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Metabolism of [3H]androstenedione was studied in brain tissue homogenates of opossum, bird, snake, sea turtle, urodele amphibian, teleost, shark, skate, hagfish, and lobster. Estrone, 17 beta-estradiol, or 17 alpha-estradiol was formed by central neural tissues of all species, with the exception of the opossum, hagfish, and lobster. Aromatase activity was concentrated in the forebrain, although some estrogen was synthesized by mid- or hindbrain homogenates of two lower vertebrates (teleost and skate) and the newly hatched chick. 5 alpha-Androstanedione (5 alpha-androstane-3,17-dione) or 5 alpha-dihydrotestosterone were products of metabolism in several nonmammalian vertebrates and in the invertebrate central nervous system also. 5 alpha-Reductase was found in all major brain divisions. These and previously reported comparative studies indicate that the ability to aromatize and otherwise transform androgen substrates is a primitive characteristic of the brain that has been widely conserved phylogenetically.

The exceptionally high rate of aromatization previously demonstrated in the testis of the urodele amphibian Necturus maculosus prompted us to investigate estrogen binding activity in this tissue. When crude cytosolic extracts were incubated with (3 HI estradiol prior to DNA-cellulose affinity chromatography or sucrose gradient centrifugation, no estrogen binding was detected. Under these conditions, the presence of an endogenous estrogen-conjugating system in the crude cytosol resulted in extensive conversion of radiolabeled tracer to sulfoconjugates which presumably were poor receptor ligands; however, when this enzyme was first removed from the crude cytosol by fractionation through DNA-cellulose columns, DNA-adhering components could then be labeled with radioactive estradiol. This post-labeling method revealed an estrogen binding component with the physicochemical properties of a classical estrogen receptor: high affinity (Kd”�10’’#{176});low capacity (2-5 fmol/mg protein); and affinity for estrogenic steroids only. On sucrose gradients the estrogen receptor complex eluted from DNA-cellulose had a sedimentation coefficient of 4.7-5S. Estrogen receptors having properties similar to the cytosolic form were also demonstrated in nuclear extracts prepared from testes of untreated animals, suggesting that translocation of estrogen synthesized from endogenous precursors is a normal in vivo occurrence in this species. Preliminary measurements indicate that both cytoplasmic and nuclear estrogen receptors may show seasonal changes keyed to the annual reproductive cycle. We reported earlier that estrogen binding activity in the crude cytosol of Necturus testis is located mainly in regions comprised of differentiating or fully differentiated Leydig cells. Further studies of aromatization, the estrogen receptor system, and the estrogen-conjugating enzyme in Necturus testis may provide new insights into the role of intratesticular estrogen in male reproduction.

Steroid-tipped hypothalamic probes containing either cholesterol, aldosterone, corticosterone, or betamethazone were found to significantly suppress plasma corticosterone levels in adult male Rana pipiens 24–48 hr postimplantation. Stress significantly elevated plasma corticosterone, and the stress response was inhibited by hypothalamic implants of both corticosterone and aldosterone.

In contrast to numerous other vertebrate groups in which there is biochemical evidence for aromatase activity in brain tissues, estrogen synthesis was not detectable in the CNS of a North American marsupial, the Virginia opossum. In the present study, the occurrence of aromatase activity in marsupial brain and other tissues was reinvestigated using the Australian species, Macropus eugenii and Trichosurus vulpecula . Following incubation of tissue homogenates with [7- 3 H]androstenedione and an NADH-NADPH generating system, estrogen products were isolated and finally identified by methylation and crystallization to constant specific activity. No estrogen was detectable in samples of adult brain; however, the brain of wallaby pouch young conformed to the general vertebrate pattern. Not only were small amounts of authentic estrone synthesized, but aromatization was greatest in limbic areas and activity per unit protein in all regions progressively declined with age. The ovaries of Macropus and Trichosurus as well as the adrenals of Macropus were also capable of converting labeled substrate to estrone or estradiol-17β. This study provides the first definitive evidence for synthesis of conventional eutherian estrogens in Metatheria.

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing.

The synthesis of steroids from 7α[3H]cholesterol and 7α[3H]pregnenolone by turtle ovarian tissues in vitro was studied. Pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione, testosterone, dehydroepiandrosterone, estrone, estradiol 17β, estriol, and 16-epiestriol were identified as products. All estrogens were detectable in incubates of preovulatory follicular tissue, but only small quantities of estrone were found in incubates of follicular tissue from postovulatory animals and luteal tissue. The effects of mammalian and avian gonadotropins on the metabolism of tritiated precursors were studied. Both mammalian and avian LH were stimulatory when conversion of cholesterol or pregnenolone to major steroid products was examined. In particular, enhancement of estrogen biosynthesis predominated in preovulatory follicular tissue, whereas increased progestin yield was the major effect in follicular and luteal tissue from postovulatory animals. The effects of FSH were minimal compared to the same dose of LH. Thus, a slight increase in estrogen yield was only noted when preovulatory follicular tissue was incubated with cholesterol and mammalian FSH, and neither mammalian nor avian FSH had an effect on pregnenolone conversion by follicles from postovulatory animals. Prolactin had no effect on luteal progesterone synthesis when used alone, but reduced the stimulatory effect of mammalian LH on progesterone synthesis. 11-Desoxycorticosterone was not found to be a product of the turtle ovary under normal conditions or after in vitro ACTH stimulation.

Cells obtained from turtle forebrain can be maintained in culture for at least 3 weeks. The cells are capable of aromatizing [3H]androstenedione and [3H]testosterone to estrone and estradiol and several C-19 metabolites. There are marked differences in the quality and quantity of the products formed from the two substrates. Conditions in living cells favor accumulation of 17-hydroxylated steroids. Aromatase activity as measured by estrogen yield increases with time in culture. Estrogen content of 14-day-old cultures may be enhanced or reduced by addition of natural or synthetic steroids. This system may provide a model for studying the regulation of brain aromatization, an essential step in the expression of androgen action on certain behavioral and neuroendocrine responses.

