- Email: [email protected]
The regulation of neurohypophyseal peptide gene expression in gonadal tissues
Regulatory Peptides, 45 (1993) 263-267 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
263
REGPEP 01307
The regulation of neurohypophyseal peptide gene expression in gonadal tissues Richard Ivell, Norbert Walther, Uwe Wehrenberg, Craig McArdle and Hendrik Ungefroren Institute for Hormone and Fertility Research, University of Hamburg, Hamburg (Germany)
Key words: Oxytocin; Vasopressin; Neurophysin; Aberrant transcript; COUP transcription factor; Corpus luteum; Testis
Introduction
The neurohypophyseal peptide hormones oxytocin and vasopressin are responsible for the control of important endocrine systems in all mammal species examined. The effective levels of these hormones in the circulation are controlled on the one hand by regulating the release of the synthesized peptides in their major site of production, the hypothalamoneurohypophyseal system. Chronic control on the other hand is attained by up- or down-regulating the biosynthesis of the hormone precursors in the hypothalamus, in particular by altering the levels of gene transcription [ 1]. Although this transcriptional control can be studied in vivo by in situ or northern hybridization to learn about the physiological conditions under which the genes are modulated, only defined cell culture systems offer a basis with which to elucidate the intracellular mechanisms and pathways involved at the molecular level. To date cell cultures suitable for such experimentation have been lacking Correspondence to: R. Ivell, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 2000 Hamburg 54, Germany.
for studying oxytocinergic or vasopressinergic expression. To overcome this problem we have been characterizing a variety of peripheral tissues where oxytocin and vasopressin are also produced, usually as paracrine hormones with low level expression. In particular, the bovine ovary and testis have proved to be very useful in understanding the regulation of oxytocin gene expression. During the normal estrous cycle of the cow the oxytocin gene is massively up-regulated in the granulosa ceils of the preovulatory follicle accompanying the differentiation switch of these cells at ovulation from a predominantly estrogenic to a progestagenic phenotype [2]. The corpus luteum that results from the follicular cells as a consequence of this switch contains levels of oxytocin mRNA substantially greater than in the hypothalamus, and comprises a much larger number of oxytocin-producing cells [2,3 ]. Cloning of the luteal cDNA and primer extension sequencing showed that transcription was initiated from exactly the same position in the gene as in the hypothalamus, presumably subserved by the same promoter [3,4]. In vivo the oxytocin gene is down-regulated again about 3 to 4 days following
264 ovulation, so that oxytocin mRNA levels decline hereafter to reach basal levels in the mid to late corpus luteum. Virtually no oxytocin mRNA is detectable in the corpus luteum of pregnancy [2]. Granulosa cells of the weovulatory follicle are relatively easy to prepare from slaughterhouse tissue, and can be culturedin serum-free media in vitro for several days with a cell purity of almost 100~o [5,6]. We are how employing this cell culture system to unravel the control mechanisms of the oxytocin gene.
Which signal transduction systems are involved in cells endogenously expressing the oxytocin gene? In vivo granulosa cells do not see normal serum but are bathed in follicular fluid, a medium determined and controlled by the follicle cells themselves. Correspondingly, it was found that addition of fetal calf serum to cultures, permanently down-regulated the oxytocin gene and no effectors were able to reverse this influence [7]. In serum-free conditions both insulin or IGF-I and gonadotropins were able synergistically to up-regulate the oxytocin gene [6,8]. The gonadotropins could be substituted by forskolin, but insulin or IGF-I was still essential. This indicates that the oxytocin gene requires both tyrosine kinase activation and cAMP production.
