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Biology of Reproduction 62, 839-846 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Ruminant Relaxin in the Pregnant One-Humped Camel (Camelus dromedarius)1

Sabine Hombach-Klonisch2,,a, Mahmoud Abd-Elnaeimb, Julian A. Skidmorec, Rudolf Leiserd, Bernd Fischera, and Thomas Klonischa

a Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, D-06097 Halle (Saale), Germany b Department of Anatomy, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt c The Camel Reproduction Centre, Nakhlee, Dubai, United Arab Emirates d Institute of Veterinary Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, D-35392 Giessen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have determined the cDNA sequence of preprorelaxin in the pregnant one-humped camel by employing reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Camel preprorelaxin consisted of 600 base pairs (bp) encoding a protein of 199 amino acids (aa) with a signal peptide of 25 aa (75 bp), a B domain of 28 aa (84 bp), a C domain of 121 aa (366 bp), and an A domain of 24 aa (72 bp). The N terminus of the C domain of camel prorelaxin contained the unique proline-rich repetitive sequence (-RPAP)3-(-K/RPAL-)2, and within the B domain the classical -GRELVR- receptor binding motif was found. Camel preprorelaxin showed highest homology with porcine (74.6%) and equine (65.4%) relaxin. The ovary and the uteroplacental unit were a dual source of relaxin in the pregnant dromedary. Within the ovary, weak expression of relaxin was detected in large luteal cells of the mature corpus luteum. In the ovarian follicles, immunoreactive relaxin, but not relaxin mRNA, was detected in the granulosa and theca interna cell layer. Beginning at around Day 93 of gestation and coinciding with increasing interdigitation of the fetal villus with the underlying maternal endometrium, uterine luminal epithelial cells in the uteroplacental tissue expressed relaxin. Weak expression of immunoreactive relaxin, but not relaxin mRNA, was observed in villous trophoblast cells. Pseudostratified trophoblast cells at the base of the placental villi and multinucleate giant cells did not express relaxin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterodimeric polypeptide hormone relaxin is a member of the insulin-like superfamily and attains the highest plasma levels during pregnancy [1]. Within the order of ruminantia, a functional relaxin has not been determined. Instead, the structurally closely related relaxin-like factor (RLF) may serve as substitute for relaxin in domestic ruminants and has been cloned in the cow [2], the sheep [3], and the goat [4]. RLF is able to bind to receptors specific for relaxin and RLF [5].

Recently, the llama and alpaca, two members of the family of Camelidae, have been shown to express a relaxin during pregnancy, and relaxin has been implicated in both species to be an indicator of pregnancy [6].

All Camelidae are ruminants and have developed a unique reproductive anatomy and physiology. Unlike the other ruminantia, the placenta of the dromedary or one-humped camel lacks the synepitheliochorial cotyledon/caruncular placental structure but, instead, displays diffuse, epitheliochorial placentation much as in pigs and horses [7,8]. The female one-humped camel does not exhibit cyclic sexual activity [9], and copulation is necessary for ovulation to occur [10, 11].

In the present study, we present for the first time a unique nucleic acid sequence of a ruminant relaxin and demonstrate the expression of relaxin within the ovary and the uteroplacental tissue of the pregnant one-humped camel (Camelus dromedarius).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Tissues and RNA Isolation

Uteroplacental and ovarian tissues from the one-humped camel (Camelus dromedarius) at approximately Days 93, 102, 170, 196, 213, 240, 300, 340, and 372 of gestation, as determined by measurement of the crown-rump length of the fetus, were collected at the Cairo (Egypt) abattoir. Camel liver tissue served as control tissue. No animal was operated upon or killed specifically for this study. Tissues were snap-frozen in liquid nitrogen and stored at -80°C until used and fixed in Bouin's solution. Total RNA was isolated with Trizol reagent (Life Technologies, Eggenstein, Germany) from uteroplacental tissue at Day 300 of gestation. For the cloning of camel relaxin, mRNA was isolated from 75 µg of total uteroplacental RNA using oligo(dT)-coated magnetic beads according to the manufacturer's instructions (Dynal, Hamburg, Germany). The amount of mRNA isolated was determined by spectrophotometry at 260 and 280 nm [12].

