Evolution of the reproductive endocrine system in chordates

The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the presence an endocrine system comparable to the pituitary-gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have any pituitary hormone genes except the thyrostimulin gene. Thyrostimulin is a heterodimeric glycoprotein hormone consisting of α and β subunits, and is present in various organs of vertebrates. Analyses of a phylogenetic tree and a synteny suggest that amphioxus' thyrostimulin is an ancestral type of the glycoprotein hormones in chordates. In addition, genes for sex steroidogenic enzymes belonging to the CYP family were found in the genome sequences. The conversion pathway of sex steroids from cholesterol to estrogen, androgen, and major sex steroids was also identified in the gonads of amphioxus in vitro. Furthermore, we demonstrated the expression of genes encoding thyrostimulin and sex steroidogenic enzymes by an in situ hybridization technique. Here, we discuss the evolution of hormones and reproductive functions in the neuroendocrine control system of chordates.     

Regulation of 17β-hydroxysteroid dehydrogenases in cancer: regulating steroid receptor at pre-receptor stage

Increased intracelullar hormone concentration levels have been shown to be the cause of several endocrine-related cancers including breast, prostate, endometrial, ovarian, cervix, testicular, thyroid, and osteosarcoma. Deregulated expression of steroidogenic enzymes in these tumors seems to be the source of a positive balance in active steroids that bind to the corresponding nuclear receptor, thus ultimately stimulating cell proliferation. Among these enzymes, 17β-hydroxysteroid dehydrogenases catalyze the interconversion between 17-ketosteroids and 17-hydroxysteroids on the last steps of sex hormones biosynthesis and metabolism. To date, 14 isoforms have been identified in vertebrates although only 13 are present in humans. Development and clinical evaluation of specific inhibitors to block their activity is currently under progress especially against the best characterized members 1 to 5. Selectivity and potency of these drugs constitute the main challenge in this new approach to cancer and steroid-dependent diseases treatment at the "pre-receptor level". Here we review the current state of knowledge regarding the deregulation of the expression of some of these enzymes in endocrine-related tumors.     

Endocrine mediation of vertebrate male alternative reproductive tactics: the next generation of studies

In many species of animals, males may achieve reproductive successvia one of several alternative reproductive tactics. Over thepast decade or so, there has been a concerted effort to investigateendocrine mechanisms that underlie such discrete behavioral(and often morphological) variation. In vertebrates, the firstgeneration of studies focused on potential organizational oractivational effects of steroid hormones (Moore, 1991; Mooreet al., 1998). Some of these studies have made it clear that,in addition to circulating hormone levels, one must also considerother aspects of the endocrine system, including hormone receptors,binding globulins and potential interactions among endocrineaxes. In this paper, I review recent work on endocrine mechanismsand suggest possibilities for future investigation. I highlighthow individual variation in sensitivity to environmental conditions,particularly with respect to various stressors, may accountfor the existence of alternative male reproductive phenotypes.Along these lines, I briefly explain the logic behind our workwith male phenotypes of longear sunfish (Lepomis megalotis)that is aimed at determining the tissue-specific distributionand activity of two enzymes that are common to androgen andglucocorticoid metabolism. A major goal of our work is to examinethe potential role of steroidogenic enzymes in the transductionof environmental information to influence the expression ofalternative male reproductive phenotypes.     

Temperature-dependent sex determination in reptiles: Proximate mechanisms,ultimate outcomes,and practical applications

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an “all or none” process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes—rather than the crganized/default system characteristic of vertebrates with genotypic sex determination—that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoaing for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants. © 1994 Wiley-Liss, Inc.     