Characterization of the cis elements present in the oxytocin gene promoter and the luteal transcription factors involved in the control of gene expression Computer comparison of the oxytocin genes, especially the 5' upstream promoter regions from several mammalian species highlights several highly conserved blocks of nucleotides, particularly in the 200 nucleotides upstream of the translational start site [9]. Besides the Goldberg-Hogness box required for transcriptional initiation by RNA polymerase II, most noticeable are a series of repeats including the motif ..TGACC.. which is a common feature of sev-
eral of the steroid hormone receptor binding elements [ 10,11 ]. Because of the involvement of estradiol in the hypothalamic up-regulation of the oxytocin gene in vivo it was speculated that these sites may function as estrogen responsive elements [12,13]. However, we were able to show in a heterologous system transfected with different constructs of the bovine oxytocin gene that the bovine promoter does not respond to estradiol [ 14]. Also in vivo, although the granulosa cells derive from estradiol-containing follicles, oxytocin gene regulation in culture cannot be influenced by this steroid [15]. More recently we have shown using PCR analysis that there are no transcripts encoding the estrogen receptor in these cells (Kascheike and Walther, unpublished data). In gel retardation experiments where the bovine oxytocin gene promoter is used to probe protein extracts of cell nuclei from early corpora lutea, it could be shown by competition experiments with specific oligonucleotides that not only the cis element described above at -162 to -152 (Fig. 1C) is very important in regulation, but that it is recognized by a transcription factor very similar if not identical to the C O U P transcription factor (COUP-TF) [11]. This factor is a member of the steroid hormone receptor superfamily but has no known ligand. DNA methylation footprinting of this region of the oxytocin gene indicated that two conserved G residues in the middle of this sequence which are not involved in estrogen receptor binding, are important for regulation in the bovine ovary [ 11 ], and are reported as being necessary for C O U P - T F binding [ 16]. Finally, it could be shown that antibodies directed against the C O U P - T F specifically disrupted the in vitro DNAprotein complex formation (Wehrenberg, Ivell and Walther, unpublished data). Analysis of the time course of appearance of C O U P - T F and its complexing ability with the oxytocin promoter suggested that it is expressed whenever the oxytocin gene is downregulated, and probably competes with another transcription factor for binding to the same site on the DNA. This other factor is expressed concomitantly with oxytocin gene up-regulation and is therefore
265
A.
Oxvtocin
Gene
Vasopressin
Gene
Exons
+, 0
I
ABC ...... H-t;..-
........
2
8
4
CB
6
10~12
I I
14
16
18
20kb
'
B.
C.
A
-<-_t_t__-_2-_t__: .......
#q2
e
A
C"~
I
~'G
G TCA
-
-
liiiii] -
TGA
C C
-161 C AITIAIAIC [ T
_168 -, 1, ,6 8
,Pl,FRlc
NT IZlZlZ T
-164 IG G TIGIAIC C
t
ERE
Bovine Sheep Mouse Rat Human
CCTTGACC _161-,68 -
-161
RTRA
AITIAIA C C T T G
Bovine Sheep
0,T Ac II0 c
Mouse Rat Human
G GIT G A C C
G
Oxytocin gene expression in the bovine testis
Consensus
C CTTGACC
-174 GGAITGACCT G~C~c 168
corpus luteum were able to form tissue-specific complexes with these D N A repeat elements. This poses the question of whether such elements may not be involved also in the activation of genes. The cultured bovine granulosa cell system has provided us already with much useful information concerning the cis elements and transcription factors, as well as the signal transduction systems involved in oxytocin gene regulation. The next steps are to fill in the gaps in these pathways, and to see whether these findings may be of more general application to oxytocin gene expression in other tissues.
C
TGA CC YTGA CC
COUP-TF element
Fig. 1. Schematic presentation of the bovine oxytocin gene and its flanking regions. (A) The vasopressin/oxytocin gene locus indicating the close neighbourhood and inverse orientation of the two genes. (B) The 5' upstream promoter region of the bovine oxytocin gene indicating the positions of the two repetitive elements identified by sequence and luteal nuclear protein binding [11]. (C) Detail of the -162 to -152 region of the upstream promoter conserved across five species as indicated. Upper panel shows homology to the classic estrogen response element (ERE); the lower panel the better homology to the COUP-TF binding sequence.