To date, biochemical evidence has been presented for hypophysial aromatization in only one species, a teleost fish, although the pituitary glands of several mammals have been reported to be aromatase negative. To reinvestigate this problem, established clonal strains of rodent pituitary cells (GH3, GH4C1, and AtT20/D16) were incubated at 37 C for 6-48 h in serum-less medium containing [7-3H]androstenedione. Radiolabeled metabolites were isolated by solvent extraction, thin layer chromatography, and phenolic partition. The authenticity of the estrogenic products in both cells and incubation medium was verified by methylation and recrystallization to constant specific activity. Measurement of androgen metabolites was also validated by recrystallization of selected samples. Authentic estrone and 17 beta-estradiol were identified in cultures of the two PRL- and GH-secreting clones, and there were strain differences in the quantity of estrogen produced (GH3 greater than GH4C1). Under the same conditions, aromatization was not detectable in the ACTH-secreting line (AtT20/D16). A time-yield analysis of androgen metabolism in GH4C1 cells showed that aromatization was linear for 12 h after labeling, but that substrate was diverted mainly to 5 alpha-reducing pathways. Large amounts of highly polar metabolites accumulated 24 and 48 h after the addition of [3H]androgen, and subsequent hydrolysis revealed that these were sulfo- and glucuronoconjugates. The metabolic fate of estrogen in GH4C1 cultures was investigated indirectly by adding a radioinert estrone trap together with the radiolabeled androgen substrate and was also tested in separate cultures by adding [3H]estrone and [3H]estradiol directly. Although the two estrogens were interconverted, there was no evidence that formed or added estrogen was extensively metabolized or conjugated. We conclude that the expression of aromatase activity in hypophysial cells is not a property of all transformed lines but may be dictated by the secretory cell type. Although low relative to other metabolites, estrogen yields in cultured GH cells resemble production in primary cultures derived from other tissues known to be estrogen targets, including the hypothalamus.

In Necturus maculosus the organization of the interstitial tissue varies according to the stage of spermatogenesis. Leydig cells at various stages of differentiation and myoid cells are always present in this tissue. The Leydig cells are undifferentiated at all phases of germ cell activity and only hypertrophy following spermiation and degeneration of Sertoli cells. These Leydig cells are structurally analogous to mammalian Leydig cells. They do not form part of the lamina propria of the seminiferous lobules and hence cannot be referred to as lobule-boundary cells previously described in the urodele testis (Lofts, '74). When the Leydig cells hypertrophy, numerous unmyelinated axons appear in the interstitial tissue. These axons, often devoid of Schwann-cell cytoplasm, occur in close proximity to Leydig cells. Because the levels of both Substance P and neurotensin increased in the testis of Necturus maculosus as Leydig cells differentiated, we concluded that these neural elements may regulate Leydig-cell function locally, through the release of neuropeptides.

The subcellular distribution patterns of aromatase, 5α- and 5β-reductase in the hypothalamus/preoptic area of Japanese quail were studied using standard methods of centrifugation, and fractional const

The ability to convert androgen to estrogen (aromatization) is a constant feature of gonadal and neural tissues in all major vertebrate groups. In experiments reported here, the existence of this pathway was investigated in the protochordate amphioxus ( Branchiostoma lanceolatum ). Following incubation with [ 3 H]19-hydroxyandrostenedione, gonadal homogenates contained authentic estrone and estradiol-17β, as determined by derivative formation and recrystallization to constant specific activity. Cephalic (“brain”) and other segments were aromatase negative. The results indicate that a potential for estrogen biosynthesis in the gonads predates that in other tissues and arises prior to the evolution of true vertebrates.

In the dogfish sharkSqualus acanthias different germ cell stages are topographically segregated within the testis. Using this species we have developed methods for the isolation and culture of Sertoli cells from premeiotic, meiotic and post-meiotic stages of spermatogenesis and present preliminary evidence for stage-dependent variations in cell morphology and behavior, thymidine incorporation, protein synthesis and steroidogenesis. The goal of future studies is to determine how maturational changes are regulated in Sertoli cells and, in turn, to elucidate Sertoli cell-germ cell interactions.

The present study tests the hypothesis that brain aromatase is an “ancient” property of nervous tissue and may be identified in homologues of the limbic system in a non-mammalian vertebrate, the turtle Chrysemys picta. Tissue homogenates(180 mg wet weight/2 ml) were incubated with [7α-3H]androstenedione and cofactors for 60 min at 37 C. Estrone (E1) was isolated and characterized by thin layer chromatography, methylation and recrystallization to constant specific activity. No estradiol-17β was detected. Aromatase was found only in the forebrain but was diffusely distributed throughout this major brain division. No other neuralor non-neural tissues, including mid- and hindbrain structures, testis, and ovary, synthesized detectable quantities of E1 in our system. The strio-amygdaloid complex of both sexes synthesized more E1 per unit weight than the preoptic-hypothalamic area (POA-HTH) or other forebrain structures. It is possible that the conversion of androgen to estrogen has biological significance in th...

The intratesticular site of estrogen biosynthesis is still somewhat controversial. Since the testis of the urodele amphibian Necturus maculosus has exceptionally high aromatase activity and is organized into a distinct glandular region that can be readily separated from another component made up mainly of seminiferous lobules, a study was undertaken to correlate function and structure of these two regions. When microsomal preparations were incubated with radiohabeled substrates, two key enzymes in estrogen biosynthesis, aromatase and 1 7a-hydroxylase, were found to be concentrated in the glandular tissue. Also, spectral measurements showed that eytochrome P450 speciesthatbind androstenedione and progesterone, respective steroidal substrates for aromataseand 17cs-hydroxylase, were distributed between the two zones in ratios similar to their steroidogenic activities. The relative yields of radioimmunoassayable estrogen and androgen from cultured cells derived from each zone was further evidence fot the higher steroidogenic capacity of the glandular tissue. Observations with the electron microscope indicate that active steroid synthesis in the glandular tissue could be attributed solely to numerous, highly differentiated Leydig cells, which are characterized by enormous volumes of smooth endoplasmic reticulum and mitochondria with tubular cristae. The presence of structural heterogeneities in the cell population in this region further suggests there may be functional differences among the various Leydig cell types. Although differentiating Leydig cells were seen surrounding the seminiferous lobules, they had little smooth endoplasmic reticulum. Based on structural features, it is improbable that differentiating Leydig cells, Sertoli cells, or elements other than differentiated Leydig cells in either the glandular or lobular tissues are steroidogenically active. The functional significance of testicular estrogen is still unknown. Because of the uniquely high Leydig cell aromatase aetivity, the specialized anatomy of the urodele testis, and an annual cycle in which temporal changes are protracted, we conclude that Necturus is a useful animal model for further investigation of this problem.

