Localization of 17beta-hydroxysteroid dehydrogenase and characterization of testosterone in the brain of the male frog.

Several enzymes involved in the formation of steroids of the pregnene and pregnane series have been identified in the brain, but the biosynthesis of testosterone has never been reported in the central nervous system. In the present study, we have investigated the distribution and bioactivity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) (EC 1.1.1.62; a key enzyme that is required for the formation of testosterone and estradiol) in the brain of the male frog Rana ridibunda. By using an antiserum against human type I placental 17beta-HSD, immunoreactivity was localized in a discrete group of ependymal glial cells bordering the telencephalic ventricles. HPLC analysis of telencephalon and hypothalamus extracts combined with testosterone radioimmunoassay revealed the existence of two peaks coeluting with testosterone and 5alpha-dihydrotestosterone. After HPLC purification, testosterone was identified by gas chromatography/mass spectrometry. Incubation of telencephalon slices with [3H]pregnenolone resulted in the formation of metabolites which coeluted with progesterone, 17alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone. The newly synthesized steroid comigrating with testosterone was selectively immunodetected by using testosterone antibodies. These data indicate that 17beta-HSD is expressed in a subpopulation of gliocytes in the frog telencephalon and that telencephalic cells are capable of synthesizing various androgens, including dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone.

Localization of the mouse gene releasing sex-limited expression of Slp.

To probe genetic variation in the regulation of sexual dimorphism, we have characterized the mouse protein Slp, coded by the gene sex-limited protein (Slp). Slp expression in many strains is limited to males and is androgen-dependent. However, female expression is also observed in rare strains, due to nonlinked gene(s) termed regulator of sex-limitation (rsl). In this report we demonstrate that female expression of Slp results from homozygous recessive allele(s) at a single autosomal locus that maps to a 2.2-centimorgan interval on chromosome 13. This conclusion was supported by extensive genetic analyses including the use of polymorphic microsatellites to type numerous backcross progeny and a recombinant inbred series and to identify the congenic interval in three independently derived congenic strains. Four attractive candidate genes were identified by the localization of rsl. Interestingly, rsl was found not only to enable expression in females but to also increase expression in males. The findings suggest that the expression of Slp and perhaps other sexually dimorphic proteins is regulated by two pathways, one that is dependent upon rsl but not androgens and another that is rsl-independent but requires androgens.

Efficacy of Stanozolol on bone regeneration: in vitro and in vivo study

La ricerca di nuove strategie per la rigenerazione ossea rappresenta un focus di interesse centrale per migliorare la gestione di casi clinici complessi nell’ambito della chirurgia orale e maxillo-facciale. Uno degli approcci più utilizzati in tale contesto si basa sull’utilizzo di molecole con proprietà osteoinduttive e molte sostanze sono state fino ad oggi sperimentate. E’ noto in letteratura che gli androgeni svolgono un ruolo chiave nella regolazione della morfogenesi ossea e nel mantenimento della sua omeostasi durante il corso della vita. Questo lavoro di tesi nasce dall’ipotesi che la somministrazione locale di tali ormoni, eventualmente combinata a materiali da innesto, possa favorire la guarigione di difetti ossei. Stando a questa premessa, sono stati valutati gli effetti dello steroide sintetico Stanozololo sulla rigenerazione ossea in diversi settings sperimentali. La tesi è strutturata secondo un percorso che segue le fasi della ricerca, attraverso sperimentazioni in vitro e in vivo; ogni capitolo può essere approcciato come uno studio a sé stante, corrispondente ad una determinata tappa dell’iter sperimentale. Sulla base di questi intenti, viene fornito inizialmente un quadro d’insieme circa gli effetti degli androgeni sull’osso. A seguire, è presentata una sperimentazione in vitro nella linea cellulare SaOS-2. Infine, è proposta un’innovativa metodologia di analisi per lo studio della rigenerazione ossea nel modello di ratto, ove viene testata la somministrazione locale di Stanozololo combinato a materiale da innesto.

Relações entre comportamento vocal, imunidade e níveis plasmáticos de corticosterona e testosterona em sapos (Rhinella granulosa)

