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te or glucuronide moiety, involving sulphatase or glucuronidase enzymes, spectively. In mammals, this occurs most extensively in the liver as a way of a

HSD) in species from several differe to

and in the skin of Salmo trutta

production is unclear.

The gonads of many vertebrates both synthesise and further metabolise major steroids (Ozon and Stocker, 1974; Kime and Hews, 1980; Huf et al., 1989; Cuevas et al., 1992). Conversion of T to 11-KT may actually occur following biosynthesis of T in the testis in these species, as it does in Salmo salar (Idler et al., 1971). 11-Ketotestosterone is also produced by both gonadal and fat body tissue of males and females of a urodele amphibian Triturus cristatus carnifex (Lupo Di Prisco et al., 1971; 1972).

The second way in which a steroid molecule may be altered is by conjugation with either a sulpha

re

deactivating and solubilising steroids (Kime, 1987; Norris, 1997) for later excretion in faeces or urine (Heistermann et al., 1993; Wasser et al., 1996; Velloso et al., 1998). Steroid conjugation is also known to occur in the mammalian placenta, gonads and adrenals (Scott and Vermierssen, 1994). Most non-mammalian vertebrates also conjugate some steroids in the liver to facilitate excretion (Kime, 1987).

However, conjugation of steroids for reasons other than deactivation has been described in some non-mammalian vertebrates. In teleosts, the role of conjugated steroids as

(Scott and Vermierssen, 1994; Stacey et al., 1994; Stacey and Sorensen, 1986). Conjugated steroids are known to stimulate milt production (Stacey et al., 1989; Van Der Kraak et al., 1989) and spawning behaviour (Sherwood et al., 1991; Sorensen and Stacey, 991; Carolsfeld et al., 1997a; b) in fish. These conjugated steroids are largely gonadal in

t al., 1986) and gills (Kime and Ebrahimi, 1997) have also been

plicated in the conjugation of steroids for use as pheromones.

are differences between the vertebrate classes in the dominant

f the goldfish Carassius

uratus (Scott and Sorensen, 1994), both glucuronidated and sulphated steroids are

1

origin (Stacey et al., 1986; Van Den Hurk et al., 1987; Scott and Vermierssen, 1994), rather than being excretory products generated by the liver as occurs in mammals (Kime, 1987). However, whether gonadal conjugation involves newly synthesised steroids prior to their release into the circulation, or whether the gonads are conjugating already circulating steroids, and so acting much like an additional peripheral tissue, is unclear. In teleosts, skin (Stacey e

im

Among other aquatic vertebrates the testis of the elasmobranch Squalus acanthias

(Cuevas et al., 1992), the Australian lungfish, Neoceratodus forsteri (Joss et al., 1996) and the liver of the amphibians Pleurodeles waltlii (Ozon and Breuer, 1966) and

Dicoglossus pictus (Ozon and Stocker, 1974) produce conjugated steroids, although it is

not yet clear if any of these molecules function as pheromones.

Additionally, there

conjugates that are formed. Tissues from mammalian species usually form steroid sulphates (Ruokonen and Vihko, 1974; Payne, 1980; Dehennin, 1993), while glucuronidated steroids predominate in teleost species that use conjugates as pheromones (Kime, 1980; Schoonen and Lambert, 1986a; Van Den Hurk et al., 1987; Sherwood et al., 1991). However, some fish species partition steroid conjugate production. For example, in the trout Oncorhynchus mykiss glucuronides are produced by the liver for excretion in faeces, while sulphates are released in the urine (Vermierssen and Scott, 1996). In the testis of S. acanthias (Cuevas et al., 1992) and the ovary o

a

produced, for which separate paracrine regulatory, excretory or pheromonal functions are proposed. Recently, differential routes of steroid excretion and different roles for

particular steroid molecules have also been documented in some mammalian species (Wasser et al., 1996; Velloso et al., 1998).

6.1.3 Variation in the patterns of steroid metabolism

Numerous in vitro and in vivo studies of steroid metabolism in vertebrates have documented variations in the patterns of production of conjugates and derivatives according to the reproductive condition of the animal (Ozon and Fouchet, 1974; Kime, 1987; Schlinger et al., 1989; Borg et al., 1992). In ectothermic vertebrates such variations can be mediated by the effects of temperature on enzyme activity (Kime, 1979; Kime and Hyder, 1983; Manning and Kime, 1985; Lofts, 1987) (Refer to Chapter 5 Section 5.4.6

r more information). Seasonal variations in several other factors, including receptor fo

density, which is itself up or down regulated in response to steroid concentrations in lizards (Paolucci and Di Fiore, 1994; Cardone et al., 1998), steroid substrate concentration (Kime and Hews, 1980; Kime and Abdullah, 1994) and binding protein concentration, could also conceivably alter ratios of free to conjugated steroids, although such factors are often not considered.

