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Oxytocin and vasopressin gene expression during gestation and lactation
Molecular Brain Research, 4 (1988) 1-6 Elsevier
1
BRM 70080
Research Reports
Oxytocin and vasopressin gene expression during gestation and lactation Hans H. Zingg and Diana L. Lefebvre Laboratory of Molecular Endocrinology, Royal Victoria Hospital, McGill University, Montreal, Que. (Canada) (Accepted 22 March 1988) Key words: Vasopressin; Oxytocin; Messenger ribonucleic acid; Hypothalamus; Pregnancy; Lactation; Northern blot; Poly(A) tail
Levels of rat hypothalamic oxytocin (OT) and vasopressin (AVP) mRNA were determined during gestation and lactation using a densitometric hybridization assay. Pregnancy induced a gradual rise in OT mRNA, reaching, by day 18, a level exceeding control by a factor of 2.5. Throughout the lactation period OT mRNA remained elevated at levels corresponding to 3 times that of control. Surprisingly, the dynamics of AVP mRNA accumulation paralleled closely the profile observed for OT mRNA throughout the time period studied. We conclude that in late pregnancy and lactation the expression of both, the OT and the AVP gene is stimulated in parallel by mechanisms operating at a pretranslational level, involving increased gene transcription or mRNA stabilization, or both. Further characterization of the two mRNA species revealed that both mRNAs are endowed with very long poly(A) tails of >200 nucleotides, under conditions of both, normal and stimulated states of gene expression. The role, if any, of the prolonged poly(A) tails in mRNA stability remains to be determined. INTRODUCTION
precursor molecule contains 3 exons, termed A, B, and C, with O T being encoded by the first exon 15'
The hypothalamic neuropeptide oxytocin (OT) plays an important role in both, parturition and lactation. During parturition, O T regulates the contractions of uterine smooth muscle and, during lactation, OT triggers milk ejection by its action on myoepithelial cells of the m a m m a r y gland (for review, see refs. 8, 14, 26). In addition, O T fulfills neurotransmitter functions and is involved in the mediation of mothering behaviour 3°'4°. O T is synthesized in magnocellular neurons located in the supraoptic and paraventricular nuclei of the hypothalamus, as well as in parvicellular neurons located in the caudal paraventricular nucleus, the anterior commisural nucleus and the perifornical region 2J2'38. Moreover, OT-like immunoreactivity has also been demonstrated in the ovary, in the testis and in the adrenal 7't6"29'44. O T is biosynthetically derived from a 16-kDa precursor molecule which consists of the nonapeptide O T and, in addition, the oxytocin-associated neurophysin (Np I). In man, rat and cow, the gene encoding the O T
33,36
The gene coding for the related peptide vasopressin (AVP) has a very similar organization and shows significant sequence homology with the O T gene in all three species examined, especially within exon B 15,33,36. However, the 5' p r o m o t e r regions of the two genes show very little homology, suggesting independent regulation. Much work has been done on the regulation of A V P m R N A accumulation in hypothalamic neurons. We and others have shown a significant rise in hypothalamic A V P m R N A in response to dehydration ~'39'5°. Furthermore, in a subset of paraventricular parvicellular neurons, A V P gene expression is under negative control by glucocorticolds 37"45'47. By contrast, comparatively little is known of the regulation of hypothalamic O T m R N A . In the present study, rat O T and A V P gene expression has been investigated throughout pregnancy and lactation. Our data indicate a 2-fold rise in O T m R N A accumulation, which precedes the actual on-
Correspondence: H.H. Zingg, Laboratory of Molecular Endocrinology, Royal Victoria Hospital, room H 7.63, 687, Pine Avenue West, Montreal, Oue., H3A IA1 Canada. 0169-328X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
set of labor, followed by a sustained 3-fold elevation throughout lactation. The study further indicates that the time course of A V P m R N A accumulation follows very closely that observed for O T m R N A . MATERIALS AND METHODS
Animals Timed pregnant, lactating and control female Sprague-Dawley rats (150-200 g) were obtained from Charles River, Canada. They had free access to laboratory chow and tap water and were kept in a temperature- and humidity-controlled environment. Animals were killed by decapitation and the hypothalamus (50-70 mg) was removed, immediately frozen and stored at - 7 0 °C.