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.

Interrenal function was studied in the freshwater turtle Chrysemys picta by competitive protein-binding analysis of plasma corticosterone in vivo and corticosterone synthesis by interrenal cell suspensions in vitro. Although circulating levels of corticosterone in intact, unanesthetized turtles (0.22 μg/100 ml) are lower than in other vertebrates, hypophysectomy reduced and ACTH increased these levels. Synthetic ACTH1–24 was more potent in vivo than pACTH, and in vitro 10–1000 pg/ml gave a log dose increase in corticosterone production. Interrenal cell suspensions were also responsive to crude homogenates of turtle pituitary. Bleeding was a potent stimulus to interrenal secretion in vivo mediated in part via endogenous corticotropin release.

Aromatization and 5 alpha-reduction are known to be required for the full expression of testosterone actions in neuroendocrine tissues. Although aromatase and 5 alpha-reductase activities in brain and pituitary can be experimentally manipulated by castration and steroid replacement, naturally occurring variations during seasonal reproductive cycles have not been examined in any species. Goldfish (Carassius auratus) were selected for study because they exhibit exceptionally high levels of aromatase in both brain and pituitary, although 5 alpha-reductase levels resemble the vertebrate norm. Four animals of each sex were tested monthly through three breeding seasons (2.5 yr). Using previously validated techniques, the enzymes were assayed by product formation from [3H]androstenedione in homogenates of anterior hypothalamus-preoptic area (AHPOA), remaining telencephalon (TEL), whole pituitary, ovary, and testis. Seasonal variations in aromatase were most dramatic in the AHPOA of female fish, exhibiting a peak in April and May that was 6-fold higher than the nadir in July. As judged by changes in the appearance and weight of the gonads, maximal aromatase coincided with the spawning season, whereas low enzyme levels corresponded to reproductive inactivity. Seasonal variations were similar but of a lesser magnitude in the TEL of females and in the AHPOA and TEL of males (2- to 3-fold, peak to nadir). Both ovarian and testicular aromatase showed cyclic changes; however, activity was much lower than that in brain at all times of the year (4.5, 1.2, and 47.0 pmol/mg protein, maximal values in ovaries, testes, and AHPOA, respectively). Pituitary aromatase varied from 5-22 pmol/mg protein, but was not consistently correlated with season. Cyclic changes in 5 alpha-reductase were distinctly different from those in aromatase, with maximal values in both brain and pituitary occurring when fish were reproductively inactive. In general, circulating sex steroids were high when aromatase was high and low when reductase was maximal; however, there was no apparent causal relationship suggested by temporal changes in a given steroid. Variations in testosterone metabolism, by regulating the quantity and quality of active hormone in close proximity to receptor sites, may be responsible for the changes in feedback sensitivity and behavioral responsiveness that are known to occur in seasonal breeders.

The full expression of testosterone (T) actions in neuroendocrine tissues requires aromatization and 5α-reduction to estradiol (E2) and 5α-dihydrotestosterone (DHT), respectively. Recently, we documented striking changes in aromatase and 5α-reductase during the annual reproductive cycle of goldfish ( Carassius auratus ). To investigate possible regulatory effects of sex steroids, goldfish were implanted with hormone-filled silastic capsules for 2–5 weeks. Conversion of [ 3 H]androstenedione to estrone or 5α-androstanedione by homogenates of anterior hypothalamus/preoptic area, remaining telencephalon, and whole pituitary (PIT) was used to estimate aromatase and 5α-reductase, respectively. Gonadosomatic index and plasma E2, T, and DHT were monitored as an index of reproductive status and capsule effectiveness. In reproductively inactive fish in which plasma steroids and aromatase were basal (October), E2 or T increased aromatase activity in brain of both sexes but stimulated activity in PIT of females only; DHT was not effective. In a subsequent experiment initiated close to the spawning peak and prior to the seasonal decline in plasma steroids and brain aromatase (April), T increased or maintained brain aromatase in a time-dependent manner. 5α-Reductase activity was unaffected by steroid treatment in both reproductively active and inactive fish. These results indicate that variations in circulating steroids are responsible, at least in part, for changes in brain aromatase during the annual reproductive cycle of goldfish and provide the first evidence for steroid control of pituitary aromatase. The steroid specificity of the induction suggests that an estrogen receptor mechanism is involved. Product (E2)-induced increases in aromatization during the spawning period may be part of a positive feedback loop leading to neural E2 accumulation and culminating in ovulation/spermiation and breeding behavior.

When added to primary cultures of adult turtle (Chrysemys) brain, dibutyryl cyclic AMP increases estrogen yields from [3H]testosterone in a dose- and time-dependent manner, although rat FSH, rat LH, and dibutyryl cyclic GMP are relatively ineffective. A phosphodiesterase inhibitor (MIX) by itself increases estrogen yields from the substrate and potentiates the response to dibutyryl cyclic AMP.