Old experiments and new trends in avian sex differentiation

Summary The influence of steroid hormones on sex differentiation was first demonstrated in birds in 1935. Steroid female hormones injected in vivo into male embryos determined a partial or total feminization of gonads and genital ducts. Male hormones determined only the sex reversal of the ducts. Some substances of the group of androgens, such as dehydroandrosterone, had a paradoxical effect; they feminized males and masculinized females. Similar effects were observed later by several authors in all groups of vertebrates. In placentary mammals, only genital ducts were transformed. Castration of avian embryos also demonstrated the role of embryonic sexual hormones on genital ducts. These results, first obtained in vivo, were confirmed by experiments in vitro. Since then numerous studies have been undertaken on the nature of the hormone responsible for the regression of müllerian ducts in embryos of birds and other groups of vertebrates. Some authors assumed that these substances are proteins; many offered new evidence for the role of steroid sexual hormones during sex differentiation. Thus the problem appeared more complicated than it was thought at first. In recent years, synthesis of steroid sexual hormones have been demonstrated in young embryos during or even before sex differentiation; and enzymes that catalyze the synthesis of these hormones, such as hydroxysteroiddehydrogenase, also have been discovered. Further research has been oriented toward the characterization of steroid hormones by techniques of immunochemistry and labeled isotopes confirming the results obtained by other techniques. Specific proteins are being isolated in the effectors; they work as receptors of steroid hormones. Nuclear receptors of estradiol have been discovered in the embryonic gonads and in the cloacal wall at the time of sexual differentiation. Thus a mechanism can be conceived in which proteins and steroid hormones play mutual roles in the process of sex differentiation. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978.     

Annual variations of adrenal gland hormones in the lizard <Emphasis Type="Italic">Podarcis sicula</Emphasis>

The adrenal gland regulates metabolism and maintains normal electrolyte balance. Adrenal hormones are equivalent in all vertebrates; the chromaffin tissue produces adrenaline and noradrenaline and the steroidogenic tissue produces most of the steroid hormones present in mammals. Podarcis sicula belongs to the Squamata family of lizards and it is the most abundant lizard species in southern Italy. This species shows a reproductive annual cycle and the presence of seasonal variations in the activity of the hypothalamus-hypophyseal-thyroid axis. To investigate the existence of an annual cycle of lizard adrenal gland, we have measured plasma concentrations of corticosterone, ACTH, noradrenaline and adrenaline. We have shown that corticosterone rapidly increased from January to March to reach a peak value that persisted until July, then, it slowly decreased until December. ACTH levels increased from January to May and slowly decreased from July to December. Noradrenaline levels were higher in March and then decreased until December. On the contrary, adrenaline levels increased from March to July and slowly decreased until December. Our results demonstrate the existence of an annual cycle of the lizard adrenal gland activity. This is very interesting because its activity is important to rightly regulate the reproductive status of the Podarcis sicula lizard.     

Gut-islet endocrinology-some evolutionary aspects

Immunological and biological studies have shown that many of the mammalian gastroenteropancreatic (GEP) hormones have counterparts in lower vertebrates. Hormonal localization in cyclostomes and fishes suggests that insulin was phylogenetically the first islet hormone, followed by somatostatin, glucagon and, last, pancreatic polypeptide (PP). Some of the GEP peptides are present in the central and peripheral nervous system of lower vertebrates as well as mammals. GEP hormone-like substances resembling insulin, somatostatin, glucagon, PP, gastrin, secretin, VIP, substance P and enkephalin also occur in protostomian invertebrates (Annelida, Arthropoda, Mollusca), particularly in their nervous system. These findings indicate that the vertebrate hormones may have originated in neural tissue before the development of the vertebrate line of evolution.     

Variation of Loci Encoding Homologous Enzymes in the Evolutionary Series of Vertebrates

A study of variability of 11 allozyme loci (sAat, G3pdh, Gpi, sIdh, Ldh-A, Ldh-B, sMdh, sMe, sSod, Pgdh, and Sdh) in the evolutionary series of vertebrates from Cyclostomat to Mammalia revealed that (1) in vertebrates, these loci encoding multimeric enzymes are characterized by different heterozygosity levels, the extremes of which (represented by loci Ldh-A and Pgdh) differ from each other more than by a factor of 4; (2) classes of vertebrates markedly differed from one another in genetic variation; lower Tetrapoda are characterized by the highest level of genetic polymorphism, the classes representing the margins of the phyletic line—primitive (Cyclostomata and Chondrchthyes) and advanced (Aves and Mammalia)—have minimum heterozygosity levels, whereas Osteichthyes are characterized by intermediate heterozygosity level; (3) in the evolutionary series of vertebrates, heterozygosity varies rather independently in the groups of loci characterized by low, medium, and high variability. These patterns are explained in the context of intraorganismic factors: integration of mono- and polygenic traits (primarily, body size and ontogeny rate) and evolutionary specialization.     

Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family

ABSTRACT

Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of dCK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters of the dCK/dGK enzymes encoded by these genes. The two dCK enzymes in G. gallus have broader substrate specificity than their human or X. laevis counterparts. Additionally, the duplicated dCK enzyme in G. gallus might have become mitochondria. Based on our study we postulate that changing and adapting substrate specificities and subcellular localization are likely the drivers behind the evolution of vertebrate dNKs.     

Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.     

THE MOLECULAR EVOLUTION OF PITUITARY HORMONES

1. The pituitary hormones can be divided into 4 families; within each the members are structurally related and have probably evolved from a common ancestor by a process of gene duplication and divergence. 2. Recent structural studies have revealed much about the evolution of proteins. The roles of point mutation, gene duplication and partial gene duplication in molecular evolution have been highlighted, and the nature of the evolutionary forces involved has been extensively debated. The information available about the evolution of proteins in general provides a background for consideration of pituitary hormone evolution. 3. The structure and function of the mammalian neurohypophysial hormones (oxytocin and vasopressin) has been studied in detail. Related (structurally similar) peptides have been found in the neurohypophyses of lower vertebrates and have been Characterized in many instances. Several schemes have been proposed for the evolution of these hormones. 4. The vasopressins of the pig and its relatives show a genetic polymorphism. The roles of neurohypophysial hormones in lower vertebrates are very varied and not fully understood. 5. The ACTHs and MSHs are members of a second family of pituitary hormones. They are polypeptides of moderate size. Studies on amino-acid sequences have been carried out for ACTHs and MSHs from several mammals. α-MSH is identical in all cases studied in detail, but β-MSH and ACTH vary to some extent. There is considerable sequence homology between the hormones in this family - indicating a common phylogenetic origin and several gene duplications. 6. Dogfish MSH is the only non-mammalian hormone of the ACTH-MSH family to have been studied in detail. Two MSHs have been isolated from this species; both resemble the a-MSH of mammals in amino-acid sequence. ACTH-like and MSH-like hormones exist in many other vertebrate groups, but have not been characterized fully. 7. Structure-function relationships have been widely studied in the ACTH-MSH family, and have some interesting evolutionary implications. Polymorphism of P-MSHs is found in some mammals. 8. A third family of protein hormones includes pituitary prolactin and growth hormone, and placental lactogen. These are proteins of moderate size which have been shown to be widely distributed among the vertebrates. Species specificity can be recognized with regard to biological, immunological and structural properties. 9. Amino-acid sequences have been determined for growth hormones and prolactins from several mammals. There is sequence homology between growth hormone and prolactin. Human placental lactogen closely resembles human growth hormone. A phylogenetic tree has been constructed for this protein family. Rates of evolution within the group are rather variable. 10. The fourth family of pituitary hormones (FSH, LH, TSH and some related placental hormones) are all glycoproteins and have a subunit structure. Extensive sequence studies have been carried out on the hormones from some mammals, and show that there is considerable homology between the various subunits. The α-subunits of human TSH, LH and HCG (and probably FSH) are very similar. The β-subunits are different, but homologous. Evolution of this family clearly took place by a series of gene duplications followed by gene divergence. Schemes whereby this could have occurred have been discussed. Related hormones occur in lower vertebrates, but have not been fully characterized. Some lower vertebrates may possess only one gonado-trophin. 11. The pituitary hormones provide an interesting range of evolutionary problems, and are useful models for the study of molecular evolution. The evolutionary processes involved in their diversification have been discussed, with particular reference to the co-evolution of hormones and their receptors. Neutral mutations and gene duplications may have played a role in providing co-existing variation of hormones and receptors. 12. A speculative model for the evolution of neurohypophysial hormones is proposed, as an example of how molecular evolution may have operated in this and other hormone groups. 13. Homologies have been proposed between the various families of pituitary hormones, and between pituitary proteins and other entero-secretory proteins. The pituitary protein hormones were probably elaborated from smaller molecules by a process of partial gene duplication.     

