A bovine oxytocin transgene in mice: expression in the female reproductive organs and regulation during pregnancy, parturition and lactation

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Abstract

Transgene bovine oxytocin 3.5 (bOT3.5) consists of the bovine oxytocin structural gene flanked by 0.6 kbp of upstream and 1.9 kbp of downstream sequences. We have examined the expression of bOT3.5 in the female reproductive organs, and we show tissue-specific and physiological regulation dependent on the stage of pregnancy and lactation. In the ovary, no transgene expression could be detected during the estrus cycle, or during pregnancy. However, high levels of transgene RNA were found at day 1 of lactation. Expression dropped 10-fold by day 2 of lactation, and was undetectable thereafter. Interestingly, the expression of bOT3.5 in the mouse ovary at the beginning of lactation mimics that of the endogenous OT gene in the bovine ovary. Expression of the bOT3.5 transgene correlates with a parturition defect that results in considerable maternal mortality.

Introduction

The peptide hormone oxytocin (OT) is synthesised as a propeptide in neuronal cell bodies of the hypothalamus. This precursor is subject to cleavage and other modifications as it is transported axonally to the posterior pituitary (Brownstein et al., 1980). The mature peptide products–the nonapeptide hormone OT and a putative carrier molecule termed neurophysin (NP)–are stored in axon terminals until neural inputs, governed by physiological stimuli, elicit their release. OT is considered to be a central component of the systems that regulate birth and nursing. OT travels through the blood stream and, through an interaction with OT receptors located in the uterus and mammary gland, stimulates smooth muscle contraction, leading to parturition or milk ejection.

In many species OT is also synthesised in small amounts in some peripheral tissues (Murphy et al., 1993) where, it is presumed, the hormone acts in a paracrine manner on adjacent cells. In ruminants, however, luteal OT is synthesised in large quantities as part of a positive feedback loop with endometrial prostaglandins that is thought to regulate luteolysis (Flint et al., 1994). Luteal OT and OT mRNA levels fall drastically during the first few days of pregnancy (Ivell et al., 1985, Jones and Flint, 1988, Ivell et al., 1990). However, at the end of pregnancy, and particularly after the onset of labour, luteal OT gene expression and OT synthesis are activated (Ivell et al., 1995). The function of luteal OT expression at term, and the factors responsible for its induction at parturition, are unknown (Ivell et al., 1995).

Little is known about the molecular mechanics of the cell-specific and physiological regulation of OT gene expression. This is particularly so for the bovine oxytocin (bOT) gene which, whilst being of great agricultural interest, represents a somewhat problematic experimental model. In order to define novel and more tractable models for the study of ruminant reproductive physiology, we have generated transgenic mice bearing the bOT gene (Ang et al., 1991, Ang et al., 1993, Ang et al., 1994, Ho et al., 1995). We have described transgenic mice bearing the transgene bOT3.5, which consists of the bOT structural gene flanked by 0.6 kbp of upstream and 1.9 kbp of downstream sequences (Ho et al., 1995). The bOT3.5 demonstrates appropriate neuron-specific and physiological regulation, being expressed in OT magnocellular neurons in the mouse supraoptic nucleus (SON) and paraventricular nucleus (PVN). Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increases the abundance of bOT transcripts in the SON, but not the PVN, in parallel with the endogenous mouse OT RNA. In addition, male mice showed transgene expression in testis and lung.

We have now asked if the bOT3.5 transgene can mediate the expression of the bOT gene in the female reproductive organs of host mice.

Section snippets

Animals

Animals were cared for in accordance with NIH guidelines. The generation of transgenic mice bearing the bOT3.5 transgene has been described in Ho et al. (1995). Two independently derived lines were examined in this study (bOT3.5 line 1 and 2). Transgenic mice were maintained on a (CBA/J X C57BL/10) background. Only Southern blot positive heterozygous transgenic animals were used for expression analysis. Sexually mature virgin female transgenic mice were checked for normal cycling by daily

Expression of the bOT3.5 transgene in female mice

Northern blotting was used to detect transgene expression in a number of female bOT3.5 transgenic mouse tissues at different stages of the estrus cycle, pregnancy, parturition and lactation. Two independently derived bOT3.5 lines were examined (bOT3.5 Line 1 and 2). Transgene expression was compared with the expression of the endogenous OT and VP RNAs in both transgenic and wild-type mice. Then the ovary, placenta, uterus and mammary gland were examined. All tissues from both lines were

Discussion

We have described a bOT transgene, bOT3.5, consisting of the bOT structural gene flanked by 0.6 kbp of upstream and 1.9 kbp of downstream. The bOT3.5 shows cell-specific expression in the mouse ovary, within which it is subject to physiological regulation.

We have previously shown that bOT3.5 RNAs are expressed abundantly in the testes of male transgenic mice (Ho et al., 1995). Immunocytochemistry has revealed transgene-derived NP in Sertoli cells. Similarly a larger transgene, bOT, consisting

Acknowledgements

The staff in the IMCB Animal Holding Unit are thanked for expert animal care. We thank Dr Helen Nicholson (Department of Anatomy, University of Bristol) for her critical reading of the manuscript.

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