The urodele amphibian Necturus maculosus has a zoned testis, which is advantageous for separating Leydig cells from germinal elements and for studying stage-dependent biochemical changes. Using [3H]testosterone (T) in a standard binding assay and dextran-coated charcoal (DCC) or Sephadex LH-20 to separate free and bound steroids, we identified an androgen-binding protein (ABP) in Necturus testis cytosols. This protein was of high affinity (Kd = 10(-9) M) and was saturable (Bmax = 10(-9) M) and specific for androgen (T; 5 alpha-dihydrotestosterone, DHT) but could be distinguished from the androgen receptor of Necturus testis by its relative abundance (300-550 fmol/mg protein), short half-time of dissociation (3 min at 22 degrees C), inability to adhere to DNA-cellulose, and absence from nuclear extracts. Additionally, when analyzed on sucrose gradients, the ABP of Necturus testis sedimented at 6-7 S in both low or high ionic strength buffers. In that estradiol (E2) is a poor competitor for T-binding, this protein resembles a sex steroid-binding protein previously identified in urodele serum but differs from the ABP and testosterone-estradiol-binding globulin (TEBG) of rodents, humans, goldfish, and sharks. It is differentially distributed within the testis, with the highest levels in immature lobular regions composed of Sertoli cells and germ cells in premeiotic stages and lower levels in regions composed primarily of Leydig cells. The cellular source and function of this protein in Necturus testis remain to be determined.

By investigating steroid conversions in the bullfrog Rana catesbeiana, the present study tests the hypothesis that androgen metabolism by the central nervous system (CNS) is a primitive vertebrate characteristic. Parts of the brain containing the amygdala, preoptic area, and hypothalamus (AMY/HTH), remaining forebrain (RFB), midbrain and hindbrain (MB/HB), and also the ovary, testis, muscle, and fat were homogenized and incubated with [7α-3H]androstenedione and cofactors for 60 min at 25°. Products were isolated and characterized by thin-layer chromatography and recrystallized to constant specific activity; estrone (E1) derived from brain incubates was also methylated. E1, testosterone (T), and 5α-dihydrotestosterone (DHT) were the metabolic products in brain incubates. In addition to E1, ovarian homogenates synthesized estradiol-17β (E2-17β) and estradiol-17α (E2-17α), but no aromatase activity was detectable in the testis, fat, or muscle. T was the primary metabolite in the testis. Radioimmunoassayable T, DHT, E1, and E2 were present in the plasma of male and female bullfrogs. Concentration of aromatase in the HTH/AMY of the bullfrog is consistent with a similar distribution of enzyme activity in the limbic system or its homologs in a reptile and mammals. Centers for the control of reproduction and cells which bind sex steroids have been identified in the same brain areas in other anuran species. The experiments reported here indicate that the conversion of androgen to estrogen and other neutral metabolites by the brain is a primitive tetrapod characteristic and suggest that metabolism is an integral component of brain-steroid interactions which has been conserved during the evolution of vertebrates.

An investigation was carried out to determine whether isolated interrenal cells of the turtle could be used as a biossay for reptilian cortocotropin. The action of synthetic ACTH, ACTH analogs, and naturally occurring corticotropins of four vertebrate classes were compared. A typical sigmoid log dose response (LDR) curve was elicited with 1–10 6 pg/ml of ACTH 1–24 and pACTH (3rd IWS), and crude extracts of homologous chelonian or avian adenohypophyses gave similar responses. Potency in ACTH 1–24 equivalents was as follows: pACTH, 10 pg/μU; chelonian pituitary, 80 pg/μg (wet wt); avian pituitary, 160 pg/μg (wet wt). Corticotropic activity was present in Rana pituitary, but the maximum response was half that obtained with the other corticotropins and relative potency could not be estimated. Molar potency of ACTH analogs tested on isolated turtle cells decreased in the following order: ACTH 1–24 > ACTH 1–16 > ACTH 1–13 ∼ ACTH 7–13 > β-MSH > ACTH 4–10 . The maximum response obtained with ACTH 1–16 was 80% of that with ACTH 1–24 , but insufficient quantities of other analogs were available for complete LDR curves. Molar potency comparisons suggest that, in the reptile, amino acids 11–24 but not 1–6 are important for receptor binding. Further, the portion of the molecule essential for maximal receptor activation spans amino acids 1–16 and 17–24. The intrinsic activities of mammalian, avian, and reptilian corticotropin are approximately equivalent but twice that of amphibian corticotropin in a reptilian interrenal system.

The successful dispersion of the recent vertebrates has depended ultimately upon the adaptation of their reproductive cycles to the environments in which they evolved. Since almost all habitable environments show some degree of fluctuation, adaptation to these environments has necessitated some control of the reproductive cycle to produce offspring at the time of the year most advantageous to their survival. Such control of the vertebrate reproductive cycle requires that the organism be able to sense changes in the environment and modify its physiology accordingly. This has been accomplished through modulation of the neuroendocrine system, which is basically the same from fish to mammals with only minor variations. It is likely that the ancestral group from which the vertebrates arose already possessed a pituitary gland (Barrington, 1968), but there is no way of knowing whether or not this ancestral pituitary had any control over the reproductive processes, as there are no protochordates with a recognizable pituitary. While there is some evidence of a relationship between the neural complex and the gonads of ascidians (Georges, 1976), attempts to trace the antecedents of the vertebrate pituitary to this specialized structure in tunicates (Berrill, 1955) or the wheel organ and Hatschek’s pit of Amphioxus (Barrington, 1964) have been singularly unsuccessful (reviews by Dodd, 1955; Charniaux-Cotton and Kleinholz, 1964; Bern and Nandi, 1964; Barrington, 1968; Olssen

Aromatase has been identified in the teleostean, avian, and mammalian pituitaries, although its cellular location(s) is not yet certain. To address this question, experiments were performed in tilapia (Oreochromis mossambicus), a species which has been well characterized with respect to the intraglandular distribution of the different pituitary cell types. To estimate aromatase, glands were microdissected into rostral pars distalis (RPD), proximal pars distalis (PPD), and neurointermediate lobe (NIL) and organs were cultured in the presence of [3H]androstenedione for 16–24 hr. [3H]Estrogen products were isolated and quantified after ether extraction, hydrolysis with glucuronidase-sulfatase, thin-layer chromatography, and phenolic partition. Authentic estrone or estradiol-17β were produced by all pituitary regions and also by the urophyseal region of the spinal cord. Aromatase was two to five times higher in PPD than in RPD or NIL and similar to activity in adjacent hypothalamus-preoptic area (HPOA). Much lower estrogen yields were obtained in cultures of cerebellum, urophysis, and other cord regions. Since the PPD contains most of the somatotropes, these data are consistent with earlier studies implicating GH3/GH4 cell strains as an enriched enzyme source, although its presence in other cell types cannot be ruled out. The unusually high and localized aromatase in tilapia pituitary renders this species a useful model for studying the targets and functional importance of estrogen as a parahormone in the pituitary.