Construction and characteristics of transgenic tobacco <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. plants expressing <Emphasis Type="Italic">CYP11A1</Emphasis> cDNA encoding cytochrome P450<Subscript>SCC</Subscript>

In steroidogenic animal tissues cytochrome P450_SCC catalizes the conversion of cholesterol into pregnenolone, a common metabolic precursor of all steroid hormones. To study the possibility of functioning of mammalian cytochrome P450_SCC in plants and the mechanism of its integration in the plant steroidogenic system, transgenic plants of tobacco Nicotiana tabacum L. were developed carrying cDNA of CYP11A1 encoding cytochrome P450_SCC of bovine adrenal cortex. Pregnenolone, a product of the reaction catalyzed by cytochrome P450_SCC, was discovered in the steroid-containing fraction of transgenic plants. Transgenic plants are characterized by a reduced period of vegetative development (early flowering and maturation of bolls) and increased productivity. The contents of soluble protein and carbohydrates in leaves and seeds of transgenic plants are essentially higher than the contents of these components in leaves and seeds of control plants.     

Evolutionary Origin of the Mitochondrial Cholesterol Transport Machinery Reveals a Universal Mechanism of Steroid Hormone Biosynthesis in Animals

Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria via a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In adrenal glands and gonads, this process is accelerated by hormones, leading to the production of high levels of steroids that control tissue development and function. A hormone-induced multiprotein complex, the transduceosome, was recently identified, and is composed of cytosolic and outer mitochondrial membrane proteins that control the rate of cholesterol entry into the outer mitochondrial membrane. More recent studies unveiled the steroidogenic metabolon, a bioactive, multimeric protein complex that spans the outer-inner mitochondrial membranes and is responsible for hormone-induced import, segregation, targeting, and metabolism of cholesterol by cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1) in the inner mitochondrial membrane. The availability of genome information allowed us to systematically explore the evolutionary origin of the proteins involved in the mitochondrial cholesterol transport machinery (transduceosome, steroidogenic metabolon, and signaling proteins), trace the original archetype, and predict their biological functions by molecular phylogenetic and functional divergence analyses, protein homology modeling and molecular docking. Although most members of these complexes have a history of gene duplication and functional divergence during evolution, phylogenomic analysis revealed that all vertebrates have the same functional complex members, suggesting a common mechanism in the first step of steroidogenesis. An archetype of the complex was found in invertebrates. The data presented herein suggest that the cholesterol transport machinery is responsible for steroidogenesis among all vertebrates and is evolutionarily conserved throughout the entire animal kingdom.     

Brassinosteroid biosynthesis and inactivation

The term brassinosteroids (BRs) refers to the growth-promoting plant steroidal hormones. Various developmental programs including but not limited to cell elongation, stress tolerance, and skoto-/photo-morphogenesis are controlled by subnanomolar concentrations of BRs. Accordingly, BR mutants that are defective in BR biosynthetic or signaling pathways usually display dwarfism. Characterization of numerous BR dwarf mutants isolated from Arabidopsis , pea, tomato, and rice greatly contributed to our understanding of BR biology. Recently, an enzyme that mediates the final step in the BR biosynthetic pathways has been characterized by two different groups. The brassinolide synthases (Cytochrome P450s 85A2 and 85A3) are multifunctional enzymes that catalyze the last three consecutive steps in BR biosynthetic pathways, namely, C-6 hydroxylation, dehydrogenation, and Baeyer-Villiger type oxidation. In addition, many of the previously unknown steps have been genetically characterized. This review aims to summarize the knowledge that has been developed during the last 2–3 years in this field of BR biosynthesis and inactivation research.