probably an activator. At the present time we have no information as to the identity of this factor except that it shares some characteristics with COUP-TF. About 1200 and 2500 nucleotides upstream of the site discussed above are two repetitive elements (Fig. 1B) belonging to the ruminant dispersed repeat family [ 11]. These elements occur throughout the bovine genome and it has been speculated that they may have some retroposon-like properties. Surprisingly, we could show that nuclear proteins from early
In the testis, the Sertoli cells occupy the same functional role vis-a-vis the germ cells that the granulosa cells have in the ovary. Both cell types are immediately apposed to the gametes inside a compartment defined by a semi-permeable basement membrane. Both cell types produce the hormones inhibin and MIF, and both cell types appear to produce oxytocin. In the bovine testis, this evidence was provided by in situ hybridization where clear signals could be seen within the seminiferous tubules [17]. More convincing evidence however has been provided by studies with transgenic mice [ 17]. In these mice the bovine oxytocin gene has been transfected into the germ line, and although there was no specific hypothalamic expression there was consistent oxytocin gene expression due to the bovine transgene within the Sertoli cells of the mouse testis. The endogenous mouse oxytocin gene is undetectable in these cells. This would suggest that the cell-type specificity of expression is an attribute of the bovine gene residing presumably as a cis element in the DNA sequence, whereas the mouse Sertoli cells provide transcription factors necessary for oxytocin gene expression. Immunohistochemistry showed that the bovine transgene was also translated in the Sertoli cells via a precursor polypeptide with both neurophysin and oxytocin moieties (Ivell, unpublished
266
data). Regarding the regulation of the bovine oxytocin gene in the testis, both in the transgenic mouse and in the bull, gene expression was up-regulated after puberty. Interestingly, just as in the granulosa cells of the ovary, oxytocin gene expression is reciprocally related to that for the inhibin alpha gene. At luteinization the inhibin-alpha gene is down-regulated in the granulosa cells as the oxytocin gene is being switched on. In the Sertoli cells at puberty it is the same, inhibin-alpha is prepuberally expressed, oxytocin postpuberally (Ungefroren and Ivell, unpublished data).
Gonadal expression of oxytocin and vasopressin genes in other species In other species neither vasopressin nor oxytocin genes are appreciably expressed in gonadal tissues [18]. Transcripts are detectable, especially using PCR-based assays [18,19]. However, a problem in these other species is that sometimes the genes may be expressed as non-functional aberrant transcripts [20-22]. Recently, we described vasopressin gene transcripts in the rat testis, which were lacking the nonapeptide-encoding exon 1. Instead they contained a piece of nucleic acid deriving from some 10 kb further upstream in the gene locus [20]. There was no open reading-frame and consequently no translation could occur. Whether this is a common feature of other peripherally expressed neuropeptide genes remains to be evaluated, but it warrants caution in the interpretation of such peripheral gene expression where the transcripts are not characterized in detail.
Acknowledgements We should like to thank Werner Rust, Martina Jansen and Bettina Bartlick for excellent technical assistance. Special thanks are due to Dr. David Murphy, Ang Hwee Luan and their partners in Singapore for all their work regarding the development
of the transgenic mice, and to Mike Millar and Dr. Richard Sharpe in Edinburgh for advice in setting up the immunohistochemistry. We should also like to acknowledge the generous financial support of the Deutsche Forschungsgemeinschaft (projects Ho 388/6-3 and Ho 388/6-10) as well as Prof. F. Leidenberger for providing excellent research facilities.