50 mM sodium phosphate (pH 6.5), 0.8 mM NaCI, 0.5% sodium dodecyl sulfate (SDS), 1 mM E D T A at 42 °C for 24 h. After washing 3 times for 10 rain in 12 mM NaCI, 0.1% SDS, 5 mM sodium phosphate (pH 6.5), 1 mM E D T A at 40 °C, membranes were exposed to X-ray film (Kodak XAR-5) at - 7 0 °C for 1-3 days using an intensifying screen. Probes were removed from the blots by washing in 0.1 x SSC (15 mM NaC1, 1.5 mM sodium citrate) at 80 °C for 20 rain. Autoradiographic signals were quantitated by densitometric scanning as described 49 using software developed by Hoefer Scientific, C A (GS 350 Data System). Statistical evaluation of data was performed by analysis of variance followed by the DuncanKramer multiple range test 22.
Oligonucleotide probes Preparation of RNA Hypothalami c fragments were homogenized in 3 ml of 4 M guanidine thiocyanate, 25 mM sodium acetate (pH 7.0), 0,7 mM fl-mercaptoethanol. The extract was layered on top of 2 ml 5.7 M CsCI, 25 mM sodium citrate (pH 5.5) and centrifuged at 47,000 rpm for 24 h in a fixed-angle rotor (Sorval TFT 65.13). The R N A pellet was resuspended in 0.3 M sodium acetate and ethanol precipitated. Using this method, total R N A recovery was shown earlier to be 82 -+ 5% 49. In one experiment, the poly(A) + tail was removed from the m R N A by treatment with R N A s e H (Pharmacia; final concentration 2 U/25/A) following hybridization to a > 10-fold molar excess of oligo d(T)12_18 (Pharmacia) as described by Vournakis et al. 42. Following dissolution of the pellet in 1-M glyoxal, 50% dimethyl sulfoxide (DMSO), 10 m M phosphate buffer (pH 7.0), the R N A was glyoxalated at 50 °C for 1 h. R N A size standards were produced using the p G E M riboprobe transcription system (Promega Biotech).
Northern blot analysis Following glyoxalation, samples were frozen on dry ice and loaded immediately after thawing on a 1.5% agarose gel made up in 10-raM phosphate buffer (pH 6.5). After electrophoresis (200 V, 4 h), R N A was transferred to a nylon membrane (Gene Screen, Dupont), baked for 2 h at 90 °C and hybridized to labeled oligonucleotide probes (500,000 cpm/ml). Hybridization occurred in 50% formamide,
The specific probes used in this study consisted of two 27 nucleotides long oligomers, complementary to portions in exon C of the rat O T and A V P genes.
A
1
B
2
1 2 3 4
28 S
18S
AVP
- OXY
Fig. 1. A: specificity of oligonucleotide probes. Lane i: Northern blot analysis of 50ag of total rat hypothalamic RNA (equivalent to 1/2 rat hypothalamus) probed with the AVP-specific 27-mer oligonucleotide 'VP-27'. The RNA was extracted, electrophoresed, transferred to a membrane and hybridized to 3' end labeled VP-27 as described under 'Methods'. The membrane was exposed for 36 h to Kodak XAR-5 film using an intensifying screen. Lane 2: rehybridization of the same membrane to the OT-specific oligonucleotide probe 'OT-27". The origin (O) and the positions of ribosomal 28S RNA and 18S RNA are indicated. B: poly(A) tail removal from AVP and OT mRNA. Fifty ~tg of total hypothalamic RNA was treated with RNAse H (2U) in the absence (lanes l and 3) or presence (lanes 2 and 4) of 5/~g of oligo d(T). The membrane was first hybridized to the AVP-specific VP-27 probe (lanes 1 and 2) and then to the oxytocin-specific oligonucleotide OT-27 (lanes 3 and 4). Size markers: O, origin; 1,28S RNA; 2, 18S RNA; 3-5, in vitro synthesized RNA size markers of 898,723 and 479 nucleotides, respectively,
The oligonucleotides were produced by solid phase synthesis on an Applied Biosystems DNA synthesizer. The oligomer OT-27 has the sequence 5'-GCGCTC G G A G A A G G C A G A C T C A G G G T C - 3 ' and is complementary to the portion of the OT mRNA encoding residues 117-125 of pre-prooxytocin 15. VP-27 has the sequence 5 ' - G G C C C G T C C A G C T G C G T G GCGTTGCTC-3' and is complementary to the portion of the AVP mRNA-encoding residues 133-142 of pre-propressophysin 15. Probes were 3' end labeled with a-[32p]deoxycytosine triphosphate using terminal deoxynucleotidyl transferase. The labeling reaction (25/A) contained 5 pmol oligonucleotide, 15/A a-[32p]dCTP (ICN Biochemicals, 3000 Ci/mmol), 20 U terminal deoxynucleotidyl transferase (Pharmacia) in 2 mM MgC12, 0.14 M cacodylic acid (adjusted with KOH to pH 7.2). Under these conditions, 5-6 residues of dCTP were added to each oligonucleotide during a 2-h reaction at 37 °C, yielding a specific activity of >12.106 cpm/pmol. Quantitation of total poly(A) + RNA per lane was done by hybridization of the blots to a >10-fold molar excess of oligo d(T)12_ls (Pharmacia) labeled to a final specific activity of 4 x 104 cpm//~g, exactly as described by Lowe et al. 24.