Rapid Amplification of cDNA ends (RACE)- and Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Cloning

We employed rapid amplification of the 5'- and 3'-cDNA ends (5'- and 3'-RACE-PCR) and RT-PCR to clone the cDNA for camel preprorelaxin from mRNA of uteroplacental origin. PCR primers employed for amplification of the full-size cDNA of preprorelaxin (Table 1) flanked the putative single intron present at the N terminus of the C domain of relaxin to preclude any genomic DNA amplification (Fig. 1). Approximately 500 ng of mRNA were used for first-strand cDNA synthesis employing the Superscript reverse transcriptase kit and 500 ng/ml of oligo(dT) primer (both Life Technologies). PCR reactions were carried out in 50 µl solution containing 1 µl cDNA, 5 µl 10-strength Advantage cDNA polymerase mix buffer, 100 µM dNTP, 10 pmol of each primer (Table 1), and 2.5 U Advantage cDNA mix polymerase (Clontech, Heidelberg, Germany). A cDNA fragment of camel relaxin was initially amplified using the degenerate oligonucleotide primer pair 1 and 2 (Table 1), designed according to a relatively conserved amino acid region at the N terminus of the B domain and the C-terminal part of the A domain of porcine, human, and rat relaxin cDNA. The PCR cycles consisted of initial denaturation for 3 min at 95°C, followed by 40 cycles of denaturation at 95°C and annealing at 56°C, both for 1 min each, an elongation step for 2 min at 72°C, and a final extension cycle for 10 min at 72°C. The 3'- and 5'-RACE-PCR reactions were performed according to the manufacturer's instructions (Life Technologies) employing the gene-specific primers 3–10 and a universal primer (primer 11; Life Technologies) listed in Table 1. RACE-PCR reactions were run for 35 cycles at an annealing temperature of 65°C. Finally, the complete cDNA of the preprorelaxin of the dromedary was amplified by RT-PCR for 40 cycles at an annealing temperature of 68°C using the specific primers 3 and 4 located at both ends of the coding sequence of camel preprorelaxin (Table 1). All PCR products were separated on a 1% low-melting point agarose gel, purified by Magic column extraction, and cloned into the pGEM-T vector (both Promega, Heidelberg, Germany). Sequence analysis of the PCR-amplified cDNA clones was performed employing the PRISM dye Deoxy Terminator cycle sequencing kit (Perkin Elmer, Foster City, CA).


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  		TABLE 1. List of primers employed in the RT-PCR or 5'- and 3'-RACE-PCR for the cloning of camel preprorelaxin



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  		FIG. 1. Schematic representation of the results of the nucleic acid sequence of camel preprorelaxin. Numbered arrows indicate the primers specified in Table 1 used for cDNA cloning by RT- and RACE-PCR

Digoxigenin (DIG) Labeling of cRNA Probes and In Situ Hybridization

Synthesis of DIG-labeled cRNA has been described previously [13]. For nonradioactive in situ hybridization, paraffin-embedded dromedarian uteroplacental sections (5 µm thick) at various stages of gestation (Table 2) attached to glass slides coated with 2% aminopropyltriethoxysilane (APTEX; Sigma, Deisenhofen, Germany) were processed according to the procedure described by Lewis and Wells [14] employing a 1:1000 dilution of an anti-DIG alkaline phosphatase-conjugated Fab-antibody (Boehringer Mannheim, Indianapolis, IN) in 1% BSA. Specific signals were visualized using the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (both Sigma). After counterstaining with hematoxylin, the slides were mounted in glycerol gel and examined under brightfield microscopy.


