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Endocrinology Vol. 138, No. 5 1857-1862
Copyright © 1997 by The Endocrine Society

Articles

Expression and Region-Specific Regulation of the Oxytocin Receptor Gene in Rat Brain1

Christophe Breton2 and Hans H. Zingg3

Laboratory of Molecular Endocrinology, Royal Victoria Hospital, McGill University, Montreal, Quebec, H3A 1A1, Canada

Address all correspondence and requests for reprints to: Dr. Hans H. Zingg, Laboratory of Molecular Endocrinology, Royal Victoria Hospital, 687 Pine Avenue West, Room H7.63, Montreal, Quebec H3A 1A1, Canada. E-mail: ZINGG{at}RVHRI.LAN.MCGILL.CA


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neuropeptide oxytocin (OT) exerts its various neurotransmitter functions via specific OT receptors (OTRs) that have been localized to distinct brain regions, including the ventromedial hypothalamus, the bed nucleus of stria terminalis, the amygdala, the subiculum, the hippocampus, and the olfactory nuclei. In the present study, we have characterized OTR gene expression by Northern blot and by semiquantitative RT-PCR in these brain regions and studied its regulation in response to estrogen (E2), progesterone, and the antiestrogen tamoxifen. We find that all regions analyzed express two messenger RNA (mRNA) bands (6.7 and 4.8 kb) that hybridize to a rat OTR complementary DNA probe and that correspond in size to two of the three OTR mRNA bands expressed in rat uterus. Analysis by RT-PCR, with two different primer pairs, did not reveal any structural differences between the coding regions of uterine and brain OTR mRNA. E2 treatment and gestation led to an 8-fold and a 6.5-fold increase in OTR mRNA levels, respectively. Progesterone was without effect, if administered alone, and did not influence the E2-induced rise in OTR mRNA. The E2 effect was restricted to E2-sensitive regions, such as the hypothalamus, and was not observed in the subiculum or the olfactory nuclei. Tamoxifen had a dual effect: on the one hand, it acted as a partial agonist in raising OTR mRNA levels in the hypothalamus of ovariectomized animals; on the other hand, it suppressed the E2-induced OTR mRNA rise in E2-sensitive brain regions.

Although the present data do not exclude the possible existence of OTR subtype(s) in brain, they show that the uterine-type OTR gene is expressed in all major OTR-containing brain regions. Moreover, they show that region-specific regulation of OTR gene expression underlies the previously observed region-specific steroid regulation of central OT binding sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PEPTIDE hormone oxytocin (OT) mediates a variety of peripheral and central functions (1, 2). Whereas the peripheral actions include uterine contractions, milk ejection, PRL release, and natriuresis, the central functions are related mainly to female reproductive behavior, including induction of sexual receptivity (3), maternal behavior (4, 5, 6, 7, 8), and pair bonding (9). Additional proposed central functions of OT comprise modulation of cardiac vagal output (10), memory consolidation (11), and social/affiliative behavior (9, 12). Evidence for a biological role of endogenously released OT stems from the findings that onset of specific maternal and sexual behaviors can be delayed or blocked by central administration of OT antiserum, OT antagonists (4, 13, 14, 15), or OT receptor (OTR) antisense oligonucleotides (16). These observations suggest that central OT effects are mediated via specific OTRs. Evidence for the presence of central OTRs indeed has been provided by receptor autoradiography (17, 18, 19, 20), as well as by in situ hybridization (21, 22, 23, 24). These studies established the presence of OTRs in specific brain areas, including the ventromedial hypothalamus (VMH), the olfactory bulb (specifically the anterior olfactory nucleus), the anterior aspects of the bed nucleus of stria terminalis (BNST), the ventral subiculum, and the central amygdaloid nuclei. These different areas all have been implicated in mediating OT’s varied central functions. For example, the VMH and the BNST mediate OT-induced sexual and maternal behaviors (25). OT-induced inhibition of olfactory processing is thought to facilitate maternal behavior by suppressing the perception of the (normally aversive) smell of pups (26). The hippocampal site of action seems to be important for OT’s attenuating effect on memory consolidation (11, 27), and the amygdala are involved in mediating the effect on vagal output (10).