OT and AVP mRNA levels during pregnancy and lactation, blots of total hypothalamic RNA were prepared and hybridized to labeled OT- and AVP-specific oligonucleotide probes. As illustrated in Figs. 2 and 3, pregnancy induced a gradual rise in hypothalamic OT mRNA accumulation. By day 18, OT mRNA levels exceeded control levels by a factor of 2.5. Throughout the ensuing lactation period, OT mRNA levels remained elevated, at levels corresponding to 3 times that of control. The dynamics of AVP mRNA accumulation closely paralleled the profile observed for OT mRNA. At day 21 of pregnancy, AVP mRNA levels exceeded control levels by a factor of 1.9 and, during lactation, AVP mRNA accumulation rose further and reached, like OT mRNA, a level which exceeded control by a factor of 3. These changes were specific inasmuch as the levels of total poly(A) mRNA did not undergo any significant changes during the time period studied (Fig. 3). DISCUSSION Hypothalamic OT and AVP gene expression was investigated during pregnancy and lactation by
RESULTS
1
2
3
4
5
Characterization of oxytocin and vasopressin mRNA The oxytocin specific oligonucleotide probe OT-27 hybridized to a single band of hypothalamic RNA (Fig. 1A). Using RNA size markers, the size of the band was estimated at 750 bases. Hybridization with the vasopressin-specific probe VP-27 yielded a different, slightly larger band of approximately 820 bases in size (Fig. 1A). Removal of the poly(A) tail by the oligo dT/RNAse H method reduced the size of the OT mRNA transcript to 530 bases (Fig. 1B). Hypothalamic OT mRNA, therefore, contains a relatively large poly(A) tail of over 200 nucleotides. Similarly, poly(A) removal reduced the size of AVP mRNA to 600 bases (Fig. 1B). Thus, like hypothalamic OT mRNA, all hypothalamic AVP mRNA is endowed with a poly(A) tail structure exceeding 200 nucleotides in length.
Quantitative analysis of mRNA accumulation To further evaluate the quantitative differences in
28S
--
1 8 S '-
Fig. 2. OT mRNA levels during pregnancy and lactation. Northern blot analysis of hypothalamicRNA extracted from 7and 21-daypregnant rats (lanes 1,2), 7 and 28 day lactating rats (lanes 3, 4), and control (lane 5). Each lane contains the equivalent of 1/2 rat hypothalamus. The membrane was probed with 3'-labeled OT-27 and exposed for 36 h to Kodak XAR-5 with an enhancer screen.
400
• Oxytocin rnRNA
*T*
o Vasopressin mRNA
• POlYIAI + RNA tO
/ / ~
~¢,
300
200
S < Z 1 O0
0
I 0 I
I 7'
/,, 14
I 18
Days of Pregnancy
I 21 I I
I 7
~
I 98 I
Days of Lactation
Fig. 3. Ouantitation of OT and AVP mRNA during pregnancy and lactation. Six Northern blots were hybridized sequentially with labeled OT-27, VP-27 and oligo d(T). Autoradiographic bands were quantitated by densitometric scanning and values were expressed as percentage of control. Each data point represents the mean + S.E.M. of 6 independent determinations. Asterisks denote values which differ significantly from control (*P < 0.05; **P < 0.01).