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  		TABLE 2. Homology of camel preprorelaxin and single relaxin domains with relaxin molecules of other species

Immunohistochemistry

Immunohistochemical staining was performed on 5-µm-thick paraffin-embedded uteroplacental sections mounted on slides coated with 2% APTEX (Sigma). After dewaxing, the slides were treated with 35 µg/ml proteinase K for 30 min at 37°C for the detection of immunoreactive cytokeratin and relaxin. Endogenous alkaline phosphatase activity was inactivated by incubation in 20% acetic acid at 4°C for 20 sec. The tissue sections were blocked with 3% BSA in 0.5 M Tris-buffered saline (0.5 M TBS) for 30 min to saturate nonspecific binding sites. The primary mouse monoclonal antibody (Dako, Hamburg, Germany) to cytokeratin (MNF-116) at a concentration of 1:250 or the rabbit polyclonal anti-relaxin antiserum R6 (generously provided by Prof. B.G. Steinetz, New York University Medical Center, NY) at 1:15 000–1:60 000 was diluted in single-strength TNMT (0.1 M Tris-HCl [pH 7.5], 0.1 M NaCl, 2 mM MgCl2, 0.05% [v:v] Triton X-100) containing a combination of 1% BSA plus 3% nonimmune goat serum. Sections were incubated overnight at 4°C in a moist chamber and then washed 3 times for 10 min each in 0.1 M TBS. Negative controls were performed in all cases by omitting the primary antibody and, for relaxin immunohistochemistry, by incubating with rabbit nonimmune serum as primary antibody instead of rabbit polyclonal anti-relaxin antiserum R6. The alkaline phosphatase anti-alkaline phosphatase (APAAP)-technique was employed for immunodetection of cytokeratin and camel relaxin. Sections were first incubated for 1 h at room temperature in single-strength TNMT containing 1% BSA with an alkaline phosphatase-conjugated goat anti-mouse antibody (Dianova, Hamburg, Germany) at 1:100 (for cytokeratin) or with an alkaline phosphatase-conjugated goat anti-rabbit (Dianova) at 1:100 (for camel relaxin). Sections were washed and incubated for 30 min at room temperature with a mouse APAAP (Dako) for cytokeratin or a rabbit-APAAP (Sigma) for camel relaxin, both at 1:100 in 0.5 M TBS. The incubations with alkaline phosphatase-conjugated antibodies and APAAP complexes were repeated once, and specific immunoreactivity was visualized with the AP-substrate Histo Mark Red (KPL, Gaithersburg, MD). Finally, sections were counterstained with hematoxylin, mounted with glycerol gel, and examined under brightfield microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Employing RT- and RACE-PCR on mRNA from uteroplacental tissue at Day 300 of gestation, we cloned the preprorelaxin cDNA of the one-humped camel consisting of 600 bp encoding a preprorelaxin of 199 amino acids (aa; Fig. 1; Table 1). Using either mRNA or water as template yielded no amplification products (data not shown). According to the known cleavage sites in other relaxin molecules [1], camel preprorelaxin encoded a signal peptide of 25 aa (75 bp), a B domain of 28 aa (84 bp), a C domain of 121 aa (366 bp), and an A domain of 24 aa (72 bp) (Figs. 1 and 2). The location of the cysteine residues in the A and B domains was similar to that of other relaxins (Fig. 2) [1]. Within its B domain, camel relaxin contained the classic receptor binding motif (-GRELVR-; Fig. 2). The N terminus of the C domain contained the unique 20-mer repetitive proline-rich amino acid motif (-RPAP)3-(-K/RPAL-)2, showing camel preprorelaxin to be the largest relaxin cloned so far (Fig. 2). The deduced amino acid sequence of camel preprorelaxin shared highest overall homology with porcine (74.6%) and equine (65.4%) relaxin and, among the different relaxin domains, showed highest sequence conservation in the signal peptide (Table 2). The small amounts of total RNA available did not allow for Northern analysis.



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  		FIG. 2. Nucleic acid sequence and deduced amino acid sequence of camel preprorelaxin cloned from uteroplacental tissue at Day 300 of pregnancy, using RT- and RACE-PCR. The primer binding sites are indicated. Primer numbers correspond to the primers in Table 1. Four amplicons from three independent amplifications were sequenced in both directions by PRISM dye deoxy terminator cycle sequencing to exclude sequencing errors. Identical sequencing results were obtained in all cases

Nonradioactive in situ hybridization was performed on paraffin-embedded ovarian and placental tissues at Days 93, 102, 170, 196, 213, 240, 340, and 372 days of gestation. Within the ovarian tissue, weak specific hybridization signals for relaxin mRNA (Fig. 3C) as well as for immunoreactive relaxin (Fig. 3E) were observed in the corpora lutea of all stages studied. Ovarian follicles, stromal cells, and sections treated with the sense DIG-cRNA probe were devoid of hybridization signals (Fig. 3D). Despite the lack of relaxin transcripts, the follicular granulosa and theca interna cell layer displayed specific immunostaining for relaxin (Fig. 3A).



