Vasopressin and oxytocin gene expression in rat testis.

The vasopressin (VP) gene is expressed as three different transcripts in the rat testis. Using polymerase chain reaction (PCR) analysis we have been able to identify a VP RNA that is identical in exonic structure to that found in the hypothalamus. However, the abundance of this form is very low, and it cannot be detected by Northern blotting. Two VP RNAs with a novel structure, as shown using exon-specific probes, are present in higher abundance. By differential hybridization, sequencing of a cDNA clone, and PCR we have deduced the structure of these novel transcripts. Both of the novel testicular VP RNA species share two exons with the classical hypothalamic RNA. However, the testicular VP gene-derived RNA lacks the first exon of the hypothalamic transcript, the exon that contains the sequence information for the VP nonopeptide hormone. Instead, it has novel sequence that are derived from at least two unique testis-specific exons, one of which is located 7-10 kilobase up-stream of the brain-specific start of transcription. These two unusual transcripts are probably derived by alternative splicing of at least two up-stream exons. Sequence and polysome analyses indicate that the testicular VP RNAs are probably not translated. Northern blotting revealed that the VP gene-derived RNA species are tightly regulated during postnatal development, becoming apparent by 40 days of age, although they subsequently fail to respond to a variety of physiological perturbations. Oxytocin gene transcripts are not detectable by Northern hybridization, but the authentic hypothalamic-type RNA can be detected in the rat testis using PCR analysis.

Testicular oxytocin gene expression in seminiferous tubules of cattle and transgenic mice.

We are using transgenic mice to study the regulation of the bovine vasopressin (VP) and oxytocin (OT) genes. Prompted by the observation that mice bearing a bovine OT transgene express bovine OT RNA in their testes, we investigated the expression of the VP-OT locus in normal mice and cattle. Normal wild-type mice do not have detectable levels of either VP or OT RNA in their testes. Normal cattle are also devoid of detectable VP transcripts, but have relatively high levels of testicular OT RNA. Additionally, OT, but not VP, peptide is detectable by HPLC. In situ hybridization to RNA in bovine testicular tissue sections localized OT transcripts to seminiferous tubules, with a distribution similar to that of alpha-inhibin, suggesting expression in Sertoli cells. Interestingly, the bovine OT RNAs in the transgenic mouse testes were also shown by in situ hybridization to have the same distribution. These data suggest that the cis-acting regulatory sequences responsible for expression of the OT gene in bovine Sertoli testis reside within the limits of the transgene used in this study. Further, the trans-acting factors present in murine testicular cells are able to recognize these elements, although they do not express the endogenous mouse OT gene in this tissue.

Activation of promoters for cellular lipogenic genes by hepatitis B virus large surface protein.

Hepatitis B virus large surface protein has the unusual property of accumulating in a particulate form within a preGolgi compartment, leading to marked proliferation of intracellular membranes. We show here that large surface protein activates the promoters for two lipogenic genes that code for farnesyl diphosphate synthase and fatty acid synthase. This activation is transduced, in part, by the transcription factor NF-Y. Although NF-Y is also necessary for the transcriptional induction of chaperone proteins residing in the endoplasmic reticulum by unfolded proteins, other inducers of chaperone synthesis do not activate the promoters for farnesyl diphosphate synthase and fatty acid synthase. Our results suggest the presence of a novel signaling pathway from the endoplasmic reticulum to the nucleus that causes the intracellular membrane proliferation seen in the hepatocytes of persons with accumulated large surface protein particles.

Expression of a rat vasopressin transgene in rat testes.

The rat vasopressin gene contains two transcriptional promoters; the activity of one is confined to the hypothalamus, while the other is testis specific. To define the sequences mediating the cell-specific expression of the vasopressin gene, we introduced rat vasopressin transgenes into the rat germ line. Neither transgene 1.5-V beta gal-0.2, which consists of the entire vasopressin structural gene containing a 3 kbp beta-galactosidase reporter element in exon III, flanked by 1.5 kbp upstream of the start of hypothalamic transcription and 0.2 kbp downstream of the polyadenylation site, nor transgene 3-V beta gal-0.2, which consists of the entire VP structural gene containing a 3 kbp beta-galactosidase reporter element in exon III, flanked by 3 kbp upstream of the start of hypothalamic transcription and 0.2kbp downstream of the polyadenylation site, were expressed in the hypothalamus. This contrasts with a previously described transgene consisting of the rat vasopressin structural gene containing a reporter in exon III, flanked by 5 kbp of upstream and 3 kbp of downstream sequences, which is expressed in vasopressinergic hypothalamic neurons. Both the 3-V beta gal-0.2 and 1.5-V beta gal-0.2 transgenes were expressed in testicular germ cells using a promoter located within the beta-galactosidase reporter element. Transgene RNA was most abundant during the late stages of meiosis. Rats bearing vasopressin-beta-galactosidase transgenes provide new models for the study of the mechanisms whereby an epigenetic choice is made between the use of a germ cell or a somatic promoter, and the stage-specific transcriptional regulation of a germ cell promoter during spermatogenesis.

A testis-specific promoter in the rat vasopressin gene.

In the rat testis, the vasopressin gene is transcribed into precursor RNAs that are processed into a number of mature transcripts. One of these transcripts has a structure identical to that of the hypothalamic RNA that encodes the vasopressin prepropeptide, but is present at such low levels that it can only be detected by the polymerase chain reaction. Other vasopressin-like RNAs are derived from differential splicing events that join transcribed sequences between 3 and 9 kilobases upstream of the hypothalamic transcription start site to exons corresponding to II and III of the hypothalamic-type RNA. Here we describe the sequence of a testis-specific promoter and the exon structure of its transcription unit. We show that an in vitro synthesized RNA corresponding to the longer testicular vasopressin gene-derived transcript is not able to act as a template for protein synthesis in two different cell-free lysates. As attempts to localize the vasopressin-gene derived RNAs to particular cell types in the testis by in situ hybridization have consistently failed, we have used indirect methods. Three different procedures were used to effect germ cell depletion in adult male rats. Acute heat treatment of the testis, chronic ingestion of hydroxyurea, and chronic vitamin A deficiency all reduced the level of the aberrant testicular vasopressin-gene derived RNAs, indicating that their expression is closely associated with the integrity of germ cells and ongoing spermatogenesis.