Exposure of hamsters to short days increases sensitivity to the negative feedback effects of testosterone (T) but decreases responsiveness to the behavioral effects of the hormone. Since T is metabolized in the brain to 5 alpha-dihydrotestosterone (DHT) and estradiol, which differentially affect gonadotropin secretion and sex behavior, it is reasonable to postulate that daylength can modulate neural responses by quantitative or qualitative alterations in T metabolism and subsequent receptor binding of active hormone. Experiments reported here focused on aromatization and the nuclear accumulation of estrogen receptors. Adult male hamsters were maintained for 6-12 wk in long (14:10 LD) or short (8:16 LD) daily photoperiods. Both intact and castrated animals were used to assess direct effects of short days versus changes due to short-day-induced testicular regression. Discretely dissected regions of the brain (preoptic area, POA; hypothalamus, HTH; and corticomedial amygdala, CMA) or limbic blocks (LIM) comprised of all three regions were assayed for estrogen-synthesizing activity (aromatase) and estrogen-binding activity (receptors). Aromatase was estimated in vitro by conversion of [7-(3)H] androstenedione to [3H] estrogen and in vivo by measuring increases in nuclear estrogen receptor levels after injection of aromatizable androgen. Receptor-binding activity was assayed in crude cytosolic and nuclear extracts by incubating samples with [3H] estradiol +/- 100-fold excess inert estradiol, and separating free and bound steroids by Sephadex LH-20 gel filtration. When aromatase was assayed in homogenates prepared from discrete brain regions of individual hamsters, significantly lower activity was found in the HTH of short-day animals than in long-day controls. This effect was seen in both intact and castrated animals, which indicates that it was not mediated by the testis. Decreased enzyme activity in the POA and CMA of short-day hamsters was not significant, nor was there an effect of castration independent of short days. Low levels of nuclear estrogen receptors were present in LIM of intact males, but these were reduced after castration or concomitant with testicular regression after short-day exposure. This suggests that the hamster testis normally secretes estrogen or aromatizable androgen. A single injection of estradiol or aromatizable androgen (T or androstenedione) increased nuclear receptors in LIM of castrated animals. Cytosolic receptors were not different in short-day vs. long-day hamsters, nor were there differences in nuclear receptor levels after a single estradiol injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Aromatization and 5α-reduction appear to be essential for the expression of certain androgen actions in the central nervous system. Although 5α-reductase activity occurs in neural tissues of invertebrates and all major vertebrate groups, brain aromatase has not been traced below elasmobranch. Using the lamprey ( Petromyzon marinus ), androgen metabolism was investigated in brain, gonad, and other tissues by incubating homogenates with [ 3 H]androstenedione and cofactors. After extraction, chromatography, and phenolic partition, products were identified by dilution with authentic unlabeled steroid and recrystallization to constant specific activity. Estrone was formed in ovary, testis, and kidney but not in liver, muscle, or tissue-free control incubates. Testicular samples also synthesized estradiol-17β. Identification of estrogen in brain incubates was not definitive. All tissues except muscle had 5α-reductase activity as shown by accumulation of 5α-androstandione. No 5α-dihydrotestosterone or estradiol-17α were identified in any samples. The presence of 5α-reductase in cyclostome brain is consistent with the widespread phylogenetic distribution of this enzyme although the occurrence of central aromatase activity in Agnatha has yet to be demonstrated.

The distribution of aromatase and 5 alpha-reductase was investigated in the brain, spinal cord, and pituitary of adult goldfish (Carassius auratus) and toadfish (Opsanus tau) of both sexes. Tissue homogenates were incubated with [3H]androstenedione in the presence of an NADPH-generating system and, following validation of assay conditions, radiolabeled products (estradiol, estrone, 5 alpha-androstanedione), were measured as an index of enzyme activity. Neuroendocrine tissue of both species produced exceptionally large amounts of estrogen, thus confirming previously observed differences between teleosts and other vertebrates. By contrast, 5 alpha-reductase levels resembled the vertebrate norm. In general, aromatase was concentrated in the pituitary and various forebrain regions, especially the hypothalamus/preoptic area; however, estrogen yields from the medulla and anterior spinal cord of toadfish were high compared to adjacent midbrain, hindbrain, and cord regions. This same neural region in toadfish, but not in goldfish, is known to control a sex dimorphic behavior, the courtship boatwhistle. In contrast to aromatase, 5 alpha-reductase was more uniformly distributed throughout the brain, although somewhat higher activity was obtained in the pituitary. High levels of aromatase in the neuroendocrine tissues of teleosts recommend them as animal models for further studying the enzyme, its regulation, and its role in governing androgen-dependent responses in central targets.

In the spiny dogfish (Squalus acanthias), germ cells and adjacent steroidogenic elements are topographically segregated within the testis according to stage of development. In the experiments reported here, we have taken advantage of this favorable anatomical arrangement to demonstrate stage-specific variations in steroidogenic enzymes and estrogen receptor number. The testes of Squalus collected in July-November were sectioned transversely and further subdivided into three zones as follows: I) germinal bed plus immature lobules with spermatogonia; II) lobules with primary or secondary spermatocytes; III) lobules with spermatids or mature spermatozoa. The morphology of these zones was verified by light microscopy and, in a separate study, by electron microscopy. Through the course of spermatogenesis, Sertoli cells increased dramatically in size and in the abundance of steroidogenic organelles. By contrast, interstitial tissue was sparse in all stages of development, and only relatively undifferentiated Leydig-like cells were present. Microsomes prepared from each zone were incubated with [3H] progesterone, [3H]17 alpha-hydroxyprogesterone, or [3H]androstenedione to evaluate androgen and estrogen biosynthetic potentials. Based on product formation, 17 alpha-hydroxylase and C-17,20-lyase activities increased progressively from less mature to more mature regions (zone III greater than II greater than I), whereas aromatase was greatest in regions undergoing meiosis or early spermiogenesis (zone II). These enzymes were not detected in semen, although C21 substrates were converted to unidentified polar metabolites in high yield. Estrogen receptors were concentrated in immature zones (zone I greater than II greater than III), and the percentage of occupied receptors revealed the same distribution. Semen and epigonal tissues were receptor negative. We conclude that Sertoli cells are responsible for steroidogenesis in Squalus testis and that hormone production is keyed to the spermatogenic cycle. The data are consistent with an important role for androgens during spermatid maturation and/or after sperm release, but reveal that estrogen actions are expressed primarily during early spermatogenic stages. This report demonstrates the usefulness of unconventional animal models for obtaining new information of general relevance.

