Vasopressin gene transcripts in the bovine corpus luteum are defective

Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,

255

53 (1987) 255-258 Ltd.

MCE 01769

Rapid Communication

Vasopressin

gene transcripts

in the bovine corpus luteum are defective

Steven D. Morley and Richard Institut

(Received

Key words: Corpus luteum;

Ivell

ftirZellbiochemie und klinrsche Neurobiologie, Uniuersitiit Hamburg, Hamburg. F.R.G.

Vasopressin;

mRNA,

11 August

1987; accepted

vasopressin;

Transcriptional

17 August

1987)

regulation;

Lambda

gtll

cloning

Summary cDNA clones corresponding to vasopressin gene transcripts were isolated from a hgtll library made using mRNA extracted from a bovine corpus luteum of the early non-pregnant cycle. None of the characterized clones included the vasopressin-encoding exon A sequence, instead two of these clones included sequence from the first intron. Together these data and controls indicate that vasopressin gene transcription in this tissue does not yield translatable mRNA and that positive RNA-DNA hybridization signals are not necessarily evidence for local biosynthesis of the neuropeptide.

Introduction The nonapeptide hormones vasopressin and oxytocin, once thought to be synthesized exclusively in the hypothalamus, have recently been detected at low levels using a variety of immunological and chromatographic systems in several peripheral tissues, such as the adrenal, the testis and the ovary (review in Clements and Funder, 1986; Ivell, 1987). From the point of view of regulation it is important to know whether the hormones are synthesized locally in these tissues, or sequestered from the blood stream. In studies undertaken to investigate this aspect, detection of the specific mRNA by nucleic acid hybridization has often been considered a sufficient criterion to verify local synthesis. As part of a study of gene expression in the bovine corpus luteum, we isolated from a luteal library cDNA clones encoding Address for correspondence: Hormone and Fertility Research, 54, F.R.G.

0303-7207/87/$03.50

Dr. R. Ivell, Institute for Grandweg 64, 2000 Hamburg

0 1987 Elsevier Scientific

Publishers

Ireland,

parts of the vasopressin gene, which was earlier shown by dot blots to be expressed at a low level in this tissue; Northern analysis had been negative (Ivell and Richter, 1984). None of the clones isolated contained sequences corresponding to the first, vasopressin-encoding exon of the gene. Instead, it appears that the first intron is not fully spliced out, and therefore the copied mRNA does not represent a translatable transcript. Materials and methods mRNA was extracted from a whole corpus luteum from day 5 of the bovine cycle, by the guanidinium thiocyanate procedure (Chirgwin et al., 1979) converted into double-stranded cDNA (Gubler and Hoffman, 1983) and size-fractionated by passage over Sephacryl S-500. The cDNA fractions were gathered to give large (> 1800 bp), medium (2000 > 800 bp) and small (1000 > 300 bp) pools, which were separately packaged into bacteriophage. To avoid insert length effects during packaging, the library was plated out such that Ltd.

256

TABLE

1

NUMBERS OF POSITIVE PLAQUES IN THE BOVINE LUTEAL cDNA LIBRARY REACTING WITH EITHER THE OXYTOCIN (OT)-SPECIFIC OR THE VASOPRESSIN (VP)-SPECIFIC 3’ PROBES Probe

Number screened

OT-3’ VP-3’

6ooooo 1000000

of plaques

Number positive ca. 4000 9

of plaques

B HYPOTHALAMIC mRNA

-=.x_ 5'

j II :

,' ),' y----yg_.

Percent

0.67 o.ooo9

each pool was represented according to its abundance in the cloned cDNA (viz, small, 78%; medium, 15%; large, 7%). cDNA was inserted into hgtll via EcoRI linkers according to published procedures (Huynh et al., 1985). Phage were packaged (Gigapack in vitro packaging system, Stratagene, California, U.S.A.), plated out at high density without induction of the encoded fusion proteins, and screened by in situ hybridization on nitrocellulose replica filters (ca. 50000 phage per 150 mm plate). The filters were hybridized either using the 3’ oxytocin (OT)-specific fragment of the bovine oxytocin gene (Ivell and Richter, 1984) or the equivalent fragment from the vasopressin gene specific for this product (Ivell and Richter, 1984). The number of positive signals (Table 1) agrees fully with the estimates obtained previously by dot blot analysis (Ivell and Richter, 1984). Small-scale preparation of the vasopressin-positive clones indicated two with apparently full-length inserts (ca. 650 bp). These two and a third shorter (ea. 350 bp) insert were subcloned into Bluescribe (Stratagene, California, U.S.A.) plasmids or into M13mp8 (Messing and Vieira, 1982) for further restriction and sequencing analysis by both enzymatic and chemical methods (Sanger et al., 1977; Maxam and Gilbert, 1980). Results and discussion The shorter of the three clones (pBV-C2) contained no 5’ sequence nor the first splice junction (Fig. 1C). The two longer clones suprisingly also contained no sequence corresponding to the first 5’ exon; instead both included about 150 hp (pBV-C4, 155 bp; pBV-C6, 132 bp) of the first intron upstream of the 3’ first splice junction. The

