embryo itself.
In the investigation presented here, the complete rabbit haptoglobin cDNA was sequenced. Another goal of the study was to investigate whether haptoglobin is part of the preimplantation maternalembryonic signalling. Consequently, the time schedule of haptoglobin mRNA expression and protein distribution in the oviduct and uterus, as well as in the embryo, during the preimplantation period was studied. To investigate whether haptoglobin is detectable at the embryomaternal interface like several other functional proteins (Herrler et al., 2002), the extra-embryonic matrices, i.e. the zona pellucida, were analysed for the presence of haptoglobin protein. In addition, haptoglobin mRNA expression and protein synthesis was determined in all preimplantation embryos. In this investigation we did not differentiate between the known isoforms of the
-subunits, which will be part of further functional investigations.
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Materials and methods |
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mRNA expression analysis
All animal experiments were conducted in accordance with the principles for the care and use of research animals and were run with the permission of the District Government at Cologne, Germany (AC 39 15/95). Liver, oviducts, uteri and embryos were recovered from female New Zealand White rabbits ranging in age from 6 to 8 months. They were housed individually in air-conditioned rooms (25°C, 45% relative humidity) under a 12 h light, 12 h dark cycle (0700 to 1900 h) and were fed with a commercial diet and water ad libitum.
To stimulate multiple follicular growth, rabbits received one injection of 100 IU pregnant mare's serum gonadotrophin (PMSG) s.c. (Intergonan®; Intervet, Tönisvorst, Germany), except those animals used for tissue sampling before ovulation (+6 and +9 h). For ovulation induction, 75 IU HCG (Primogonyl®; Schering, Berlin, Germany) were administered i.v. directly after mating by two fertile bucks. To terminate further PMSG activity and follicular growth, Neutra-PMSG® (Intervet) was administered i.v. in parallel to HCG application (i.e. 72 h after PMSG injection). Six hours (6 h), 9 h (9 h), 24 h (day 1), 48 h (day 2), 72 h (day 3), 96 h (day 4), 120 h (day 5) and 144 h (day 6) following mating, samples were collected. The total reproductive tract was removed. Embryos were collected by flushing oviducts (days 1, 2 and 3) and/or uteri (days 3, 4, 5 and 6) with 510 ml Dulbeco's phosphate-buffered saline (PBS) (Sigma, Deisenhofen, Germany). Embryos were washed three times in PBS, pooled in 500 µl RNAzol (WAK Chemie, Bad Soden, Germany; days 1 and 2, n=40; days 3 and 4, n=20; days 5 and 6, n=10) and snap-frozen in liquid nitrogen. Oviducts were split into ampulla and isthmus, removing the middle piece and fimbriae. Uteri were opened at the mesometrial side and endometrium was sampled by scraping off the surface tissue. Endometrial as well as oviductal samples were submerged in 500 µl RNAzol (WAK Chemie) and snap-frozen in liquid nitrogen. Samples were stored at 20°C prior to analysis. At least three animals were analysed per day of pregnancy. Liver samples served as positive control. PCRs primers 1 and 2 were used as described above. To determine the size of the PCR products a 100 bp ladder (GeneRuler Fermentas, St Leon-Rot, Germany) was used.
Western blot analysis
Animals were treated as described above. Six to 144 h following mating, uteri and oviducts were flushed with 150 mM ammonium bicarbonate buffer. The first millilitre was collected separately and was centrifuged following embryo recovery (1000 g, 10 min). The supernatant was snap-frozen and used for determination of haptoglobin secreted by the oviduct and endometrium.