In all vertebrates gonadal secretions, through their actions on the central nervous system (CNS), control sex and sex-related behaviors and also regulate production of gonadotropin. Recently, major advances have been made in characterizing the molecular events which underlie these processes. Discussed in this paper are the nature and source of androgens and estrogens; their sites of action within the CNS as determined by steroid implant and autoradiographic studies; some manifestations of brain-steroid interactions, both “triggering” and “organizational”; evidence supporting a conventional genomic mechanism versus alternative modes of action; the critical role of androgen metabolizing enzymes in regulating the quality and quantity of active hormone in close proximity to targets; and some determinants of steroid access to the CNS. Information from nonmammalian vertebrates is reviewed in relation to the major body of knowledge derived from common laboratory species. The intent of this paper is to provide a biological perspective and to stimulate interest in the use of unconventional animal models for future studies.

During the reproductive cycle of the little brown bat (Myotis lucifugus), the display of sexual behavior is out of synchrony with maximal gonadal development, suggesting that changes in plasma hormone levels per se are not the primary determinants of neural responsiveness. In the present study, we investigated pathways of androgen metabolism in the brain of the bat and compared brain aromatase activity in spring and in autumn. Tissue homogenates were incubated with [3H] androstenedione in the presence of an NADH/NADPH-generating system, and radiolabeled products isolated and their authenticity verified by recrystallization to constant specific activity. Aromatase was identified in preoptic/hypothalamic and adjacent limbic areas, and 5 alpha-reductase was detected in all major brain divisions. No gender-based differences in brain aromatase were seen in spring just prior to emergence from hibernation; however, activity was greater in males than in females when measured during maximal breeding activity in autumn. In addition, seasonal changes were evident in the preoptic/hypothalamic regions of both sexes (spring greater than autumn), although estrone yields from limbic tissues remained constant. Thus, in seasonally breeding species, the rate at which circulating androgen is converted to estrogen or other biologically active metabolites in the brain itself may be an important determinant of behavioral or feedback responsiveness.

Hypothalamic implants of corticosterone and aldosterone were equally effective in suppressing plasma corticosterone level in the desert iguana, Dipsosaurus dorsalis , 96 hr after implantation.

Plasma testosterone was measured at stages during the reproductive cycle of control male turtles ( Chrysemys picta ) and following hypophysectomy and/or mammalian FSH or LH injection. Testosterone levels were highest in April and December when large Leydig cells were observed in the interstitium but the epithelium was inactive. During active spermatogenesis (July, October) plasma steroid levels were basal, and not significantly reduced by hypophysectomy. While LH injections had no effect, FSH was a potent stimulus to plasma testosterone in both intact and hypophysectomized turtles. Histologic observations during the testicular cycle and after hypophysectomy indicated that the tubular epithelium was dependent on endogenous pituitary hormone secretion but the interstitium did not atrophy during the experimental period. Injections of FSH in May increased testicular weight and caused disappearance of vacuoles and granules from apical portions of the Sertoli cell with a consequent reduction in cell height. Neither FSH nor LH had obvious effects on the morphology of the interstitium in control or hypophysectomized turtles either in July or November.

SummaryOvarian cysts were experimentally induced by injecting hypothyroid rats with human chorionic gonadotropin. Cystic ovarian tissue synthesized in vitro a large quantity of 20α-hydroxypregn-4-en-3-one but estrogen synthesis was low.

SummaryA comparison of 20α-hydroxypregn-4-en-3-one and corticosterone reveals that their fluorescence properties are similar in concentrated sulphuric acid. Since rodent ovaries secrete 20α-hydroxypregn-4-en-3-one, this steroid may interfere with the assay of corticosterone by fluorimetric techniques; however, interference may be reduced by the use of a petroleum ether wash. It is suggested that a fluorescence method may be used as a sensitive assay for 20α-hydroxypregn-4-en-3-one.

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.