References 1 Gainer, H., Altstein, M., Whitnall, M.H. and Wray, S., The biosynthesis and secretion of oxytocin and vasopressin. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven Press, New York, 1988, pp. 2265-2282. 2 Ivell, R., Brackett, K.H., Fields, M.J. and Richter, D., Ovulation triggers gene expression in the bovine ovary, FEBS Lett., 190 (1985) 263-267. 3 lvell, R. and Richter, D., The gene for the hypothalamic peptide hormone oxytocin is highly expressed in the bovine corpus luteum: biosynthesis, structure and sequence analysis, EMBO J., 3 (1984) 2351-2354. 4 Ivell, R. and Richter, D., The oxytocin gene and its expression in the hypothalamus and ovary. In J.A. Amico and A.G. Robinson (Eds.), Oxytocin: Clinical and Laboratory Studies, Elsevier, Amsterdam, 1985, pp. 115-123. 5 McArdle, C.A. and Holtorf, A.P., Oxytocin and progesterone release from bovine corpus luteum cells in culture: effects of insulin-like growth factor I, insulin and prostaglandins, Endocrinology, 124 (1989) 1278-1286. 6 Holtorf, A.P., Furuya, K., Ivell, R. and McArdle, C.A., Oxytocin production and oxytocin messenger ribonucleic acid levels in bovine granulosa cells are regulated by insulin and insulin-like growth factor I: dependence on developmental status of the ovarian follicle, Endocrinology, 125 (1989) 26122620. 7 Luck, M.R., Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture, J. Exp. Zool., 251 (1989) 361-366. 8 McArdle, C.A., Kohl, C., Rieger, K., Gr6ner, I. and Wehrenberg, U., Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production, Mol. Cell. Endocrinol., 78 (1991) 211-220. 9 Ivell, R., Hunt, N., Abend, N., Brackmann, B., Nollmeyer, D., Lamsa, J.C. and McCracken, J.A., Structure and ovarian expression of the oxytocin gene in sheep, Reprod. Fertil. Dev., 2 (1990) 703-711. 10 O'Malley, B.W. and Tsai, M.J., Molecular pathways of steroid receptor action, Biol. Reprod., 46 (1992) 163-167.
267 11 Walther, N., Wehrenberg, U., Brackmann, B. and Ivell, R., Mapping of the bovine oxytocin gene control region: identification of binding sites for luteal nuclear proteins in the 5' non-coding region of the gene, J. Neuroendocrinol., 3 (1991) 539-549. 12 Burbach, J.P.H., Adan, R.A.H., Van Tol, H.H.M., Verbeeck, M.A.E., Axelson, J.F., Van Leeuwen, F.W., Beekman, J.M. and Ab, G., Regulation of the rat oxytocin gene by estradiol: examination of promoter activity in transfected cells and of messenger ribonucleic acid and peptide levels in the hypothalamo-neurohypophyseal system, J. Neuroendocrinol., 2 (1990) 633-639. 13 Richard, S. and Zingg, H.H., The human oxytocin gene promoter is regulated by estrogens, J. Biol. Chem., 265 (1990) 6098-6103. 14 Adan, R.A.H., Walther, N., Cox, J.J., Ivell, R. and Burbach, J.P.H., Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters, Biochem. Biophys. Res. Commun., 175 (1991) 117-122. 15 Furuya, K., McArdle, C.A. and Ivell, R., The regulation of oxytocin gene expression in early bovine luteal cells, Mol. Cell. Endocrinol., 70 (1990) 81-88. 16 Wang, L.H., Tsai, S.Y., Cook, R.G., Beattie, W.G., Tsai, M.J. and O'Malley, B.W., COUP transcription factor is a member
17
18
19
20
21
22
of the steroid receptor superfamily, Nature, 340 (1989) 163166. Ang, H.L., Ungefroren, H., De Bree, F., Foo, N.C., Carter, D., Burbach, J.P.H., Ivell, R. and Murphy, D., Testicular oxytocin gene expression in seminiferous tubules of cattle and transgenic mice, Endocrinology, 128 (1991) 2110-2117. Ivell, R., Vasopressin and oxytocin gene expression in the mammalian ovary and testis. In S. Jard and R. Jamison (Eds.), Vasopressin, Colloque INSERM, Vol. 208, Libbey Eurotext, Paris, 1991, pp. 31-38. lvell, R., Furuya, K., Brackmann, B., Dawood, Y. and KhanDawood, F., Expression of the oxytocin and vasopressin genes in human and baboon gonadal tissues, Endocrinology, 127 (1990) 2990-2996. Foo, N.C., Carter, D., Murphy, D. and Ivell, R., Vasopressin and oxytocin gene expression in rat testis, Endocrinology, 128 (1991) 2118-2128. Morley, S.D. and Ivell, R., Vasopressin gene transcripts in the bovine corpus luteum are defective, Mol. Cell. Endocrinol., 53 (1987) 255-258. Ivell, R., All that glisters is not gold - common testis gene transcripts are not always what they seem, Int. J. Androl., 15 (1992) 85-92.