For dissection, embryos were washed three times in PBS and transferred into a clean culture well. All remaining fluid was removed. The embryo was then punctured and the blastocyst fluid collected. The collapsed embryo was floated in a drop of PBS, and the embryo separated from the embryonic coats. Coats and embryo were washed and sampled separately. For each investigation, five embryos, 10 coats or blastocyst fluid of 20 embryos were pooled separately, snap-frozen and stored at 20°C until further analysis. Tissue samples were collected by separating ampulla and isthmus. The endometrium was scraped off with a scalpel. All samples were snap-frozen directly in liquid nitrogen. Protein content (except of embryonic coats) was determined by DC Protein Assay (Bio-Rad, Munich, Germany) according to Lowry et al. (1951). Twenty micrograms of protein of each sample was diluted in loading buffer [5% (v/v)
-mercaptoethanol; 30 mM Tris, pH 6.8; 1.5 M urea; 7.5% (v/v) glycerol; 0.5% (w/v) sodium dodecyl sulphate (SDS); 0.05% (w/v) Bromophenol Blue]. Samples were heated to 100°C for 5 min, centrifuged and subjected to SDSPAGE using a 5% stacking gel and a 15% resolving gel under reducing conditions. For size determination, a prestained molecular weight marker (Rainbow, high molecular weight range; Amersham Biosciences, Freiburg, Germany) was used. After electrophoretic separation, the proteins were transferred to a polyvinylidene diflouride membrane (Sartorius, Epsom, UK) by semi-dry blotting (Kyhse-Andersen, 1984
) for 45 min at 900 mA. Following transfer, the membrane was blocked over night with 5% (w/v) milk powder in PBS/Tween-20 (0.1% w/v) at 4°C. Because of the high homology between rabbit and human haptoglobin sequence, a primary antibody against human haptoglobin was used (goat anti-human haptoglobin; SP1121P; DPC Biermann, Bad Nauheim, Germany; crossreactivity to rabbit haptoglobin has been described by DPC Biermann) at a concentration of 12 µg IgG/ml PBS containing 5% milk powder. The blot was incubated at room temperature for 2 h. After three washes in PBS/0.1% Tween, the second antibody (donkey anti-goat conjugated with horseradish peroxidase, diluted 1:5000 in PBS/0.1% Tween/1% milk powder; Dako, Glostrup, Denmark) was added and left at room temperature for 1 h. After further rigorous washing, detection of the second antibody was performed using enhanced chemiluminescence (ECL) reagents (Amersham, Little Chalfont, UK; ECL: horseradish peroxidase-induced chemiluminescent reaction) and the blot was exposed to an X-ray film (X-Omat AR; IBI-Kodak Ltd, Cambridge, UK) for 3 min. At least three animals were analysed per day of pregnancy. Rabbit serum served as positive control. To verify the specificity of the antibody it was preabsorbed for 30 min with haptoglobin (60 µg haptoglobin 1-1 plus 60 µg haptoglobin 2-2 plus 12 µg anti-haptoglobin IgG/ml; H 0138 and H 9762; Sigma, St Louis, MO, USA) and applied to day 6 uterine secretions (see Figure 5, negative control).
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Results |
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Haptoglobin in maternal and embryonic tissues
Haptoglobin mRNA expression has been investigated in ampulla and isthmus from 6 h after mating to day 6 of pregnancy, in the endometrium as well as in preimplantation embryos from day 1 to day 6. Haptoglobin mRNA expression could be detected in the oviduct and in the endometrium (380 bp RTPCR product; Figure 4). In the oviduct, haptoglobin mRNA expression was detectable at each day during embryo passage (day 0 to day 3). Already 6 h after mating (the first point of time investigated, 3 h before ovulation) haptoglobin mRNA is expressed in both segments of the oviduct. From day 1 to day 3 post-conception (p.c.), when embryos are passing through the oviduct, haptoglobin mRNA expression was detectable; however, expression was slightly higher in the ampulla than in the isthmus (Figure 4A versus B). By day 4 p.c. haptoglobin mRNA expression decreased to undetectable levels. The haptoglobin mRNA expression pattern in the endometrium differs from the oviduct (Figure 4C). First expression was detected by day 4 p.c. within
30% of all investigated endometria. By day 5 p.c. haptoglobin mRNA expression was detected in all endometrial samples, and expression reached its maximum by day 6 p.c. (Figure 4C, last day investigated).