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  		FIG. 3. Bouin-fixed ovarian tissue sections of pregnant dromedary camels at Days 93, 170, 196, 213, 240, 340, and 372 of gestation were subjected to nonradioactive in situ hybridization for the detection of camel relaxin mRNA expression (C, D) or rabbit APAAP immunohistochemistry with a rabbit polyclonal antibody specific for relaxin (R6) (A, E). At all stages of pregnancy studied, weak expression of relaxin mRNA was detected in large luteal cells as shown here for Day 170 of gestation (C). Sections of ovarian follicles of all sizes and corpus luteum sections treated with the sense copy RNA were devoid of hybridization signals (D). In agreement with the in situ hybridization, immunoreactive relaxin was detected in large luteal cells of the corpus luteum (E). The granulosa (g) and theca interna (ti) cell layer of ovarian tertiary follicles displayed immunoreactivity for relaxin (A). Negative controls were performed by omitting the primary antibody (B, F). Magnifications: A, B) x109; C–F) x218 (published at 80%)

Within the uteroplacental tissue, relaxin mRNA was exclusively detected in maternal luminal epithelial cells at the feto-maternal interface. At around Day 93 of gestation, expression of relaxin transcripts in placental sections was restricted to some luminal epithelial cells (data not shown). However, uteroplacental sections at Days 170 to 372 of the approximately 380 days of gestation displayed strong expression of relaxin transcripts in all uterine luminal epithelial cells (Fig. 4A). Maternal endometrial glands, trophoblast cells, and maternal and fetal stromal cells, as well as sections treated with the sense DIG-cRNA probe were devoid of hybridization signals (Fig. 4, A and B). The uterine luminal epithelium expressing relaxin mRNA also showed strong immunohistochemical staining for immunoreactive relaxin (Fig. 4C). Although we were unable to detect relaxin mRNA in fetal villous trophoblast cells, these cells displayed weak specific staining for immunoreactive relaxin even at high dilutions (1:60 000) of the R6 relaxin antiserum (Fig. 4C), suggesting marginal expression of relaxin. In all uteroplacental tissue sections, pseudostratified trophoblast cells located at the base of the fetal villi were devoid of relaxin mRNA and immunoreactive relaxin (Fig. 4E). Uterine luminal epithelial cells and fetal trophoblast cells were further characterized by the expression of the epithelial cell marker cytokeratin (Fig. 4F). Control experiments omitting the primary antibody or replacing the relaxin antiserum with a rabbit nonimmune serum consistently gave negative results in the ovarian (Fig. 3, B and F) and uteroplacental sections (Fig. 4D).