263
REGPEP 01307
The regulation of neurohypophyseal peptide gene expression in gonadal tissues Richard Ivell, Norbert Walther, Uwe Wehrenberg, Craig McArdle and Hendrik Ungefroren Institute for Hormone and Fertility Research, University of Hamburg, Hamburg (Germany)
Key words: Oxytocin; Vasopressin; Neurophysin; Aberrant transcript; COUP transcription factor; Corpus luteum; Testis
Introduction
The neurohypophyseal peptide hormones oxytocin and vasopressin are responsible for the control of important endocrine systems in all mammal species examined. The effective levels of these hormones in the circulation are controlled on the one hand by regulating the release of the synthesized peptides in their major site of production, the hypothalamoneurohypophyseal system. Chronic control on the other hand is attained by up- or down-regulating the biosynthesis of the hormone precursors in the hypothalamus, in particular by altering the levels of gene transcription [ 1]. Although this transcriptional control can be studied in vivo by in situ or northern hybridization to learn about the physiological conditions under which the genes are modulated, only defined cell culture systems offer a basis with which to elucidate the intracellular mechanisms and pathways involved at the molecular level. To date cell cultures suitable for such experimentation have been lacking Correspondence to: R. Ivell, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 2000 Hamburg 54, Germany.
for studying oxytocinergic or vasopressinergic expression. To overcome this problem we have been characterizing a variety of peripheral tissues where oxytocin and vasopressin are also produced, usually as paracrine hormones with low level expression. In particular, the bovine ovary and testis have proved to be very useful in understanding the regulation of oxytocin gene expression. During the normal estrous cycle of the cow the oxytocin gene is massively up-regulated in the granulosa ceils of the preovulatory follicle accompanying the differentiation switch of these cells at ovulation from a predominantly estrogenic to a progestagenic phenotype [2]. The corpus luteum that results from the follicular cells as a consequence of this switch contains levels of oxytocin mRNA substantially greater than in the hypothalamus, and comprises a much larger number of oxytocin-producing cells [2,3 ]. Cloning of the luteal cDNA and primer extension sequencing showed that transcription was initiated from exactly the same position in the gene as in the hypothalamus, presumably subserved by the same promoter [3,4]. In vivo the oxytocin gene is down-regulated again about 3 to 4 days following
264 ovulation, so that oxytocin mRNA levels decline hereafter to reach basal levels in the mid to late corpus luteum. Virtually no oxytocin mRNA is detectable in the corpus luteum of pregnancy [2]. Granulosa cells of the weovulatory follicle are relatively easy to prepare from slaughterhouse tissue, and can be culturedin serum-free media in vitro for several days with a cell purity of almost 100~o [5,6]. We are how employing this cell culture system to unravel the control mechanisms of the oxytocin gene.
Which signal transduction systems are involved in cells endogenously expressing the oxytocin gene? In vivo granulosa cells do not see normal serum but are bathed in follicular fluid, a medium determined and controlled by the follicle cells themselves. Correspondingly, it was found that addition of fetal calf serum to cultures, permanently down-regulated the oxytocin gene and no effectors were able to reverse this influence [7]. In serum-free conditions both insulin or IGF-I and gonadotropins were able synergistically to up-regulate the oxytocin gene [6,8]. The gonadotropins could be substituted by forskolin, but insulin or IGF-I was still essential. This indicates that the oxytocin gene requires both tyrosine kinase activation and cAMP production.