characterization and quantitation of hypothalamic OT and AVP mRNA. The length of both transcripts following removal of the poly(A) tail is in accordance with the size predicted from the structure of their respective genes 15. Both m R N A species comprise very long poly(A) tails under conditions of both, normal and stimulated states of gene expression. It has recently been shown that proopiomelanocortin m R N A of hypothalamic, but not of pituitary origin is equally endowed with a very long poly(A) tail of over 200 nucleotides 19. Long poly(A) tails, therefore, might be a characteristic of hypothalamic or neuronal m R N A in general. The recent report that AVP m R N A of testicular origin contains a considerably shorter poly(A) tail than hypothalamic AVP m R N A adds additional support to this hypothesis xs. In view of the role of the poly(A) tail in increasing m R N A stability 13'25'2s, it remains to be determined whether the remarkable length of the poly(A) tails of hypothalamic mRNAs confers indeed increased stability to these transcripts. In the present study, we observed a rise in OT m R N A levels during late gestation. This rise in mRNA is paralleled by a concomitant increase in OT-like immunoreactivity in hypothalamic neurons 3. By contrast, during this same period, the secretion of OT into the bloodstream is not stimulated, inasmuch
as OT serum levels remain unchanged throughout pregnancy, with the exception of the hours immediately preceding delivery 9-11. Therefore, the increased biosynthesis of hypothalamic OT does not seem to be a direct consequence of increased release. Interestingly, the rise of OT m R N A levels occurs in parallel with an increase in serum estrogen levels 46. This rises the possibility that estrogens may be involved in stimulating OT gene transcription. A direct effect of estrogens is further suggested by the presence of the sequence element G G T G A C C T T G A C C at position -170 to -158 in the rat and human OT gene 5' flanking region. This element bears close resemblance to the conserved sequence of estrogen-responsive elements in the 5' flanking regions of vitellogenin genes ( G G T C A N N N T G A C C ) 21. In addition, receptor sites for estrogen have been demonstrated in neurophysin positive neurons 32,35. On the other hand, however, we and others were sofar unable to detect any changes in hypothalamic OT m R N A during the estrous cycle (ref. 17 and H . H . Z . , unpublished results). Our study further indicates that pregnancy induces a parallel rise in AVP gene expression. If administered centrally, AVP acts as a strong antipyretic agent 4. It is, therefore, interesting to note that late pregnancy is indeed associated with a strong suppression of the febrile response to exogenous pyrogens 2°'27'4s. In addition, during both human and rat pregnancy, AVP secretion into the peripheral circulation is also increased due to a lower osmotic threshold for AVP release 23. Lactation induces a further, sustained increase in both, OT and AVP m R N A levels. The activity of magnocellular neurons during lactation has been the object of detailed electrophysiological studies 43. In the rat, reflex milk ejections occur regularly at 5- to 10-min intervals and each are preceded by an abrupt synchronized increase in the firing rate of OT neurons 43. This increase in neuronal activity is strictly specific for OT-producing neurons and is not observed in AVP-producing neurons. In view of the specificity of this activation pattern, it was therefore surprising to find that stimulation of gene expression is seemingly less specific and occurs in both, OT and AVP producing neurons. These results are, however, entirely compatible with earlier in vivo incorporation data obtained by Russel et al., demonstrating a
concomitant 2-fold increase in [35S]cysteine incorporation into both O T and A V P during lactation 34. A similar parallelism between the A V P and O T systems exists with respect to the response to osmotic stimula-
changes in transcription rates or to changes in the stability of the m R N A . Both mechanisms have been shown to be operative in the control of gene expression 5. The use of specific intron probes, in vitro run-
tion. In rats, substitution of 2% saline for drinking water leads not only to an increase in A V P m R N A accumulation and A V P labeling, but also to a con-
gion linked to a reporter gene will be necessary to clarify this question.
comitant, parallel rise in O T m R N A and O T labeling6, 34.41.
ACKNOWLEDGEMENTS
on assays and analyses of the isolated p r o m o t e r re-
We conclude that in late pregnancy and lactation the expression of both, the O T and the A V P gene is
We thank Dr. P,A. Kelly for his critical review of
stimulated in parallel by mechanisms operating at a pretranslational level, involving a u g m e n t e d gene transcription or m R N A stabilization, or both. The present studies do not allow to determine whether the observed changes in m R N A levels are due to
the manuscript, Dr. D. Brousseau for oligonucleotide synthesis and Mr. C. Hodges for expert photography and art work. This work was supported by MRC, Canada. H . H . Z . is a Scholar of M R C , Canada.
REFERENCES
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Note added in proof Following submission of this manuscript, another report has appeared on hypothalamic vasopressin and oxytocin mRNA content during pregnancy and lactation. (Van Tol, H.H.M., Bolwerk, E.L.M., Liu, B. and Burbach, J.P.H., Oxytocin and vasopressin gene expression in the hypothalamo-neurohypophyseal system of the rat during the estrous cycle, pregnancy and lactation, Endocrinology, 122 (1988) 945-951 .)