A prerequisite for OT’s potential to elicit maternal behavior, however, is that the animal be primed with systemic estrogen (E2) or E2 and progesterone (P4) (4, 6). This suggests that central OTRs may be under the control of gonadal steroids. This idea is further supported by autoradiographic and in situ hybridization studies, which showed an E2-induced upregulation of OTRs in the VMH and the BNST nuclei (17, 18, 22, 28). However, not all OTR-containing brain regions are similarly affected by steroid treatment. Most notably, OT binding in the subiculum and the anterior olfactory nuclei remain unaffected by E2-administration (17, 18). OTR expression in E2-responsive areas also has been shown to be affected by endogenous changes in circulating steroid levels. Thus, OT binding in the BNST and VMH increases at parturition (9, 17), and OTR messenger RNA (mRNA) in the VMH undergoes changes during the estrous cycle that parallel changes in circulating E2 levels (23).

Although binding studies have, in general, supported the idea that only one OTR type, identical to the uterine-type, is expressed in brain (19), it remains unresolved whether the region-specific regulation of central OTRs is caused by the presence of different OTR subtypes, by differential promoter usage, or by region-specific differences in the steroid-responsiveness of OTR-expressing neurons. We previously have characterized the mRNA encoding the OTR in rat uterus (29, 30), pituitary gland (31), and kidney (32). These studies showed that expression of the rat OTR gene in the uterus gives rise to 6.7-, 4.8-, and 2.9-kb mRNA bands (33). These different mRNA species encode the same OTR but differ with respect to the length of the 3' untranslated regions. The 6.7- and 4.8-kb species, but not the 2.9-kb species, also are expressed in the pituitary gland and in the kidney (31, 32). In all these organs, E2 treatment leads to an increase in OTR gene expression (31, 32, 33). In the present study, we have characterized OTR transcripts in brain regions containing either E2-sensitive or E2-insensitive OT binding sites, and we have semiquantitatively assessed the effects of gonadal steroids on the transcript levels in distinct brain areas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cycling, timed-pregnant, and bilaterally ovariectomized Sprague-Dawley female rats (200–250 g) were obtained from Charles River Laboratories (Saint-Constant, Quebec, Canada). In cycling rats, the stage of the estrous cycle was assessed by daily vaginal smears. Animals were injected on 4 consecutive days with vehicle (oil), estradiol benzoate (0.1 mg/kg), P4, 10 mg/kg), tamoxifen (TAM, 10 mg/kg), or a combination of E2 and P4 or E2 and TAM. Three hours after the last injection, the animals were killed. In ovariectomized animals, treatment was started 1 week after ovariectomy. All procedures were approved by the Bioethics Committee of the Royal Victoria Hospital Research Institute.

Different brain regions were manually dissected as follows [nomenclature and coordinates according to Palkovits and Brownstein (30)]: 1) olfactory bulb (removed from the rest of the brain, along the rhinal fissure); 2) hypothalamus (a tissue block was dissected with the following boundaries: frontal: optic chiasm; caudal: mamilliary bodies; lateral: 2 mm at each site of the midline, 2 mm depth); 3) BNST (a slice delineated by cuts at A, 300 µm, and P, 900 µm, was frozen on a glass slide and the inferior medial quarters of both sides were dissected free; this fragment included, in addition to the BNST, also the globus pallidus, the ventral pallidum, and part of the medial forebrain bundle); 4) amygdaloid nuclei (an area containing the amygdaloid nuclei was dissected by obtaining a slice delineated by cuts at P, 900 µm, and P, 3900 µm, and bisecting the lateral inferior quarters on both sides); 5) subiculum/hippocampus region [a slice delineated by two frontal cuts at P, 3900 µm, and P, 7500 µm, was frozen on a glass slide on dry ice; the area corresponding to the neocortex was removed and kept frozen (see below); from the remaining fragment, an area containing the subiculum, the parasubiculum, and the presubiculum was dissected free]; 6) parietal cortex (the fragment used corresponded to the neocortex removed in the dissection described above).

A uterus was obtained from a pregnant rat during parturition. The uterus was slit open lengthwise to remove the remaining embryos and rinsed in phosphate-buffered saline.

All tissue fragments were frozen on dry ice and kept at -70 C until RNA extraction.