Characterization of the cis elements present in the oxytocin gene promoter and the luteal transcription factors involved in the control of gene expression Computer comparison of the oxytocin genes, especially the 5' upstream promoter regions from several mammalian species highlights several highly conserved blocks of nucleotides, particularly in the 200 nucleotides upstream of the translational start site [9]. Besides the Goldberg-Hogness box required for transcriptional initiation by RNA polymerase II, most noticeable are a series of repeats including the motif ..TGACC.. which is a common feature of sev-
eral of the steroid hormone receptor binding elements [ 10,11 ]. Because of the involvement of estradiol in the hypothalamic up-regulation of the oxytocin gene in vivo it was speculated that these sites may function as estrogen responsive elements [12,13]. However, we were able to show in a heterologous system transfected with different constructs of the bovine oxytocin gene that the bovine promoter does not respond to estradiol [ 14]. Also in vivo, although the granulosa cells derive from estradiol-containing follicles, oxytocin gene regulation in culture cannot be influenced by this steroid [15]. More recently we have shown using PCR analysis that there are no transcripts encoding the estrogen receptor in these cells (Kascheike and Walther, unpublished data). In gel retardation experiments where the bovine oxytocin gene promoter is used to probe protein extracts of cell nuclei from early corpora lutea, it could be shown by competition experiments with specific oligonucleotides that not only the cis element described above at -162 to -152 (Fig. 1C) is very important in regulation, but that it is recognized by a transcription factor very similar if not identical to the C O U P transcription factor (COUP-TF) [11]. This factor is a member of the steroid hormone receptor superfamily but has no known ligand. DNA methylation footprinting of this region of the oxytocin gene indicated that two conserved G residues in the middle of this sequence which are not involved in estrogen receptor binding, are important for regulation in the bovine ovary [ 11 ], and are reported as being necessary for C O U P - T F binding [ 16]. Finally, it could be shown that antibodies directed against the C O U P - T F specifically disrupted the in vitro DNAprotein complex formation (Wehrenberg, Ivell and Walther, unpublished data). Analysis of the time course of appearance of C O U P - T F and its complexing ability with the oxytocin promoter suggested that it is expressed whenever the oxytocin gene is downregulated, and probably competes with another transcription factor for binding to the same site on the DNA. This other factor is expressed concomitantly with oxytocin gene up-regulation and is therefore
265
A.
Oxvtocin
Gene
Vasopressin
Gene
Exons
+, 0
I
ABC ...... H-t;..-
........
2
8
4
CB
6
10~12
I I
14
16
18
20kb
'
B.
C.
A
-<-_t_t__-_2-_t__: .......
#q2
e
A
C"~
I
~'G
G TCA
-
-
liiiii] -
TGA
C C
-161 C AITIAIAIC [ T
_168 -, 1, ,6 8
,Pl,FRlc
NT IZlZlZ T
-164 IG G TIGIAIC C
t
ERE
Bovine Sheep Mouse Rat Human
CCTTGACC _161-,68 -
-161
RTRA
AITIAIA C C T T G
Bovine Sheep
0,T Ac II0 c
Mouse Rat Human
G GIT G A C C
G
Oxytocin gene expression in the bovine testis
Consensus
C CTTGACC
-174 GGAITGACCT G~C~c 168
corpus luteum were able to form tissue-specific complexes with these D N A repeat elements. This poses the question of whether such elements may not be involved also in the activation of genes. The cultured bovine granulosa cell system has provided us already with much useful information concerning the cis elements and transcription factors, as well as the signal transduction systems involved in oxytocin gene regulation. The next steps are to fill in the gaps in these pathways, and to see whether these findings may be of more general application to oxytocin gene expression in other tissues.
C
TGA CC YTGA CC
COUP-TF element
Fig. 1. Schematic presentation of the bovine oxytocin gene and its flanking regions. (A) The vasopressin/oxytocin gene locus indicating the close neighbourhood and inverse orientation of the two genes. (B) The 5' upstream promoter region of the bovine oxytocin gene indicating the positions of the two repetitive elements identified by sequence and luteal nuclear protein binding [11]. (C) Detail of the -162 to -152 region of the upstream promoter conserved across five species as indicated. Upper panel shows homology to the classic estrogen response element (ERE); the lower panel the better homology to the COUP-TF binding sequence.