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Finally, the presence of haptoglobin protein in the different compartments of the mother and embryo was investigated by western blot analysis. Serum from day 6 pregnant does served as positive control, revealing a very intense staining at 42 kDa (Figure 5F, lane 4). Preabsorbtion of the antibody with haptoglobin decreased the signal significantly, proving the specificity of the antibody (Figure 5F, lane 5). In the tissue of both oviductal segments haptoglobin was detectable (Figure 5A and B). In the tissue of the ampulla 6 h after mating (i.e. 3 h before ovulation) haptoglobin was detectable, persisting up to day 2 p.c. and decreasing thereafter. In the isthmus, the highest haptoglobin tissue levels were reached at the time of ovulation (9 h) and declined to barely detectable signals on day 2 p.c, reflecting the results detected by RTPCR. In the oviductal fluid, which surrounds the embryo from day 1 to day 3 p.c., haptoglobin was detected in high amounts from 6 h after mating up to day 2 p.c. (Figure 5C). At day 3 p.c., when the rabbit embryos are passing from the oviduct to the uterus, the haptoglobin content of the oviductal fluid clearly decreased, and stayed low until day 5 p.c.
In endometrial tissue samples (Figure 5D) haptoglobin was already detectable from day 1 to day 3 p.c., whereas on day 4 p.c. only very low haptoglobin levels were found. On day 5 p.c. haptoglobin level increased again, reaching maximum levels on day 6 p.c. In parallel with the increased haptoglobin content in endometrial tissue, substantial levels of haptoglobin were detected in uterine secretions on day 5 p.c. (Figure 5E). In the following 24 h the haptoglobin content increased remarkably, reaching levels higher than the blood serum level.
Rabbit embryos (day 6 p.c.) were investigated (Figure 5F) for the presence of haptoglobin. Although embryos do not express haptoglobin mRNA, the protein was detected in significant levels in extra-embryonic matrices (Figure 5F, lane 1). In the embryonic tissue almost no haptoglobin was found, but detectable levels were present in the blastocyst fluid (Figure 5F, lane 3).
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Discussion |
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The major goal of our study was to investigate whether haptoglobin is part of the preimplantation maternalembryonic signalling. Therefore, all compartments that are potentially involved in this maternalembryonic dialogue, i.e. oviductal and endometrial tissues and secretions as well as the embryo and its extra-embryonic matrices, were investigated at each day of the preimplantation time. Haptoglobin mRNA expression is detectable in maternal tissue only. Embryos 16 days old never expressed haptoglobin, in agreement with Thomas et al. (1990), who found that haptoglobin is expressed in the fetal rat liver not earlier than day 18 of pregnancy.
In the mother, haptoglobin mRNA is expressed by the oviduct already 3 h before ovulation. Ovulation in rabbits occurs 9.5 h following mating/HCG injection (Knobil and Neill, 1988). Haptoglobin mRNA was detected in the ampullar and isthmic part of the oviduct as early as 6 h after mating/HCG (the first point of time investigated), with the highest expression at 9 h p.c. (time of ovulation). At the same time, the highest amount of haptoglobin protein is found in ampullar and isthmic tissue, as well as in the oviductal secretions, and thus is available to both, spermatozoa and oocytes. This demonstrates that haptoglobin is present in the oviductal fluid at a time when spermatozoa undergo capacitation and the fertilization of oocytes takes place. Consequently, it can be hypothesized that haptoglobin might play a role in this fundamental reproductive process. Recently, Lavery et al. (2003)
published the detection of haptoglobin in bovine oviducts. By immunohistological investigation they detected a more intensive staining in the ampulla relative to the isthmus. This corresponds to the higher expression of haptoglobin in the ampulla of rabbits, as shown in our study. Furthermore, we could demonstrate that haptoglobin is synthesized by the oviduct. Therefore, at least some of the haptoglobin detected in oviductual fluids is not transported from the vascular system (as discussed by Lavery et al., 2003
), but expressed and secreted by the oviductal mucosa. The immunohistological pictures presented by Lavery et al. (2003)
support this by showing apical release of haptoglobin by oviductual luminal epithelial cells. Additionally, the significant amount of haptoglobin in oviductal secretions already 3 h before ovulation (Figure 5C, 6 h) excludes an origin of follicular fluid at this time. Therefore, we conclude that the oviduct expresses and secretes haptoglobin for the entire time that the gametes and embryos are present, which may intermingle with haptoglobin originating from the blood.