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  		FIG. 4. Nonradioactive in situ hybridization of Bouin-fixed uteroplacental tissue at Day 213 of gestation revealing specific hybridization signals exclusively in the maternal (m) uterine luminal epithelium (A). Glandular uterine epithelium, maternal and fetal stroma, fetal trophoblast cells, and tissue sections treated with a sense cRNA (B) were devoid of relaxin transcripts. Immunoreactive relaxin was colocalized in the uterine epithelium expressing relaxin mRNA (C, E). In the trophoblast cells lining the fetal villus (v) faint specific immunostaining for relaxin was observed (C, E), whereas pseudostratified trophoblast cells (arrows) were devoid of relaxin (E). Fetal trophoblast cells and maternal uterine epithelial cells were identified by the immunodetection of the epithelial cell marker cytokeratin (F). Sections with the primary antibodies omitted were devoid of immunostaining (D). Magnifications: A, B, E, F) x132; C, D) x262 (published at 80%)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Camelids are ruminants but only possess a three-chambered stomach, lacking the omasum that is part of the four-chambered stomach of other members of the order of ruminantia. Camelids evolved in North America and separated from primitive artiodactylids in the Eocene epoch, approximately 40 million years ago. Among the 6 species in the family of Camelidae, the dromedary (Camelus dromedarius) and the Bactrian camel (Camelus bactrianus) are domesticated inhabitants of the Old World. In South America, the family is represented by two domesticated species, the llama (Lama glama) and the alpaca (Lama pacos), and by the wild-living Guanaco (Lama guanacoe) and vicuña (Vicugna vicugna). Ruminants appear not to express a functional relaxin [15]. Despite an early report on the detection of relaxin serum immunoreactivity in the pregnant goat [16] and weak hybridization signals in Northern analysis of poly(A)-enriched RNA from caprine and bovine mid-pregnant corpus luteum [15], attempts to clone a relaxin in ruminants have been unsuccessful. In the sheep, the relaxin gene contains multiple stop codon mutations that would result in the expression of nonfunctional relaxin fragments [17]. The molecular cloning of the preprorelaxin cDNA of the one-humped camel is the first report on the expression of a relaxin in a ruminant. RT-PCR revealed a single amplification product of 600 bp, excluding alternatively spliced amplification products recently described for human relaxin mRNA species [18]. Camel preprorelaxin showed highest homology (75%) to the preprorelaxin of the pig, another member of the artiodactylids. Similar to the relaxins of the pig [19], Bryde's and mink whale [20], porpoise [21], and rhesus monkey [22], and the human H1 and H2 relaxins [23], the B domain of camel relaxin contained the classical -GRELVR- receptor binding motif. The cysteine residues within the A and B domains were highly conserved [1], and the unique proline-rich repetitive amino acid motif (-RPAP)3-(-K/RPAL-)2 most likely imposed a coiled tertiary structure on the N terminus of the C domain of camel prorelaxin. Consisting of 122 aa, the C domain of camel prorelaxin was considerably longer than the longest C domain determined so far of the tammar wallaby (111 aa) [24]. The consensus cleavage motif -KRRK- for furin [25, 26] at the C domain/A domain junction found in the prorelaxins of the pig [19], rat [27], hamster [28], and tammar wallaby [24], and the human H2 relaxin [23] contained a single substitution in the first position (E->K) of camel prorelaxin.

The ovary and the uteroplacental tissue are a dual source of relaxin in the pregnant one-humped camel. Within the ovary, immunoreactive relaxin, but not relaxin mRNA, was detected in both the granulosa and theca interna cell layer of large follicles. We have previously reported similar results in large ovarian follicles of the cyclic mare [29]. Our inability to localize relaxin transcripts in the ovarian follicle of the pregnant dromedary camel may be explained either by the lower sensitivity of the nonradioactive labeled cRNA probes under low copy expression conditions [2] or by the uptake from the follicular fluid of luteal relaxin by the camel follicular cells. A more sensitive in situ hybridization technique may help to answer this question. In pig ovarian follicles, relaxin has been localized to the theca interna cells [30, 31], and in human preovulatory follicles the theca cells have been identified as the source of relaxin transcripts [32]. Relaxin's connective tissue remodelling activity in the ovary is mediated by the release of plasminogen activator, plasminogen activator inhibitor, gelatinases, collagenases, and proteoglycans [33, 34]. In the in vitro-perfused rat ovary, relaxin induces ovulation [35].

Another source of relaxin was the corpus luteum in the ovary of the pregnant one-humped camel. In contrast to other ruminants, camelids are induced ovulators [10, 11], and the corpus luteum that develops after a sterile mating in the one-humped camel [36, 37] or the llama [38] has a life span of only 8–10 days. An intact corpus luteum appears to be essential for successful pregnancy, as administration of prostaglandin F2{alpha} to dromedary camels at any stage of gestation results in abortion [39]. Similar to the case in the pig [40] and the human [41], relaxin was expressed in large luteal cells of the mature corpus luteum of the dromedary camel. The functional role of relaxin in the corpus luteum is unknown. Relaxin, in synergy with estrogen and progesterone, has angiogenic and vasodilatory properties, and the vasodilatory effects appear to be mediated by a relaxin-induced increase in nitric oxide [42, 43]. Within the human endometrium, relaxin has recently been shown to induce the expression of vascular endothelial growth factor (VEGF) [44]. VEGF is essential for corpus luteum formation [45].