probably an activator. At the present time we have no information as to the identity of this factor except that it shares some characteristics with COUP-TF. About 1200 and 2500 nucleotides upstream of the site discussed above are two repetitive elements (Fig. 1B) belonging to the ruminant dispersed repeat family [ 11]. These elements occur throughout the bovine genome and it has been speculated that they may have some retroposon-like properties. Surprisingly, we could show that nuclear proteins from early
In the testis, the Sertoli cells occupy the same functional role vis-a-vis the germ cells that the granulosa cells have in the ovary. Both cell types are immediately apposed to the gametes inside a compartment defined by a semi-permeable basement membrane. Both cell types produce the hormones inhibin and MIF, and both cell types appear to produce oxytocin. In the bovine testis, this evidence was provided by in situ hybridization where clear signals could be seen within the seminiferous tubules [17]. More convincing evidence however has been provided by studies with transgenic mice [ 17]. In these mice the bovine oxytocin gene has been transfected into the germ line, and although there was no specific hypothalamic expression there was consistent oxytocin gene expression due to the bovine transgene within the Sertoli cells of the mouse testis. The endogenous mouse oxytocin gene is undetectable in these cells. This would suggest that the cell-type specificity of expression is an attribute of the bovine gene residing presumably as a cis element in the DNA sequence, whereas the mouse Sertoli cells provide transcription factors necessary for oxytocin gene expression. Immunohistochemistry showed that the bovine transgene was also translated in the Sertoli cells via a precursor polypeptide with both neurophysin and oxytocin moieties (Ivell, unpublished
266
data). Regarding the regulation of the bovine oxytocin gene in the testis, both in the transgenic mouse and in the bull, gene expression was up-regulated after puberty. Interestingly, just as in the granulosa cells of the ovary, oxytocin gene expression is reciprocally related to that for the inhibin alpha gene. At luteinization the inhibin-alpha gene is down-regulated in the granulosa cells as the oxytocin gene is being switched on. In the Sertoli cells at puberty it is the same, inhibin-alpha is prepuberally expressed, oxytocin postpuberally (Ungefroren and Ivell, unpublished data).
Gonadal expression of oxytocin and vasopressin genes in other species In other species neither vasopressin nor oxytocin genes are appreciably expressed in gonadal tissues [18]. Transcripts are detectable, especially using PCR-based assays [18,19]. However, a problem in these other species is that sometimes the genes may be expressed as non-functional aberrant transcripts [20-22]. Recently, we described vasopressin gene transcripts in the rat testis, which were lacking the nonapeptide-encoding exon 1. Instead they contained a piece of nucleic acid deriving from some 10 kb further upstream in the gene locus [20]. There was no open reading-frame and consequently no translation could occur. Whether this is a common feature of other peripherally expressed neuropeptide genes remains to be evaluated, but it warrants caution in the interpretation of such peripheral gene expression where the transcripts are not characterized in detail.
Acknowledgements We should like to thank Werner Rust, Martina Jansen and Bettina Bartlick for excellent technical assistance. Special thanks are due to Dr. David Murphy, Ang Hwee Luan and their partners in Singapore for all their work regarding the development
of the transgenic mice, and to Mike Millar and Dr. Richard Sharpe in Edinburgh for advice in setting up the immunohistochemistry. We should also like to acknowledge the generous financial support of the Deutsche Forschungsgemeinschaft (projects Ho 388/6-3 and Ho 388/6-10) as well as Prof. F. Leidenberger for providing excellent research facilities.
References 1 Gainer, H., Altstein, M., Whitnall, M.H. and Wray, S., The biosynthesis and secretion of oxytocin and vasopressin. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven Press, New York, 1988, pp. 2265-2282. 2 Ivell, R., Brackett, K.H., Fields, M.J. and Richter, D., Ovulation triggers gene expression in the bovine ovary, FEBS Lett., 190 (1985) 263-267. 3 lvell, R. and Richter, D., The gene for the hypothalamic peptide hormone oxytocin is highly expressed in the bovine corpus luteum: biosynthesis, structure and sequence analysis, EMBO J., 3 (1984) 2351-2354. 4 Ivell, R. and Richter, D., The oxytocin gene and its expression in the hypothalamus and ovary. In J.A. Amico and A.G. Robinson (Eds.), Oxytocin: Clinical and Laboratory Studies, Elsevier, Amsterdam, 1985, pp. 115-123. 5 McArdle, C.A. and Holtorf, A.P., Oxytocin and progesterone release from bovine corpus luteum cells in culture: effects of insulin-like growth factor I, insulin and prostaglandins, Endocrinology, 124 (1989) 1278-1286. 6 Holtorf, A.P., Furuya, K., Ivell, R. and McArdle, C.A., Oxytocin production and oxytocin messenger ribonucleic acid levels in bovine granulosa cells are regulated by insulin and insulin-like growth factor I: dependence on developmental status of the ovarian follicle, Endocrinology, 125 (1989) 26122620. 7 Luck, M.R., Greatly elevated and sustained secretion of oxytocin by bovine granulosa cells in serum-free culture, J. Exp. Zool., 251 (1989) 361-366. 8 McArdle, C.A., Kohl, C., Rieger, K., Gr6ner, I. and Wehrenberg, U., Effects of gonadotropins, insulin and insulin-like growth factor I on ovarian oxytocin and progesterone production, Mol. Cell. Endocrinol., 78 (1991) 211-220. 9 Ivell, R., Hunt, N., Abend, N., Brackmann, B., Nollmeyer, D., Lamsa, J.C. and McCracken, J.A., Structure and ovarian expression of the oxytocin gene in sheep, Reprod. Fertil. Dev., 2 (1990) 703-711. 10 O'Malley, B.W. and Tsai, M.J., Molecular pathways of steroid receptor action, Biol. Reprod., 46 (1992) 163-167.
