Prenatal programming of the reproductive axis: the role of somatostatin

During life in the womb animal and human foetuses are largely protected from substances that might adversely affect their development. However, there are certain situations in which substances that may be present in the mothernal environment in unusually high concentrations or reach the developing foetus because of some physiological disfunction in the mother, that may cause abnormalities in foetal development. These abnormalities are not always obvious at birth and may not cause a problem or lead to a disease state until maturity is reached. Because there is a long lag time between the appearance of the abnormal state and the causal agent we refer to the disease as having been 'programmed' by early life experience. Hormones like those made in our gonads can, are required to programme certain aspects of normal development, the most obvious being the secondary sexual characteristics of males and females. However, in some situations, exposure to abnormal amounts or patterns of such hormones can result in abnormal or disease states. For example, if female foetuses are exposed to abnormally high concentrations of the male hormone testosterone during development this can lead to malfunctions in the reproductive system of the offspring in adulthood that, in extreme cases, may result in infertility. These abnormal hormone concentrations might be produced by some malfunction in the mother (for example a hormone releasing tumour) and cross that cross to the unborn young. Alternatively they may originate in the environment, pass into the mother and from her to the foetus. Examples of environmental agents include the so-called 'endocrine disruptors' that bear little chemical resemblance to the hormones themselves but are able to mimic their actions in the body. Several reports in the scientific and popular press have suggested that infertility in the human population is increasing and that substances in our environment, including those we are exposed to before birth, may contribute to this. Studies, that have already been completed, have have shown that certain hormones act in the developing brain (including the parts that control the reproductive system) to organise it in such a manner that it eventually begins to behave abnormally in later life. The major aim of this project is to understand how the specialised nerve cells (neurones) that control female reproduction become disrupted by exposure to testosterone before birth. These specialised cells synthesise Gonadotrophin releasing hormone (GnRH). As we cannot use the human brain to make these determination we use a good animal model of the situation, which is the case of this proposal is the female sheep that has been exposed, as a foetus, to an abnormally high concentration of testosterone. Our studies focus of a group of neurones that play a key role in controlling the GnRH neurones and release a peptide known as somatostatin. The project will identify somatostatin cells in the brain, the factors that make them active to release somatostatin and the location of receptors on which the somatostatin might act. Importantly, we will determine if there are differences between normal ewe brains and those that have been exposed to testosterone in the uterus.

General techniques used: Androgenisation of ewes: The work described in this proposal involves the programming of the ovine reproductive axis by exposure of the female foetus to an exogenous source of androgens. To achieve this the oestrous cycles of the potential mothers will be synchronised with vaginal progesterone sponges that will be removed after 10 days, and 24 hours later a fertile (raddled) ram introduced to the flock. Mated ewes will be identified by the coloured rump marking from the raddle. Pregnant ewes will be injected from day 30-90 of pregnancy with testosterone propionate or DHT (i.m.100mg/injection; twice per week). Generation of artificial oestrous cycles: It is key to these studies that the steroid concentrations in animals are carefully controlled and this is best achieved by removal of the endogenous source of gonadal steroids by ovariectomy and replacing progesterone and oestrogen in physiological concentrations using implants. Oestrogen is replaced with silastic capsules of crystalline oestradiol benzoate (manufactured in the laboratory) and progesterone using commercially available CIDR's). Specific techniques used in individual objective sections: The first objective is to determine if somatostatin neurones play a role in reproductive behaviour and the programming of the absence of this behaviour in androgenised animals. Artificial oestrous cycles will be generated in control and androgenised animals as described above. Identification of activated somatostatin neurones will use the technique of immunocytochemistry using free floating sections of tissue that will be cut on a freezing microtome at 30 ?m. reproductive behaviour will be assessed in ewes in the presence of a vasectomised teaser ram. The second objective is to determine if the LH and GH surges are functionally linked and this will involve the collection of blood samples from the jugular vein and the LH and GH concentrations in these samples will be determined by radioimmunoassay. Objective 3 is to determine if in utero testosterone exposure can influence the number and oestrogen responsiveness of gonadotrophs and somatotrophs in the ovine pituitary gland using immunocytochemistry. Mapping the somatostatin receptors in the brain (objective 4) will use the technique of in situ hybridization histochemistry The recent publication of sequences for somatostatin receptors 1 and 5 will allow us to deign specific oligonucleotide probes that will be obtained from a commercial supplier, labelled with S35 and hybridized with hypothalamic sections (15?m) that have been freeze thaw mounted into vectabond subbed slides. We also aim to clone somatostatin receptors 2-4 using published sequences for the human and rodent somatostatin receptors 2-4 to design specific primers and using rt-RCR to isolate the ovine cDNA sequences. The mRNA sequence will be obtained using a standard dideoxynucleotide method using a ABI Prism 3100 genetic analyser. In order to achieve objective 6 (to determine where in the hypothalamus somatostatin acts to alter GnRH and GH release) guide cannulae will be placed into the third ventricle using stereotaxic surgery. The head of the anaesthetised animal will be held firmly in a specially designed stereotaxic frame and needle placed in one of the lateral ventricles in order to introduce a radioopaque dye. X rays in both a dorso-ventral and lateral plane will be used to visualise the third ventricle and calculations made from these x rays to guide the correct placement of a guide cannula in this ventricle. The guide cannula will be held firmly in place using dental cement and protected by a stainless steel ring. Substances can be infused into this guide cannula from a syringe attached to a portable pump that is held firmly in place by a specially designed harness.