Northern blot analysis
Total RNA was extracted with 4 M guanidium thiocyanate and purified by ultracentrifugation through 5.7 M CsCl. One mg total RNA was enriched for polyadenylated RNA by absorption to oligo(dT) cellulose. The RNA was separated by agarose gel electrophoresis and transferred to Hybond-N membranes (Amersham, Life Sciences, Arlington, IL), as previously described (32). Blots were hybridized to a probe consisting of a 257-bp complementary DNA (cDNA) fragment encoding the N-terminal part of the rat OTR (residues 1–85) (29). Probes were labeled by the random-primer labeling method (32).

RT-PCR
Two different primer pairs (F3/R8 and F1C/R9) were used, as previously described (32). The F3/R8 pair was designed to amplify a 1103-bp cDNA fragment encompassing the entire coding region of the rat OTR (nucleotides 1952–3707, according to Ref.29). The F1C/R9 primer pair amplified only a smaller 373-bp region of the coding region, which encompassed the splice site of the large intron (intron 2) that interrupts the coding region in the rat OTR gene (29).

RNA from different brain regions, as well as from pituitary glands, was extracted using Trisol (GIBCO/BRL, Gaithersburg, MD). Two µg RNA were reverse transcribed into cDNA using 2 µg random hexamers and 200 U Moloney murine leukemia virus RT (GIBCO/BRL) (32). The methods for semiquantitative analysis of the PCR amplification products has been described and validated previously (32). In short, Southern blots of PCR products were hybridized to a [32P]-labeled 20-mer oligonucleotide probe complementary to a region internal to both the F3/R8 and F1C/R9 amplification products, and the resulting autoradiograms were scanned densitometrically (32). As an internal control, GAPDH mRNA also was quantitated by RT/PCR (32) using a primer pair designed to amplify a 470-bp region of the GAPDH. PCR amplifications involved 35 cycles for OTR cDNA and 25 cycles for GAPDH cDNA under conditions outlined in (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OTR mRNA present in different brain regions was characterized by Northern blotting and RT/PCR. The regions analyzed included parietal cortex, the hypothalamus, the BNST, the olfactory bulb, an area including subiculum and hippocampus, and an area containing the amygdaloid nuclei. Whereas only low levels of OT binding sites have been described in parietal cortex, the remaining brain regions analyzed were known to be enriched for specific OT binding sites (19, 24, 34, 35). As shown in Fig. 1Go, all brain regions rich in OT binding sites expressed two mRNA bands that hybridized to a rat OTR cDNA probe. These two bands were of the same size in all OTR-containing areas analyzed and were estimated at 6.7 and 4.8 kb. These bands corresponded in size to two of the OTR mRNA bands previously detected in rat uterus (reference (29) and Fig. 1Go). A third OTR mRNA band of 2.9 kb that is abundantly present in rat uterus was either absent (hypothalamus and subiculum) or barely detectable in OTR-expressing brain regions. We have shown earlier that these three mRNA bands encode the same OTR and differ with respect to the lengths of the 3' untranslated region, probably resulting from the differential choice of polyadenylation sites within the less-than-5-kb 3' untranslated region (29). The biological significance (if any) of this differential polyadenylation remains to be elucidated. Under the conditions used, no additional bands were detected that could be indicative for splicing variants and/or additional OTR subtypes.



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  		Figure 1. Northern blot analysis of poly(A)+-enriched RNA extracted from the uterus of a parturient rat (lane 1, 5 µg RNA) or from different rat brain regions of intact female rats as indicated (20 µg RNA). The blot was hybridized with an OTR-specific cDNA probe. Exposure times: 18 h for lanes 1, 4, 5; 2 days for lanes 2 and 3; 6 days for lanes 6 and 7.