1
BRM 70080
Research Reports
Oxytocin and vasopressin gene expression during gestation and lactation Hans H. Zingg and Diana L. Lefebvre Laboratory of Molecular Endocrinology, Royal Victoria Hospital, McGill University, Montreal, Que. (Canada) (Accepted 22 March 1988) Key words: Vasopressin; Oxytocin; Messenger ribonucleic acid; Hypothalamus; Pregnancy; Lactation; Northern blot; Poly(A) tail
Levels of rat hypothalamic oxytocin (OT) and vasopressin (AVP) mRNA were determined during gestation and lactation using a densitometric hybridization assay. Pregnancy induced a gradual rise in OT mRNA, reaching, by day 18, a level exceeding control by a factor of 2.5. Throughout the lactation period OT mRNA remained elevated at levels corresponding to 3 times that of control. Surprisingly, the dynamics of AVP mRNA accumulation paralleled closely the profile observed for OT mRNA throughout the time period studied. We conclude that in late pregnancy and lactation the expression of both, the OT and the AVP gene is stimulated in parallel by mechanisms operating at a pretranslational level, involving increased gene transcription or mRNA stabilization, or both. Further characterization of the two mRNA species revealed that both mRNAs are endowed with very long poly(A) tails of >200 nucleotides, under conditions of both, normal and stimulated states of gene expression. The role, if any, of the prolonged poly(A) tails in mRNA stability remains to be determined. INTRODUCTION
precursor molecule contains 3 exons, termed A, B, and C, with O T being encoded by the first exon 15'
The hypothalamic neuropeptide oxytocin (OT) plays an important role in both, parturition and lactation. During parturition, O T regulates the contractions of uterine smooth muscle and, during lactation, OT triggers milk ejection by its action on myoepithelial cells of the m a m m a r y gland (for review, see refs. 8, 14, 26). In addition, O T fulfills neurotransmitter functions and is involved in the mediation of mothering behaviour 3°'4°. O T is synthesized in magnocellular neurons located in the supraoptic and paraventricular nuclei of the hypothalamus, as well as in parvicellular neurons located in the caudal paraventricular nucleus, the anterior commisural nucleus and the perifornical region 2J2'38. Moreover, OT-like immunoreactivity has also been demonstrated in the ovary, in the testis and in the adrenal 7't6"29'44. O T is biosynthetically derived from a 16-kDa precursor molecule which consists of the nonapeptide O T and, in addition, the oxytocin-associated neurophysin (Np I). In man, rat and cow, the gene encoding the O T
33,36
The gene coding for the related peptide vasopressin (AVP) has a very similar organization and shows significant sequence homology with the O T gene in all three species examined, especially within exon B 15,33,36. However, the 5' p r o m o t e r regions of the two genes show very little homology, suggesting independent regulation. Much work has been done on the regulation of A V P m R N A accumulation in hypothalamic neurons. We and others have shown a significant rise in hypothalamic A V P m R N A in response to dehydration ~'39'5°. Furthermore, in a subset of paraventricular parvicellular neurons, A V P gene expression is under negative control by glucocorticolds 37"45'47. By contrast, comparatively little is known of the regulation of hypothalamic O T m R N A . In the present study, rat O T and A V P gene expression has been investigated throughout pregnancy and lactation. Our data indicate a 2-fold rise in O T m R N A accumulation, which precedes the actual on-
Correspondence: H.H. Zingg, Laboratory of Molecular Endocrinology, Royal Victoria Hospital, room H 7.63, 687, Pine Avenue West, Montreal, Oue., H3A IA1 Canada. 0169-328X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
set of labor, followed by a sustained 3-fold elevation throughout lactation. The study further indicates that the time course of A V P m R N A accumulation follows very closely that observed for O T m R N A . MATERIALS AND METHODS
Animals Timed pregnant, lactating and control female Sprague-Dawley rats (150-200 g) were obtained from Charles River, Canada. They had free access to laboratory chow and tap water and were kept in a temperature- and humidity-controlled environment. Animals were killed by decapitation and the hypothalamus (50-70 mg) was removed, immediately frozen and stored at - 7 0 °C.