267 11 Walther, N., Wehrenberg, U., Brackmann, B. and Ivell, R., Mapping of the bovine oxytocin gene control region: identification of binding sites for luteal nuclear proteins in the 5' non-coding region of the gene, J. Neuroendocrinol., 3 (1991) 539-549. 12 Burbach, J.P.H., Adan, R.A.H., Van Tol, H.H.M., Verbeeck, M.A.E., Axelson, J.F., Van Leeuwen, F.W., Beekman, J.M. and Ab, G., Regulation of the rat oxytocin gene by estradiol: examination of promoter activity in transfected cells and of messenger ribonucleic acid and peptide levels in the hypothalamo-neurohypophyseal system, J. Neuroendocrinol., 2 (1990) 633-639. 13 Richard, S. and Zingg, H.H., The human oxytocin gene promoter is regulated by estrogens, J. Biol. Chem., 265 (1990) 6098-6103. 14 Adan, R.A.H., Walther, N., Cox, J.J., Ivell, R. and Burbach, J.P.H., Comparison of the estrogen responsiveness of the rat and bovine oxytocin gene promoters, Biochem. Biophys. Res. Commun., 175 (1991) 117-122. 15 Furuya, K., McArdle, C.A. and Ivell, R., The regulation of oxytocin gene expression in early bovine luteal cells, Mol. Cell. Endocrinol., 70 (1990) 81-88. 16 Wang, L.H., Tsai, S.Y., Cook, R.G., Beattie, W.G., Tsai, M.J. and O'Malley, B.W., COUP transcription factor is a member
17
18
19
20
21
22
of the steroid receptor superfamily, Nature, 340 (1989) 163166. Ang, H.L., Ungefroren, H., De Bree, F., Foo, N.C., Carter, D., Burbach, J.P.H., Ivell, R. and Murphy, D., Testicular oxytocin gene expression in seminiferous tubules of cattle and transgenic mice, Endocrinology, 128 (1991) 2110-2117. Ivell, R., Vasopressin and oxytocin gene expression in the mammalian ovary and testis. In S. Jard and R. Jamison (Eds.), Vasopressin, Colloque INSERM, Vol. 208, Libbey Eurotext, Paris, 1991, pp. 31-38. lvell, R., Furuya, K., Brackmann, B., Dawood, Y. and KhanDawood, F., Expression of the oxytocin and vasopressin genes in human and baboon gonadal tissues, Endocrinology, 127 (1990) 2990-2996. Foo, N.C., Carter, D., Murphy, D. and Ivell, R., Vasopressin and oxytocin gene expression in rat testis, Endocrinology, 128 (1991) 2118-2128. Morley, S.D. and Ivell, R., Vasopressin gene transcripts in the bovine corpus luteum are defective, Mol. Cell. Endocrinol., 53 (1987) 255-258. Ivell, R., All that glisters is not gold - common testis gene transcripts are not always what they seem, Int. J. Androl., 15 (1992) 85-92.