 
Further structural analysis of brain OTR mRNA was undertaken using RT/PCR in conjunction with two different primer pairs. Both pairs encompassed an intron splice site located in the region encoding the putative third extracellular loop of the receptor molecule. This analysis was carried out with RNA extracted separately from all five OTR-containing brain regions studied, as well as with total brain RNA. For each extract and for each primer pair, this analysis resulted in a single band that was identical to the one obtained with pituitary or uterine RNA and corresponded to the size that was predicted from the OTR gene sequence (Fig. 2Go). Whereas primer pair F3/R8 amplified the entire coding region and gave rise to a 1.1-kb band, the F1C/R9 pair amplified a subregion centered around the intron splice site present in the coding region, resulting in a 373 bp-band. In both cases, the PCR Southern blot (Fig. 2Go) and the ethidium bromide stained agarose gels (not shown) displayed no additional bands that could be indicative of splicing variants. These findings cannot exclude the existence of OTR mRNA variants or subtypes not detectable with the methods used here. They support, however, the idea that the known uterine OTR type is expressed in all OTR-containing brain regions analyzed here.



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  		Figure 2. Southern blot analysis of RT-PCR products using total RNA extracted from the uterus of a parturient rat (lane 2, 50 ng RNA) or different rat brain areas of intact female rats (lanes 3–10, 2 µg RNA), as indicated. Lane 1, Control reaction with no RNA added. As indicated on the left, two different primer pairs were used, giving rise to amplification products of 1,103 bp (upper panel) or 373 bp (lower panel). Blots were hybridized to an oligonucleotide probe complementary to a region internal to the amplification product (32).

 
Earlier binding studies demonstrated a strong E2-inducibility of OTR binding sites in some brain regions (e.g. the hypothalamus) but not in others (e.g. the subiculum and olfactory nuclei). These observations prompted us to investigate to what extent the differential sensitivity to E2-induction may be caused by a difference in the E2-inducibility of OTR mRNA in these brain regions. To this end, we used a semiquantitative RT/PCR assay previously used to assess the regulation of pituitary and renal OTR gene expression (32). As shown in Fig. 3Go, OTR mRNA accumulation in the hypothalamus was highly responsive to E2-administration in vivo (>8-fold increase) and exceeded the E2-induced increase in pituitary OTR mRNA (3.5-fold). By contrast, OTR mRNA levels in the olfactory bulb and the subiculum/hippocampus area remained unaffected by E2 administration. P4 administration alone had no significant effect on OTR mRNA levels, nor did it influence the E2-induced rise in E2-responsive brain regions (Fig. 3Go).



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  		Figure 3. Effects of administration of vehicle (open bars), P4 (stippled bars), E2 (closed bars), or a combination of E2 and P4 (hatched bars) on OTR mRNA accumulation in different rat brain areas, as well as in the pituitary of ovariectomized rats. OTR mRNA levels were determined semiquantitatively using a RT-PCR assay described previously (32). Pit., Pituitary; Hypo., hypothalamus; Olf., olfactory bulb; Sub./Hip., subiculum/hippocampus area. Each bar represents the mean ± SE of the results obtained from four different animals. Values that differ significantly from control are indicated (+, P < 0.05; *, P < 0.01).

 
We next investigated whether these different brain areas also exhibited a differential response to physiological states associated with increased circulatory E2, such as gestation. As shown in Fig. 4Go, hypothalamic OTR mRNA levels at term were 6.5-fold higher than in nonpregnant virgin animals. This increase was similar to the one found in pituitary OTR mRNA. However, olfactory bulb and subiculum/hippocampus OTR mRNA levels remained unaffected throughout gestation.



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  		Figure 4. OTR mRNA levels in different brain areas and in the pituitary in virgin rats (open bars) and in rats at parturition (closed bars). OTR mRNA levels were determined by RT-PCR. Abbreviations are the same as in Fig. 3Go. Each bar represents the mean ± SE of the results obtained from four different animals. *, Values that differ significantly from control (P < 0.01).