In an earlier study, we identified and characterized a testicular estrogen receptor in the spiny dogfish (Squalus acanthias); however, in this species estrogen receptors were located exclusively in nuclear subfractions. We describe here a nonreceptor, sex hormone-binding protein (SBP) present in Squalus testicular cytosol and distinguishable in its physicochemical characteristics from both the estrogen receptor and serum SBP of the same species. Cytosol (100,000g supernatant) was prepared by differential centrifugation of testicular homogenates and incubated with [3H]estradiol (E2) or [3H]testosterone (T) (10-15 nM +/- 100-fold excess radioinert competitor) to determine total and nonspecific bound radioactivity. The testicular SBP had a broad specificity: E2 = T greater than progesterone greater than 5 alpha-dihydrotestosterone greater than estrone, but diethylstilbestrol was not an effective competitor. It displayed a high affinity for both E2 and T (Kd = 2.2-2.5 X 10(-9) M), sedimented at 8-10 S in both low- and high-salt sucrose gradients, and migrated more slowly than BSA during polyacrylamide disc gel electrophoresis. The testicular SBP-E2 complex was relatively stable (t1/2 = 160 min) compared to the serum SBP-E2 complex (t1/2 = less than 30 min). The testicular SBP was not found in nuclear subfractions nor did it bind to DNA-cellulose affinity columns. Its intratesticular distribution was stage-dependent: Zone III (mature spermatids) greater than Zone II (spermatocytes) greater than Zone I (stem cells and spermatogonia). Moreover, increased binding activity corresponded exactly to the hypertrophy and differentiation of Sertoli cells in the same zones, pointing to this cell as the possible site of SBP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Where aromatase and estrogen receptors are co-localized in brain and pituitary, estrogen functions as a parahormone, and estrogen levels which determine the occurrence or magnitude of a response are those in close proximity to targets. Teleost fish, a vertebrate group characterized by exceptionally high aromatase in neuroendocrine tissues, are technically advantageous animal models for studying the cellular location of aromatase, natural changes correlated with seasonal reproductive cycles, substrate-dependence of the reaction, steroid induction of enzyme activity, and possible non-genomic actions of estrogen on cultured neurons. In addition, characterization of steroid receptors reveals that the androgen receptor, like aromatase, is present in unusually high concentrations (10- to 100-fold higher than in mammalian brain). Since androgen receptors and aromatase both utilize testosterone as a ligand, their high abundance in teleost brain may be the consequence of a functional interdependence during evolution, although the primary causal factor is unknown. These studies illustrate the usefulness of unconventional species and a comparative approach for obtaining new insights into brain-steroid interactions.

Steroid hormones are primary regulators in many systems. Estrogens, androgens and progestogens regulate reproduction and attendant functions; corticosteroids control protein, water and ion balance; and the secosteroids are responsible for calcium homeostasis. Reviews of interest to comparative endocrinologists have described the physiological aspects of steroid actions in each of these subject areas.30,51,62,82,100a Although this paper is intended primarily to review sex steroid receptors in subavian vertebrates, reference is made to birds and mammals for purposes of orientation.

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.

The site of hormone synthesis in the testis of elasmobranchs has been the subject of much controversy. This is primarily due to the problem of whether Leydig cells are present or absent in the testes of many species of elasmobranchs. In previous studies we have shown that key enzymes associated with the biosynthesis of androgen increase in activity during the spermatogenetic cycle of Squalus acanthias (Canick et al., 1983). To determine the site of this activity we undertook an electron microscope study of Squalus testes to identify cells that possessed the structural correlates of steroid production. This report describes cells present in the interstitial tissue that are morphologically analogous to Leydig cells occurring in the testes of higher vertebrates. Although these cells possessed an agranular reticulum, tubulovesicular mitochondria, and lipid droplets, they were mesenchymal in appearance. We have, therefore, preferred to describe these cells as Leydig-like.

We previously reported that substantial quantities of estrogen were present in the pituitary of the marine teleost (Myoxocephalus) following perfusion of the isolated head with radiolabeled androgen. In order to determine whether this estrogen was synthesized in situ or was derived from that formed in brain areas with high levels of aromatase activity, we used an explant culture system. Pituitary and brain (preoptic area/anterior hypothalamus) were incubated separately for 20 hr with [3H]androstenedione. Alternatively, the two tissues were cultured together, either as an intact unit or after stalk section. [3H]Estrone and estradiol-17β were measured in the media and tissue fragments after ether extraction, thinlayer chromatography, and phenolic partition. The authenticity of the products was validated by reverse isotope dilution and recrystallization. Sculpin brain and pituitary in isolated cultures were independently capable of converting radiolabeled androgen to estrogen (estradiol > estrone). Aromatase activity, as indicated by estrogen production per unit wet weight or per unit protein, was greater in pituitary than in brain cultures. Both tissue types, when compared to the medium, retained estrogen against a concentration gradient. Culturing the brain and pituitary together, with or without stalk section, did not alter total estrogen yields or tissue concentration of formed estrogen. It is unlikely that aromatase activity in isolated pituitary cultures could be ascribed to fiber terminals originating in the hypothalamus; however, the cell type responsible for estrogen production remains to be determined. We conclude that, in this teleost, a significant portion of the estrogen reaching target cells in the pituitary may be synthesized in situ from circulating androgen. Hence, control of the aromatization reaction would be one means of regulating gonadotropin secretion. A reexamination of aromatase activity in the pituitary of mammals and other vertebrates is warranted.

Testing procedures which rely on paired fights are unsuitable for measuring individual aggressiveness because the outcome of initial encounters influences subsequent dominance status. In an effort to circumvent this problem, we developed a test system which quantifies aggressiveness in individual Japanese quail by measuring locomotor and pecking activity of test birds presented with the visual stimulus of a conspecific. Using this procedure, we demonstrate that aggression is specific to sexually mature males; however, the intensity of the behavior varies widely among individuals. Scores for each bird are constant with time, reproducible, independent of the gender or behavior of the stimulus bird and accurately predict the outcome of a subsequent paired fight. In addition, we show that fighting per se produces increases and decreases in aggressiveness of dominant and subordinate birds, respectively, and re-orders the rank of a group of birds. This test procedure, therefore, confirms earlier studies which indicate that dominance hierarchies are influenced by initial encounters. Finally, repeat testing is reliable and gives an absolute, not relative, measure of aggressiveness. We conclude that this behavioral test can be applied to future studies addressing the social, developmental and endocrine basis of aggressiveness and dominance in quail and other vertebrates.

Research into the evolution of gonadotropin molecules and the phylogenetic development of gonadal responses to these hormones has been hampered by the absence of a direct and quantitative test for steroidogenesis in lower vertebrates. We report here the development of a sensitive and responsive assay for gonadotropins using enzyme-dispersed testicular cells derived from a turtle ( Chrysemys picta ). Production of androgens from endogenous precursors was measured by radioimmunoassay. Cell suspensions obtained by collagenase treatment of whole testis synthesized testosterone in response to homologous and heterologous gonadotropins. All mammalian FSH's tested (ovine, rat, human) and PMS elicited dose-related increases in androgen synthesis. LH's and hCG were either ineffective or more than 100 times less potent than corresponding FSH's. Avian LH (avLH) and crude turtle pituitary (tPIT) were also active in the turtle cell system. Antibody to avLH partially neutralized responses to rat FSH, avLH, and tPIT indicating a degree of molecular correspondence among these three hormones. Although seasonal differences in gonadotropin responsiveness (maximal androgen production) were observed, it is not clear from these experiments whether Sertoli or Leydig cells are the source of secreted androgen.