Furthermore, the extra-embryonic matrix of preimpantation embryos as well as the blastocyst fluid contain haptoglobin, although the embryo by itself does not express haptoglobin. This implies an uptake of maternal haptoglobin into the extra-embryonic matrices during the passage of the embryo through the oviduct and/or the uterus. On day 2 p.c., when the so-called mucoprotein layer emerges, a second layer of the extra-embryonic matrix, originating from oviductal mucosa fluid, is formed. At the same time, high amounts of haptoglobin are detectable in the oviductal secretions. The mucoprotein layer is built up by glycoproteins (Denker, 1970; Gandolfi, 1995
), networked by lectins that are secreted especially in the isthmic part of the oviduct (Biermann et al., 1997
). One of these glycoproteins may be haptoglobin, which is glycosylated at several positions and was originally characterized by lectin-binding activity (Thie et al., 1984
; Anderson et al., 1986
). It might be recruited during the process of mucoprotein layer formation on day 2 and day 3 p.c. This would explain the high amounts of maternal haptoglobin within the extra-embryonic matrices of the embryo.
Within the endometrium the first haptoglobin mRNA expression was found by day 4 p.c in 30% of samples, in accordance with the findings of Hoffman et al. (1996). By western blot analysis, already between day 1 and day 3 p.c. a small volume of haptoglobin was detectable in endometrial samples, although at this stage no haptoglobin expression was detectable by RTPCR in that tissue. This may be explained by high endometrial proliferation during this time, which is accompanied by high vascularization. Therefore, these endometrial samples contain more blood and consequently more haptoglobin. By day 4, the endometrium starts to synthesize haptoglobin itself. The secreted protein is detectable on day 5 and day 6 within the uterine fluid. As in the oviduct, there is a high temporal correlation between the further transformation of the extra-embryonic matrices and haptoglobin expression in the endometrium. On day 5 and day 6 p.c., two new layers, the neozona and the gloiolemma (Denker and Gerdes, 1979
), are formed, at least partly by proteins of the uterine secretions. Furthermore, lectins were mainly found in the endometrium around day 5 p.c., in parallel with the formation of the new extra-embryonic matrix layers (Biermann et al., 1997
) and haptoglobin expression. As demonstrated by western blot analysis, extra-embryonic matrices of day 6 p.c. embryos contain considerable amounts of haptoglobin. As the embryo itself does not produce any haptoglobin at any stage of preimplantation, development this has to be of maternal origin. Although the presence of an embryo is not prerequisite for endometrial haptoglobin expression, as shown by Olson et al. (1997)
, embryonic development may be supported by the uptake of maternal haptoglobin.
The release of haptoglobin in the rabbit by the endometrial epithelium as shown by Olson et al. (1997), the presence of this protein in oviductal and uterine secretions (Hoffman et al., 1996
; this work) and the uptake of maternal haptoglobin by the embryo support a reproductive function during preimplantation time. In the human, haptoglobin is also detectable in uterine secretions (Beier and Beier-Hellwig, 1998
), with an increase during peri-implantation time (our unpublished data). Furthermore, in the human endometrium, haptoglobin expression and localization was found in the endometrial stroma (Sharpe-Timms et al., 2000
) and decidua (Berkova et al., 2001
), suggesting a possible function during peri-implantation time, which may be correlated to the deep interstitial implantation of the embryo in primates. The function of this protein in the early embryomaternal signalling may be an immunomodulatory one, possibly by inducing early maternal immunotolerance. This biological question is under current investigation in our laboratory.
Whether treatment, especially HCG administration, influenced the amount of haptoglobin expression has to be investigated. In general this treatment protocol is widely used in assisted reproduction to induce pregnancies. However, it is also known that HCG has a longer half live than LH, resulting in increased ovulation rate and progesterone levels (Romeu et al., 1995). Therefore, haptoglobin levels might be differnt in untreated animals.
In summary, haptoglobin is secreted prior to and at the time of ovulation by the oviducts (hereby probably influencing the gametes and fertilization). Two additional peaks of haptoglobin release have been detected in the reproductive tract, which show a temporal correlation to the transformation of the extra-embryonic matrices: in the oviduct on day 2 p.c. and in the uterus on day 5 p.c. Although the embryo itself does not express haptoglobin, high amounts of maternal haptoglobin are incorporated into the extra-embryonic matrices. Whether haptoglobin is secreted by the reproductive tract also at times following implantation or by the non-pregnant reproductive tract has yet to be investigated.
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Acknowledgements |
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Submitted on October 2, 2003; resubmitted on June 22, 2004; accepted on August 18, 2004.