 
In a last set of experiments, we tested to what extent the inducing effect of E2 could be antagonized by the ER antagonist TAM. TAM was administered alone or in combination with E2 to ovariectomized and to normal cycling animals. In ovariectomized animals, TAM alone acted as a weak E2 agonist but significantly suppressed E2-induced OTR gene expression if administered concomitantly with E2 (Fig. 5Go). This effect was only observed in E2-responsive tissues and OTR mRNA levels in olfactory bulb, and the subiculum/hippocampus area remained unaffected by these treatments. To test the effect of TAM in the presence of endogenous steroid levels present in intact normal cycling animals, the experiment was repeated in nonovariectomized animals. In normal cycling rats at proestrus, E2 administration also induced a significant, but less dramatic, increase in pituitary and hypothalamic OTR mRNA (Fig. 6Go). The reduction in the relative increase was caused mainly by the higher basal OTR mRNA levels present in control animals. Again, the steroid effect was restricted to pituitary and hypothalamic tissues and was not observed in the olfactory bulb nor in the subiculum/hippocampus area (Fig. 6Go). Probably also because of the higher basal levels of E2 in nonovariectomized animals, the weak agonistic effect of TAM was no longer observable. On the contrary, TAM suppressed both the basal levels and the E2-induced levels of OTR mRNA.



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  		Figure 5. Effects of E2 and TAM treatment on OTR mRNA in different brain regions and in the pituitary in ovariectomized rats. Animals were treated during 4 consecutive days with either oil (open bars), E2 (closed bars), TAM (stippled bars), or both E2 and TAM (hatched bars). OTR mRNA levels were determined by RT-PCR. Each bar represents the mean ± SE of the results obtained from four different animals. Signs above bars indicate values that differ significantly from control. Signs above brackets denote significant differences between the bars indicated by the bracket. +, P < 0.05; *, P < 0.01.

 


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  		Figure 6. Effects of E2 and TAM treatment on OTR mRNA in different brain regions and in the pituitary in adult intact female rats. Animals were treated during 4 consecutive days with either oil (open bars), E2 (closed bars), TAM (stippled bars), or both E2 and TAM (hatched bars). Control rats were in the proestrus phase at the day of death. OTR mRNA levels were determined by RT-PCR. Each bar represents the mean ± SE of the results obtained from four different animals. Signs above bars indicate values that differ significantly from control. Signs above brackets denote significant differences between the bars indicated by the bracket. +, P < 0.05; *, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, central OTR gene expression has been studied by characterizing OTR mRNA expressed in different rat brain regions with respect to structure and steroid regulation. On the one hand, the results indicate structural uniformity of the OTR mRNA bands expressed in different brain regions. On the other hand, they demonstrate region-specific differences with respect to steroid-mediated regulation.

Several reports provide indirect evidence for the possible existence of additional OTR subtypes in brain. For example, a particular OT antagonist was shown to be effective peripherally but not centrally (36); astrocytes may be endowed with an OTR subtype (37); two areas in the hippocampus and the amygdala that are rich in OT binding sites did not contain any immunoreactive OTR (38). Although the present studies cannot exclude the existence of OTR subtypes in the brain, they do provide evidence that the known uterine-type OTR gene is expressed in all major brain regions known to contain OT binding sites. In the absence of precise structural information, the existence of additional OTR subtypes in brain remains speculative. Low stringency hybridization and degenerate primers will be required for further attempts to identify OTR-related transcripts in brain tissues.

The presence of OTR subtypes could be caused by the expression of distinct genes that are structurally related to the OTR gene or by the generation of splicing variants of the known OTR gene. As we have shown earlier, the coding region of the rat OTR gene is interrupted by a single intron in the region encoding the third extracellular loop. The results from our RT/PCR experiments cannot exclude the possibility that an entirely different exon is spliced to the exon that encodes the transmembrane domains 1 to 6. However, the resulting transcript would be, most likely, of a different size and, therefore, would have been detected by our Northern blot analyses. On the other hand, splicing variations that would have involved even a small variation in the choice of the splice donor or splice acceptor site should have been detected by at least one of the two RT/PCR strategies that were employed in the present study. It is of note that a splicing variation of the vasopressin V2-type receptor recently has been described (39). Our present approach can, with certainty, exclude the occurrence of a similar splicing variation in centrally expressed OTR mRNA.

The need for E2 priming for the efficiency of centrally applied OT in eliciting behavioral responses has long led to the suspicion that at least some of the central OTRs are regulated by steroids. As our studies demonstrate, the expression of hypothalamic OTRs, the receptors relevant for mediating reproductive behavior, are highly induced by E2 administration. This is in accordance with previous findings using autoradiography (18, 28) or in situ hybridization (22). However, centrally injected OT is only fully effective if preceded by priming with E2 and P4. In the present studies, we did not observe any effects of P4 on OTR mRNA levels whether P4 was administered alone or in conjunction with E2. It seems, therefore, that E2 exerts a direct or indirect effect on OTR gene expression at the level of OTR mRNA accumulation, whereas P4 may act at a step further downstream and may include nongenomic actions. This interpretation is supported by observations by Schumacher et al., which suggested a nongenomic effect of P4 on OTR translocation along dendrites (40).