Although aromatase activity is exceptionally high in the teleost pituitary, it is not known which of the secretory cell types are responsible. Pituitary glands from the longhorn sculpin ( Myoxocephalus octodecimspinosus ) were sectioned transversely into “cephalic” and “caudal” fragments and cultured for 24 hr in medium containing [7- 3 H]androstenedione. Radiolabeled estrone and estradiol-17β production were measured as an estimate of aromatization. In order to determine the distribution pattern of different cell types, the in situ pituitary and dissected fragments were analyzed by standard cytological procedures. Further verification of cell function was obtained by somatostatin (SRIF) and corticotropin-releasing factor (CRF) immunocytochemistry. Estrogen yields obtained from caudal fragments in two separate experiments averaged four times higher per milligram protein than yields from matched cephalic fragments. In addition, female glands synthesized significantly more estrogen than those of males. Due to an anteroflexion of the longitudinal axis and a disposition of the gonadotropic (GTH) cells at the periphery of the gland and surrounding the neurointermediate lobe (NIL), the classical subdivisions of the teleost adenohypophysis were not strictly applicable to the sculpin. The predominance of growth hormone (GH) secreting cells in the caudal fragment suggests their participation in aromatization, a finding which is consistent with a previous study of rodent pituitary; however, a role for gonadotropes and other hypophysial cells in this transformation cannot be ruled out.

1. 1. Highly specific antisera for 11-keto- and 11β-hydroxytestosterone have been raised in sheep. 2. 2. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11β-hydroxytestosterone, testosterone, progesterone and estradiol-17β were validated for Ictalurus nebulosus plasma and Carassius auratus serum. 3. 3. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17β were the predominant steroids measured. 4. 4. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in l. nebulosus and C. auratus.

Testosterone (T) exerts its actions in brain directly via androgen receptors or, after aromatization to estradiol, via estrogen receptors. Brain aromatase activity in teleost fish is 100-1000 times greater than in mammals and would be expected to significantly reduce the quantity of androgen available for receptor binding. Experiments were carried out on the goldfish Carassius auratus to determine if androgen receptors are present in teleost brain and whether their physicochemical properties reflect elevated aromatase. Cytosolic and nuclear extracts were assayed with the use of [3H]T and charcoal, Sephadex LH-20, or DNA-cellulose chromatography to separate bound and free steroids. Binding activity was saturable and had an equally high affinity for T and 5 alpha-dihydrotestosterone (Kd, approximately 2.4 X 10(-9) M). Although mibolerone was a relatively weak competitor, the putative teleost androgen 11-ketotestosterone, methyltrienolone (R1881), estradiol, progesterone, and cortisol were poor ligands. Characteristics that distinguish this receptor from a steroid-binding protein in goldfish serum are the presence of binding activity in both nuclear and cytosolic extracts, a low rate of ligand-receptor dissociation, electrophoretic mobility, sedimentation properties in low vs. high salt, and tissue distribution (forebrain greater than or equal to pituitary greater than mid-/hindbrain). DNA cellulose-adhering and nonadhering forms were detected, but these did not differ in other variables measured. Although goldfish androgen receptors resembled those of mammals in all important physicochemical characteristics, they were unusually abundant (5-68 pmol/g tissue) compared to levels in rat brain, but comparable to levels in prostate and other male sex hormone target organs. Moreover, there were seasonal variations in total receptors, with a peak at spawning (April) 4- to 5-fold higher than values in reproductively inactive fish (July/August). This temporal pattern and magnitude of change corresponded to previously reported changes in brain aromatase. Thus, both phylogenetic and physiological correlates point to a functional interdependence between androgen receptors and aromatase in the brain. These studies in goldfish indicate that brain androgen receptors have a long evolutionary history and have been highly conserved through the vertebrate series.

Publisher Summary This chapter discusses the morphology, histology, seasonal changes, and physiology of adrenal gland in reptilia. The embryonic origins of the reptilian adrenal gland are as in other vertebrate groups; the cortical tissue is derived from a proliferation of cells from the dorsal peritoneal epithelium lining the embryonic coelom, whereas the chromaffin tissue originates from the anlagen of the prevertebral sympathetic plexuses. Unlike the amphibians, where the adrenal tissue has the form of a discrete or sometimes scattered tissue lying on the mesonephros and often embedded in the kidney tissue, the adrenal glands of reptiles are usually paired, elongated and encapsulated bodies closely associated with the main blood vessels, often adjoining the gonads. They, therefore, more closely resemble the homoiothermic vertebrates, rather than the other poikilotherms. The adrenal gland in Reptilia is completely enclosed in a thin connective tissue capsule consisting of collagenous, reticular, and elastic fibers surrounding the inner mass of cells. The latter are organized into a loosely connected network of cortical cords, which are generally orientated, as in Amphibia, longitudinally along the length of the gland. Unlike the amphibians, however, pronounced islets of chromaffin tissue are intermixed with the cortical cords similar to the avian pattern. The chromaffin and cortical tissue vary considerably, both in their relative proportions, and in their pattern of distribution throughout the gland.

Spermatogenesis is a unique developmental sequence dependent on FSH and androgen. Due to the complex organization of the mammalian testis, however, mechanistic details of regulation are largely unknown. Using the dogfish shark (Squalus acanthias) in which there is a cystic mode of spermatogenesis and a topographic separation of different germ cell stages within the testis, we have obtained new information of general relevance on stage-related biochemical and morphological changes and have proposed a model in which steroids serve as parahormonal regulators of the spermatogenic progression. In addition, techniques developed for culturing staged spermatocysts (intact Sertoli/germ cell units) and isolated, staged Sertoli cells demonstrate the usefulness of this model for studying spermatogenic regulation under defined conditions in vitro.