50 mM sodium phosphate (pH 6.5), 0.8 mM NaCI, 0.5% sodium dodecyl sulfate (SDS), 1 mM E D T A at 42 °C for 24 h. After washing 3 times for 10 rain in 12 mM NaCI, 0.1% SDS, 5 mM sodium phosphate (pH 6.5), 1 mM E D T A at 40 °C, membranes were exposed to X-ray film (Kodak XAR-5) at - 7 0 °C for 1-3 days using an intensifying screen. Probes were removed from the blots by washing in 0.1 x SSC (15 mM NaC1, 1.5 mM sodium citrate) at 80 °C for 20 rain. Autoradiographic signals were quantitated by densitometric scanning as described 49 using software developed by Hoefer Scientific, C A (GS 350 Data System). Statistical evaluation of data was performed by analysis of variance followed by the DuncanKramer multiple range test 22.
Oligonucleotide probes Preparation of RNA Hypothalami c fragments were homogenized in 3 ml of 4 M guanidine thiocyanate, 25 mM sodium acetate (pH 7.0), 0,7 mM fl-mercaptoethanol. The extract was layered on top of 2 ml 5.7 M CsCI, 25 mM sodium citrate (pH 5.5) and centrifuged at 47,000 rpm for 24 h in a fixed-angle rotor (Sorval TFT 65.13). The R N A pellet was resuspended in 0.3 M sodium acetate and ethanol precipitated. Using this method, total R N A recovery was shown earlier to be 82 -+ 5% 49. In one experiment, the poly(A) + tail was removed from the m R N A by treatment with R N A s e H (Pharmacia; final concentration 2 U/25/A) following hybridization to a > 10-fold molar excess of oligo d(T)12_18 (Pharmacia) as described by Vournakis et al. 42. Following dissolution of the pellet in 1-M glyoxal, 50% dimethyl sulfoxide (DMSO), 10 m M phosphate buffer (pH 7.0), the R N A was glyoxalated at 50 °C for 1 h. R N A size standards were produced using the p G E M riboprobe transcription system (Promega Biotech).
Northern blot analysis Following glyoxalation, samples were frozen on dry ice and loaded immediately after thawing on a 1.5% agarose gel made up in 10-raM phosphate buffer (pH 6.5). After electrophoresis (200 V, 4 h), R N A was transferred to a nylon membrane (Gene Screen, Dupont), baked for 2 h at 90 °C and hybridized to labeled oligonucleotide probes (500,000 cpm/ml). Hybridization occurred in 50% formamide,
The specific probes used in this study consisted of two 27 nucleotides long oligomers, complementary to portions in exon C of the rat O T and A V P genes.
A
1
B
2
1 2 3 4
28 S
18S
AVP
- OXY
Fig. 1. A: specificity of oligonucleotide probes. Lane i: Northern blot analysis of 50ag of total rat hypothalamic RNA (equivalent to 1/2 rat hypothalamus) probed with the AVP-specific 27-mer oligonucleotide 'VP-27'. The RNA was extracted, electrophoresed, transferred to a membrane and hybridized to 3' end labeled VP-27 as described under 'Methods'. The membrane was exposed for 36 h to Kodak XAR-5 film using an intensifying screen. Lane 2: rehybridization of the same membrane to the OT-specific oligonucleotide probe 'OT-27". The origin (O) and the positions of ribosomal 28S RNA and 18S RNA are indicated. B: poly(A) tail removal from AVP and OT mRNA. Fifty ~tg of total hypothalamic RNA was treated with RNAse H (2U) in the absence (lanes l and 3) or presence (lanes 2 and 4) of 5/~g of oligo d(T). The membrane was first hybridized to the AVP-specific VP-27 probe (lanes 1 and 2) and then to the oxytocin-specific oligonucleotide OT-27 (lanes 3 and 4). Size markers: O, origin; 1,28S RNA; 2, 18S RNA; 3-5, in vitro synthesized RNA size markers of 898,723 and 479 nucleotides, respectively,
The oligonucleotides were produced by solid phase synthesis on an Applied Biosystems DNA synthesizer. The oligomer OT-27 has the sequence 5'-GCGCTC G G A G A A G G C A G A C T C A G G G T C - 3 ' and is complementary to the portion of the OT mRNA encoding residues 117-125 of pre-prooxytocin 15. VP-27 has the sequence 5 ' - G G C C C G T C C A G C T G C G T G GCGTTGCTC-3' and is complementary to the portion of the AVP mRNA-encoding residues 133-142 of pre-propressophysin 15. Probes were 3' end labeled with a-[32p]deoxycytosine triphosphate using terminal deoxynucleotidyl transferase. The labeling reaction (25/A) contained 5 pmol oligonucleotide, 15/A a-[32p]dCTP (ICN Biochemicals, 3000 Ci/mmol), 20 U terminal deoxynucleotidyl transferase (Pharmacia) in 2 mM MgC12, 0.14 M cacodylic acid (adjusted with KOH to pH 7.2). Under these conditions, 5-6 residues of dCTP were added to each oligonucleotide during a 2-h reaction at 37 °C, yielding a specific activity of >12.106 cpm/pmol. Quantitation of total poly(A) + RNA per lane was done by hybridization of the blots to a >10-fold molar excess of oligo d(T)12_ls (Pharmacia) labeled to a final specific activity of 4 x 104 cpm//~g, exactly as described by Lowe et al. 24.