Fig. 1. A: Scheme comparing the structures of the bovine vasopressin (lower) and oxytocin (upper) genes, indicating the long region of 100% homology (black bar) due to gene conversion. B: The hypothalamic mRNA resulting from normal splicing C: Structural organization of the clones pBV-C2, pBV-C4 and pBV-C6 isolated from the bovine luteal cDNA library. V, vasopressin encoding sequence; 0, oxytocin encoding sequence; hatched bars, intron sequences,

independent status of these two clones was shown by their differing 3’ and 5’ ends. The sequences (not shown) were identical to that published previously for the h~othala~c cDNA (Land et al., 1982) and the gene (Ruppert et al., 1984). Rescreening of the library (2 X lo6 independent clones) using a 5’ specific vasopressin (VP) probe encoding the first exon, and controlling for crosshybridization with the related OT gene product (70% homology in exon A), revealed no VP exon A sequences in the cDNA library. This control also indicated that 96% of the oxytocin clones included at least part of the first exon from that gene. Restriction analysis and sequencing of other clones picked at random showed that the average insert length of the library was ca. 1000 bp, as expected from the original size selection of the cDNA. In a large number of oxytocin clones, reverse transcription had evidently proceeded without hindrance through the equivalent region in which the seven shorter VP cDNA clones were apparently terminated. The key point here is that this region, corresponding to the second, neurophysin-encoding exon is absolutely homologous between the VP and OT genes {Fig. 1A) (Ruppert et al., 1984; Ivell, 1987), including a perfect 100% sequence conservation (Fig. lA, black bar). These

257

data which demonstrate no a priori block to reverse transcription in the VP sequence, imply that the seven shorter VP cDNA clones obtained probably reflect in vivo truncated mRNAs rather than interrupted reverse transcripts. Therefore, one has to conclude that of the lo6 clones screened, all (2 out of 2) of the VP cDNAs with sequences extending through the first splice junction are untranslatable because the first intron has not been spliced out. The corresponding mRNAs would have given positive signals in dot blots, hybridization histochemistry and solution hybridization, falsely implying local synthesis of the peptide hormone which is encoded by the missing 5’ exon A (Fig. 1A). Negative Northern analyses previously attributed to lack of methodological sensitivity might now be explainable by the heterogeneous size range of the defective mRNA molecules. Thus, local synthesis can only be verified if additional information can be obtained either at the protein level, or by cDNA sequence or restriction analyses, or in some cases by the precise length on a Northern gel blot corresponding to that anticipated from the functional gene sequence. The result obtained in the present study is interesting for two further reasons. Firstly, the two long, independent VP clones both had 5’ terminations in a similar region of the intron 130-150 bp upstream of the 3’ splice site, thus not far removed from the probable position of 2’5’A lariat formation (Weissmann, 1984); this also demarcates the region of extensive gene conversion between the oxytocin and vasopressin genes (Fig. 1A) (Ruppert et al., 1984). Further, in all nine clones isolated both polyadenylation and the second splice have been correctly performed. The cloned sequences do not therefore represent the primary transcript but possibly a later intermediate. Alternatively, the RNAs may be products of transcription which has initiated at a later possibly intraintronic, cryptic start-site, similar to the situation observed for the inactive adult /3globin gene in a human embryonic erythroid cell line (Khazaie et al., 1986). Secondly, the low level of vasopressin peptide reported for the bovine corpus luteum (Wathes et al., 1983) and of its mRNA, if translatable, would imply only local paracrine effects within the ovary.

Oxytocin and its mRNA are expressed at 1000 times higher levels in the same tissue. Yet there is only a lOO-fold difference in the discriminating capacity of the two nonapeptide hormone receptors for their ligands (Jard, 1981). Consequently, vasopressin receptors, if present, would be completely occupied by oxytocin at the local concentrations present in the bovine corpus luteum. Physiologically, therefore, vasopressin production in the non-pregnant bovine corpus luteum is probably not functionally relevant. What we may be witnessing in the sequenced vasopressin cDNA clones presented here could be short-lived breakdown products of non-functional gene transcripts. Both oxytocin and vasopressin genes might be activated by a common mechanism during the differentiation of the granulosa cells, with only the oxytocin gene transcription being subsequently up-regulated. Meanwhile, the background vasopressin gene transcription is rendered ineffective either by a regulatory step blocking correct splicing or the absence of a complete transcript, both of which possibilities could result from the involvement, or lack of it, of a truns acting factor. Acknowledgements We should like to thank Werner Rust for excellent technical assistance, Professor M.J. Fields of the University of Florida for providing the day 5 corpus luteum and the Deutsche Forschungsgemeinschaft for financial support to Professor Dietmar Richter in whose laboratory this study was carried out, and to whom we are particularly grateful for his advice and encouragement. References Chirgwin, J.M., Ptzybla,

A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Clements, J.A. and Funder, J.W. (1986) Endocr. Rev. 7, 449-460. Gubier, U. and Hoffmann, B.J. (1983) Gene 25, 263-269. Huynh, T.V., Young, R.A. and Davis, R.W. (1985) In: DNA Cloning, Vol. 1, Ed.: D.M. Glover (IRL Press, Oxford), pp. 49-78 Ivell, R. (1987) In: Neuropeptides and their Peptidases, Ed.: A.J. Turner (Ellis-Hotwood, Chichester) pp. 31-64. Ivell, R. and Richter, D. (1984) EMBO J. 3, 2351-2354. Jard, S. (1981) J. Physiol. (Paris) 77, 621-628.

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