Within the uteroplacental tissue, relaxin expression in uterine luminal epithelial cells of the epitheliochorial camel placenta was first detected at around Day 93 of gestation and coincided with the onset of interdigitation of the anchoring fetal villi with the apposing maternal luminal epithelium [46]. In the pregnant llama and alpaca, the first detectable relaxin serum immunoreactivity was also reported at about Day 85 of the approximately 350 days of gestation, and the gestational relaxin profile was similar to that of the pregnant mare [6]. The uterine endometrium has previously been identified as a source of relaxin in the rabbit [47], guinea pig [48], rat [49], pig [50], and human [1]. Conceptus-derived estrogens at the implantation site appear to stimulate and maintain local endometrial relaxin synthesis [50]. Estradiol, alone and in combination with progesterone, stimulates accumulation of secretory granules containing relaxin in endometrial gland cells of the guinea pig [51], and estrogen has been shown to stimulate relaxin receptors in the myometrium of ovariectomized estrogen-primed rats [52]. At the uteroplacental interface of various species, relaxin is expressed in highly proliferative placental areas [53–55], and the ability of relaxin to stimulate uterine growth is well established [56–58]. In the rat uterus, relaxin-induced growth appears to be mediated by estrogen receptors [59]. Both embryonic and endometrial tissues of the pregnant dromedary camel exhibit high aromatase activity, and maternal estrogen concentrations are elevated in camelids during most of pregnancy [37, 60], indicating a possible role for relaxin during uteroplacental growth in camelids. Indeed, in the llama, the alpaca, and possibly the one-humped camel, relaxin is a suitable indicator for pregnancy from Day 85 of gestation onwards [6]. Induction of placental relaxin expression coincided with a fall in serum progesterone concentrations observed in the pregnant dromedary [61]. In the late pregnant beef heifer, continuous i.v. infusion of high levels of porcine relaxin resulted in a decrease in progesterone secretion [62]. It is tempting to speculate that the appearance of relaxin in the diffuse epitheliochorial placenta at the beginning of the interdigitation process between fetal and maternal tissues may assist progesterone in rendering the uterus quiescent, since both hormones are well-known myometrial inhibitors [63].

Very weak specific expression of immunoreactive relaxin was detected in villous fetal trophoblast cells. Multinucleate giant cells as well as pseudostratified trophoblast cells located at the base of the villi were devoid of relaxin. We have previously described pseudostratified fetal trophoblast cells within the equine placenta as devoid of relaxin but instead expressing maternal and paternal antigens presented on major histocompatibility complex (MHC) class I molecules [53]. In spite of employing an array of different anti-MHC class I antibodies, we have as yet not been able to detect MHC class I molecules in the camel placenta. However, the possibility that both the mare and the dromedary employ MHC-presenting fetal trophoblast cells for feto-maternal recognition and maintenance of pregnancy is intriguing, and further work is needed to clarify the role of these cells in the epitheliochorial placenta.


   ACKNOWLEDGMENTS
 
We would like would like to thank Mrs. Christine Fröhlich and Elke Bernhard for their excellent technical assistance. The authors extend their gratitude to Prof. Bernhard G. Steinetz, Nelson Institute of Environmental Medicine, New York University Medical Center, New York, NY, for generously providing the R6 relaxin antiserum. We also thank Prof. W. R. Allen, University of Cambridge, UK, for his support.


   FOOTNOTES
 
First decision: 15 October 1999.

1 S.H.-K. was supported by the Land Anhalt-Saxony in the program "Wiedereinstiegsstipendium für Frauen." Back

2 Correspondence: S. Hombach-Klonisch, Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Grosse Steinstrasse 52, D-06097 Halle (Saale), Germany. FAX: 0049 345 557 1700; sabine.hombach-klonisch{at}medizin.uni-halle.de Back

Accepted: November 15, 1999.

Received: September 9, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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