Brain areas unresponsive to E2 administration also remained unaffected by physiological stages known to be associated with increased OTR expression in other tissues. In the present study, parturition was chosen as a stage that is known to affect OTR gene expression in different tissues. We have shown earlier that at parturition, OTR mRNA levels are highly increased in uterus (33) and pituitary gland (31), whereas levels are suppressed in the kidney (32). In brain, the hypothalamus follows the pattern observed in the pituitary gland, with a strong upregulation of the OTR gene at the end of gestation, whereas brain regions that did not respond to E2 (such as amygdala, subiculum and olfactory bulb) did not show any changes at the end of the gestation period either. Comparison with earlier binding studies demonstrates that OTR mRNA levels are directly related to OT binding in different brain areas. Thus, autoradiographic studies by Insel (9) showed that, at parturition, OT binding was increased in the hypothalamus and the BNST but not in the amygdala, ventral subiculum, or anterior olfactory nucleus.

The mechanisms underlying the differential responsiveness to E2 in different brain regions remain to be determined. Although the present study does not provide any evidence for the existence of different OTR subtypes in brain, this possibility cannot be excluded at present. An alternative explanation is that the differences in E2-responsiveness are caused by differential distribution of E2 receptors. Studies by Pfaff and Keiner (41), as well as Stumpf et al. (42), have shown a dense localization of E2 receptors in the VMH and BNST areas, whereas the anterior olfactory nucleus or the central nucleus of the amygdala seem to be devoid of E2 receptors. It also remains to be determined whether E2 exerts a direct effect on OTR gene transcription or whether the effect is indirect and mediated via the increased expression of one or more other genes. The fact that no classical estrogen response element is present in the OTR promoter region (29) and that the OTR promoter region remains unresponsive to E2 (43) argues against a direct effect of E2 on OTR gene transcription.

The idea of a genomic effect of E2 is further supported by the results obtained with TAM. This nonsteroidal compound is known to have mixed partial E2 agonistic and antagonistic effects (44). Our studies revealed similar mixed agonistic and antagonistic effects with respect to central OTR gene expression in vivo. If administered together with E2, TAM acted as a partial antagonist of E2-induced OTR expression in both ovariectomized and normal cycling rats. This result is consistent with behavioral studies, which showed that TAM application to the VMH caused a significant reduction in lordosis behavior (45). However, when TAM was administered alone, the effect depended on the level of endogenously present circulating E2: If endogenous circulating E2 was present, as was the case in normal cycling rats, TAM administration had a suppressive effect on OTR mRNA levels. If administered to an ovariectomized animal with undetectable E2 levels, TAM revealed a weak agonistic effect with respect to hypothalamic OTR gene expression. The present findings support the idea that the effect of TAM not only depends on intrinsic cell- and gene-specific factors (44) but also on extrinsic factors, such as the levels of circulating E2. These effects of TAM also were observed in E2-responsive, OTR-expressing pituitary cells but not in E2-unresponsive brain areas, such as the subiculum and the olfactory bulb.

The effects of E2 and TAM on brain OTR gene expression explain the modulating effects of E2 priming and TAM application on central OT responsiveness. The studies also demonstrate that, as is the case in the periphery, the OT system is regulated to a major extent at the level of OTR gene expression. Yet, the specific mechanisms underlying the region-specific E2 inducibility of central OTR gene expression remain to be elucidated.


   Acknowledgments
 
We thank Ms. Elisa Monaco for excellent secretarial assistance and Ms. Jeana Neculcea and Ms. Caterina Russo for technical help.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada. Back

2 Present address: INSERM, Unité U401 Pharmacologie Moléculaire de Recepteurs d’Hormones Peptidiques, CCIPE, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France. Back

3 A Scientist of the Medical Research Council of Canada. Back

Received September 26, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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