6.1.4 This study

The effects of reproductive steroids on some peripheral tissues have been observed in several reptile species. For example, epididymal function (Shivanandappa and Devaraj Sarkar, 1987) and hypertrophy of the sexual segment of the kidney (Prasad and Sanyal, 1969) are both regulated by androgens, and exogenous application of E2 can stimulate the hypertrophy of cloacal glands (Cooper et al., 1986b) in lizards. Moreover, hemipenal tissue in Lacerta vivipara is insensitive to T in adult animals (Dufaure and Chambon, 1978), but in Calotes versicolor it is regulated by androgens (Ananthalakshimi et al., 1991). This tissue may, therefore, be a candidate as a site for the action of a metabolite of testosterone. However, links between gonadal steroids and their effects on peripheral tissues are often inferred rather than proven, and are, furthermore, generally assumed to be the direct effects of T or E2 rather than their metabolites.

The detection of particular steroid metabolising enzymes in target tissue cells goes some way towards strengthening the link between a steroid, its metabolites, and the

hysiological or behavioural responses attributed to it. The presence of enzymes ulphating and glucuronidating nzymes have all been detected in, or inferred by the presence of appropriate metabolites,

examined potential changes in the patterns of eripheral steroid metabolism throughout the annual reproductive cycle in reptiles, ductive condition or between sexes. This study examines the ability of

thetically by conjugation or p

including aromatase and 5α-reductase, as well as s e

in several different tissues in reptiles including the brain (Callard et al., 1977; 1978; Crews and Morgentaler, 1979; Huf et al., 1987a; Gobbetti et al., 1994; Wade, 1997; Winkler and Wade, 1998) and renal sexual segment (Crews et al., 1978). Some authors have acknowledged that observed changes in tissue morphology or physiology in response to the application of exogenous steroid may actually occur following localised metabolism of the steroid at the target tissue (Dufaure and Chambon, 1978; Abell, 1998). Indeed, variations in responsiveness to steroids may, at least partly, be due to differences in the patterns of steroid metabolism in different peripheral tissues. The presence or absence of appropriate enzymes is, in no small way, responsible for this additional level of tissue specificity.

No studies, to my knowledge, have p

according to repro

a range of reproductively important tissues to metabolise a primary steroid (T or E2) as a measure of the activity of steroid metabolising enzymes in those tissues, in the viviparous lizard Tiliqua nigrolutea and considers intersexual and seasonal differences in patterns of steroid metabolism. The possibility of a role for steroid metabolites as semiochemicals in a lizard is considered. This study was conducted prior to the experiments presented in

Chapter 5 so the possibility of an alternative to E2 as the major ovarian oestrogen in this species had not yet been raised. Testosterone and E2 were chosen as the basis for this metabolic study based on a survey of the literature.

Although not peripheral tissues, ovarian and testicular tissues were included in this study. The ability of gonadal tissue to modify T and E2 post-biosyn

derivatisation has been well documented in vertebrates (Cuevas et al., 1992; Joss et al., 1996; Scott and Vermierssen, 1996).

6.2 Materials and methods

General methods are described in Chapter 2 Section 2.1, but information specific to the work in this chapter is presented here.

6.2.1 Tissue collection

Peripheral tissues potentially capable of steroid metabolism were collected from male and female Tiliqua nigrolutea at autopsy of freshly killed individuals between April 1995 and February 1996. Male and female lizards were sampled at times of year that corresponded to distinct phases of the annual reproductive cycle (summarised in Table 6.1). Tissues collected for incubation were skin (lateral abdominal body surface), muscle (abdominal wall), liver, cloaca (surrounding the cloacal opening), adrenal and kidney (both sexes), ovary (including corpora lutea (CLs) when present), oviduct (females only), and testis, epididymis and sexual segment (SS) of the kidney (males only). Oviductal tissue was collected in gestating females from regions adjacent to developing embryos, and from an similar position in post-parturient and preovulatory animals). Kidney tissue from early spermatogenic-stage males in autumn was not collected for incubation. In males of the sympatric species, Niveoscincus metallicus, the renal SS is clearly identifiable (by hypertrophy and colour change) during the autumn mating period as the anterior third of each kidney (Jones, pers. comm.); a similar region was selected in T. nigrolutea.

Skeletal muscle was used as a (presumably) non-reproductively relevant control tissue. Gonadal tissue from each sex was included, despite its not being a peripheral tissue, because it is also a potential site of post-biosynthetic modification of primary gonadal steroids. Samples of liver, kidney and sexual segment, adrenal gland, oviduct, epididymis and cloacal tissues were preserved in Bouin’s fixative and examined histologically (details in Chapter 2 Section 2.5). Details of testicular and ovarian histology are presented in Chapter 3 Results and

Table 6.1 Sampling regime for collection of peripheral tissues