OT and AVP mRNA levels during pregnancy and lactation, blots of total hypothalamic RNA were prepared and hybridized to labeled OT- and AVP-specific oligonucleotide probes. As illustrated in Figs. 2 and 3, pregnancy induced a gradual rise in hypothalamic OT mRNA accumulation. By day 18, OT mRNA levels exceeded control levels by a factor of 2.5. Throughout the ensuing lactation period, OT mRNA levels remained elevated, at levels corresponding to 3 times that of control. The dynamics of AVP mRNA accumulation closely paralleled the profile observed for OT mRNA. At day 21 of pregnancy, AVP mRNA levels exceeded control levels by a factor of 1.9 and, during lactation, AVP mRNA accumulation rose further and reached, like OT mRNA, a level which exceeded control by a factor of 3. These changes were specific inasmuch as the levels of total poly(A) mRNA did not undergo any significant changes during the time period studied (Fig. 3). DISCUSSION Hypothalamic OT and AVP gene expression was investigated during pregnancy and lactation by
RESULTS
1
2
3
4
5
Characterization of oxytocin and vasopressin mRNA The oxytocin specific oligonucleotide probe OT-27 hybridized to a single band of hypothalamic RNA (Fig. 1A). Using RNA size markers, the size of the band was estimated at 750 bases. Hybridization with the vasopressin-specific probe VP-27 yielded a different, slightly larger band of approximately 820 bases in size (Fig. 1A). Removal of the poly(A) tail by the oligo dT/RNAse H method reduced the size of the OT mRNA transcript to 530 bases (Fig. 1B). Hypothalamic OT mRNA, therefore, contains a relatively large poly(A) tail of over 200 nucleotides. Similarly, poly(A) removal reduced the size of AVP mRNA to 600 bases (Fig. 1B). Thus, like hypothalamic OT mRNA, all hypothalamic AVP mRNA is endowed with a poly(A) tail structure exceeding 200 nucleotides in length.
Quantitative analysis of mRNA accumulation To further evaluate the quantitative differences in
28S
--
1 8 S '-
Fig. 2. OT mRNA levels during pregnancy and lactation. Northern blot analysis of hypothalamicRNA extracted from 7and 21-daypregnant rats (lanes 1,2), 7 and 28 day lactating rats (lanes 3, 4), and control (lane 5). Each lane contains the equivalent of 1/2 rat hypothalamus. The membrane was probed with 3'-labeled OT-27 and exposed for 36 h to Kodak XAR-5 with an enhancer screen.
400
• Oxytocin rnRNA
*T*
o Vasopressin mRNA
• POlYIAI + RNA tO
/ / ~
~¢,
300
200
S < Z 1 O0
0
I 0 I
I 7'
/,, 14
I 18
Days of Pregnancy
I 21 I I
I 7
~
I 98 I
Days of Lactation
Fig. 3. Ouantitation of OT and AVP mRNA during pregnancy and lactation. Six Northern blots were hybridized sequentially with labeled OT-27, VP-27 and oligo d(T). Autoradiographic bands were quantitated by densitometric scanning and values were expressed as percentage of control. Each data point represents the mean + S.E.M. of 6 independent determinations. Asterisks denote values which differ significantly from control (*P < 0.05; **P < 0.01).
characterization and quantitation of hypothalamic OT and AVP mRNA. The length of both transcripts following removal of the poly(A) tail is in accordance with the size predicted from the structure of their respective genes 15. Both m R N A species comprise very long poly(A) tails under conditions of both, normal and stimulated states of gene expression. It has recently been shown that proopiomelanocortin m R N A of hypothalamic, but not of pituitary origin is equally endowed with a very long poly(A) tail of over 200 nucleotides 19. Long poly(A) tails, therefore, might be a characteristic of hypothalamic or neuronal m R N A in general. The recent report that AVP m R N A of testicular origin contains a considerably shorter poly(A) tail than hypothalamic AVP m R N A adds additional support to this hypothesis xs. In view of the role of the poly(A) tail in increasing m R N A stability 13'25'2s, it remains to be determined whether the remarkable length of the poly(A) tails of hypothalamic mRNAs confers indeed increased stability to these transcripts. In the present study, we observed a rise in OT m R N A levels during late gestation. This rise in mRNA is paralleled by a concomitant increase in OT-like immunoreactivity in hypothalamic neurons 3. By contrast, during this same period, the secretion of OT into the bloodstream is not stimulated, inasmuch
as OT serum levels remain unchanged throughout pregnancy, with the exception of the hours immediately preceding delivery 9-11. Therefore, the increased biosynthesis of hypothalamic OT does not seem to be a direct consequence of increased release. Interestingly, the rise of OT m R N A levels occurs in parallel with an increase in serum estrogen levels 46. This rises the possibility that estrogens may be involved in stimulating OT gene transcription. A direct effect of estrogens is further suggested by the presence of the sequence element G G T G A C C T T G A C C at position -170 to -158 in the rat and human OT gene 5' flanking region. This element bears close resemblance to the conserved sequence of estrogen-responsive elements in the 5' flanking regions of vitellogenin genes ( G G T C A N N N T G A C C ) 21. In addition, receptor sites for estrogen have been demonstrated in neurophysin positive neurons 32,35. On the other hand, however, we and others were sofar unable to detect any changes in hypothalamic OT m R N A during the estrous cycle (ref. 17 and H . H . Z . , unpublished results). Our study further indicates that pregnancy induces a parallel rise in AVP gene expression. If administered centrally, AVP acts as a strong antipyretic agent 4. It is, therefore, interesting to note that late pregnancy is indeed associated with a strong suppression of the febrile response to exogenous pyrogens 2°'27'4s. In addition, during both human and rat pregnancy, AVP secretion into the peripheral circulation is also increased due to a lower osmotic threshold for AVP release 23. Lactation induces a further, sustained increase in both, OT and AVP m R N A levels. The activity of magnocellular neurons during lactation has been the object of detailed electrophysiological studies 43. In the rat, reflex milk ejections occur regularly at 5- to 10-min intervals and each are preceded by an abrupt synchronized increase in the firing rate of OT neurons 43. This increase in neuronal activity is strictly specific for OT-producing neurons and is not observed in AVP-producing neurons. In view of the specificity of this activation pattern, it was therefore surprising to find that stimulation of gene expression is seemingly less specific and occurs in both, OT and AVP producing neurons. These results are, however, entirely compatible with earlier in vivo incorporation data obtained by Russel et al., demonstrating a
concomitant 2-fold increase in [35S]cysteine incorporation into both O T and A V P during lactation 34. A similar parallelism between the A V P and O T systems exists with respect to the response to osmotic stimula-
changes in transcription rates or to changes in the stability of the m R N A . Both mechanisms have been shown to be operative in the control of gene expression 5. The use of specific intron probes, in vitro run-
tion. In rats, substitution of 2% saline for drinking water leads not only to an increase in A V P m R N A accumulation and A V P labeling, but also to a con-
gion linked to a reporter gene will be necessary to clarify this question.
comitant, parallel rise in O T m R N A and O T labeling6, 34.41.
ACKNOWLEDGEMENTS
on assays and analyses of the isolated p r o m o t e r re-
We conclude that in late pregnancy and lactation the expression of both, the O T and the A V P gene is
We thank Dr. P,A. Kelly for his critical review of
stimulated in parallel by mechanisms operating at a pretranslational level, involving a u g m e n t e d gene transcription or m R N A stabilization, or both. The present studies do not allow to determine whether the observed changes in m R N A levels are due to
the manuscript, Dr. D. Brousseau for oligonucleotide synthesis and Mr. C. Hodges for expert photography and art work. This work was supported by MRC, Canada. H . H . Z . is a Scholar of M R C , Canada.
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Note added in proof Following submission of this manuscript, another report has appeared on hypothalamic vasopressin and oxytocin mRNA content during pregnancy and lactation. (Van Tol, H.H.M., Bolwerk, E.L.M., Liu, B. and Burbach, J.P.H., Oxytocin and vasopressin gene expression in the hypothalamo-neurohypophyseal system of the rat during the estrous cycle, pregnancy and lactation, Endocrinology, 122 (1988) 945-951 .)