A exibição do comportamento vocal em muitas espécies de anfíbios anuros é relacionada aos níveis de hormônios esteroides gonadais e interrenais. Esses hormônios poderiam mediar a relação entre intensidade de sinais e imunidade, pois estão envolvidos no desenvolvimento das características sexuais secundárias, comportamento de corte e mobilização de reservas energéticas durante a atividade reprodutiva, enquanto apresentam também efeitos imunomoduladores. Nesse sentido, o objetivo deste trabalho foi explorar as relações entre comportamento reprodutivo, imunidade e níveis plasmáticos de testosterona e corticosterona em machos do sapo do semiárido brasileiro, Rhinella granulosa, em atividade reprodutiva e após manipulação hormonal. A precipitação foi o principal determinante ambiental para o aumento dos níveis de testosterona e corticosterona circulantes em machos de R. granulosa, estimulando o comportamento de vocalização. As relações fisiológicas encontradas indicam que os altos níveis plasmáticos de testosterona nos primeiros dias após a chuva devem promover o início do turno vocal, porém a corticosterona deve modular o esforço de vocalização. De forma geral, a exacerbação do comportamento vocal de R. granulosa tem efeitos negativos sobre a imunocompetência, porém alguns indivíduos que apresentam maiores concentrações plasmáticas de corticosterona apresentam concomitantemente alto esforço vocal e alta imunidade. De acordo, a aplicação transdérmica de corticosterona promoveu elevação aguda dos níveis plasmáticos deste glicocorticoide, bem como um aumento da função imune. Assim, apesar de a atenção principal ser comumente colocada no papel da testosterona na mediação de sinais honestos, nossos resultados corroboram a importância da corticosterona na mediação da expressão do comportamento de corte e imunocompetência em machos R. granulosa

Evolução de lesão neoplásica da suprarrenal : caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Androgen responsiveness to competition in humans: the role of cognitive variables

Although androgens are commonly seen as male sex hormones, it has been established over the years that in both sexes, androgens also respond to social challenges. To explain the socially driven changes in androgens, two theoretical models have been proposed: the biosocial model and the challenge hypothesis. These models are typically seen as partly overlapping; however, they generate different predictions that are clarified here. In humans, sports competition and nonmetabolic competitive tasks have been used in the laboratory setting, as a proxy for agonistic interactions in animals. The results reviewed here show that the testosterone (T) response to competition in humans is highly variable – the studies present postcompetition T levels and changes in T that depend on the contest outcome and that cannot be predicted by the current theoretical models. These conflicting results bring to the foreground the importance of considering cognitive factors that could moderate the androgen response to competition. Among these variables, we elect cognitive appraisal and its components as a key candidate modulating factor. It is known that T also modulates the cognitive processes that are relevant to performance in competition. In this article, we reviewed the evidence arising from studies investigating the effect of administering exogenous T and compare those results with the findings from studies that measured endogenous T levels. Finally, we summarized the importance of also considering the interaction between androgens and other hormones, such as cortisol, when investigating the social modulation of T, as proposed by the dual-hormone hypothesis.

The renin-angiotensin system and male reproduction: new functions for old hormones

The blood-borne renin-angiotensin system (RAS) is known best for its role in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, numerous tissues show intrinsic angiotensin-generating systems that cater for specific local needs through actions that add to, or differ from, the circulating RAS. The male reproductive system has several sites of intrinsic RAS activity. Recent focus on the epididymis, by our laboratories and by others, has contributed important details about the local RAS in this tissue. The RAS components have been localized morphologically and topographically; they have been shown to be responsive to androgens and to hypoxia; and angiotensin has been shown to influence tubular, and consequently, fluid secretion. Components of the RAS have also been found in the testis, vas deferens, prostate and semen. Angiotensin II receptors, type 1 and, to a lesser extent, type 2 are widespread, and angiotensin IV receptors have been localized in the prostate. The roles of the RAS in local processes at these sites are still uncertain and have yet to be fully elucidated, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis and fertilization. Notwithstanding this evidence for the involvement of the RAS in various important aspects of male reproduction, there has so far been a lack of clinical evidence, demonstrable by changes in fertility, for a crucial role of the RAS in male reproduction. However, it is clear that there are several potential targets for manipulating the activity of the male reproductive system by interfering with the locally generated angiotensin systems.

Pseudopregnancy in the bitch

Pseudopregnancy in the bitch is a normal phenomenon. It refers to the dioestrus phase of the bitch's reproductive cycle. It can range in severity from covert pseudopregnancy, in which the signs are barely noticeable, to severe clinical or overt pseudopregnancy. It occurs six to eight weeks following oestrous. The bitch may present with pregnancy-like behaviour including nesting and aggression, excessive mammary enlargement and contractions. These signs are an exaggerated version of the normal signs shown in a normal entire cycling bitch. The exact aetiology behind pseudopregnancy is not known. It is believed to be associated with a rapid decline in serum progesterone concentrations and the resulting surge in prolactin. Treatment may involve conservative management, medical therapy or ovariectomy/ovariohysterectomy. In the past medical therapy has included sex steroids including oestrogens, androgens and progestins. However, due to the frequent incidence and severity of side effects their use is contraindicated. Recommended forms of management incorporate the use of prolactin inhibitors such as cabergoline, metergoline and bromocriptine. These drugs reduce serum levels of prolactin and therefore reduce the development of mammary glands and decrease the behavioural signs associated with overt pseudopregnancy.

Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

Neuroendocrine control of a dynamic communication signal

Multiple physiological systems regulate the electric communication signal of the weakly electric gymnotiform fish, Brachyhypopomus pinnicaudatus. Fish were injected with neuroendocrine probes which identified pharmacologically relevant serotonin (5-HT) receptors similar to the mammalian 5-HT1AR and 5-HT2AR. Peptide hormones of the hypothalamic-pituitary-adrenal/interrenal axis also augment the electric waveform. These results indicate that the central serotonergic system interacts with the hypothalamic-pituitary-interrenal system to regulate communication signals in this species. The same neuroendocrine probes were tested in females before and after introducing androgens to examine the relationship between sex steroid hormones, the serotonergic system, melanocortin peptides, and EOD modulations. Androgens caused an increase in female B. pinnicaudatus responsiveness to other pharmacological challenges, particularly to the melanocortin peptide adrenocorticotropic hormone (ACTH). A forced social challenge paradigm was administered to determine if androgens are responsible for controlling the signal modulations these fish exhibit when they encounter conspecifics. Males and females responded similarly to this social challenge construct, however introducing androgens caused implanted females to produce more exaggerated responses. These results confirm that androgens enhance an individual's capacity to produce an exaggerated response to challenge, however another unidentified factor appears to regulate sex-specific behaviors in this species. These results suggest that the rapid electric waveform modulations B. pinnicaudatus produces in response to conspecifics are situation-specific and controlled by activation of different serotonin receptor types and the subsequent effect on release of pituitary hormones.

Endocrine regulation of dynamic communication signals in gymnotiform fish

Communication signals are shaped by the opposing selection pressures imposed by predators and mates. A dynamic signal might serve as an adaptive compromise between an inconspicuous signal that evades predators and an extravagant signal preferred by females. Such a signal has been described in the gymnotiform electric fish, Brachyhypopomus gauderio, which produces a sexually dimorphic electric organ discharge (EOD). The EOD varies on a circadian rhythm and in response to social cues. This signal plasticity is mediated by the slow action of androgens and rapid action of melanocortins. My dissertation research tested the hypotheses that (1) signal plasticity is related to sociality levels in gymnotiform species, and (2) differences in signal plasticity are regulated by differential sensitivity to androgen and melanocortin hormones. To assess the breadth of dynamic signaling within the order Gymnotiformes, I sampled 13 species from the five gymnotiform families. I recorded EODs to observe spontaneous signal oscillations after which I injected melanocortin hormones, saline control, or presented the fish with a conspecific. I showed that through the co-option of the ancient melanocortin pathway, gymnotiforms dynamically regulate EOD amplitude, spectral frequency, both, or neither. To investigate whether observed EOD plasticities are related to species-specific sociality I tested four species; two territorial, highly aggressive species, Gymnotus carapo and Apteronotus leptorhynchus, a highly gregarious species, Eigenmannia cf. virescens , and an intermediate short-lived species with a fluid social system, Brachyhypopomus gauderio. I examined the relationship between the androgens testosterone and 11-ketotestosterone, the melanocortin α-MSH, and their roles in regulating EOD waveform. I implanted all fish with androgen and blank silicone implants, and injected with α-MSH before and at the peak of implant effect. I found that waveforms of the most territorial and aggressive species were insensitive to hormone treatments; maintaining a static, stereotyped signal that preserves encoding of individual identity. Species with a fluid social system were most responsive to hormone treatments, exhibiting signals that reflect immediate condition and reproductive state. In conclusion, variation in gymnotiform signal plasticity is hormonally regulated and seems to reflect species-specific sociality.

Social and environmental regulation of signal plasticity and signal reliability in the electric fish Brachyhypopomus gauderio

The balance between the costs and benefits of conspicuous signals ensures that the expression of those signals is related to the quality of the bearer. Plastic signals could enable males to maximize conspicuous traits to impress mates and competitors, but reduce the expression of those traits to minimize signaling costs, potentially compromising the information conveyed by the signals. ^ I investigated the effect of signal enhancement on the information coded by the biphasic electric signal pulse of the gymnotiform fish Brachyhypopomus gauderio. Increases in population density drive males to enhance the amplitude of their signals. I found that signal amplitude enhancement improves the information about the signaler's size. Furthermore, I found that the elongation of the signal's second phase conveys information about androgen levels in both sexes, gonad size in males and estrogen levels in females. Androgens link the duration of the signal's second phase to other androgen-mediated traits making the signal an honest indicator of reproductive state and aggressive motivation. ^ Signal amplitude enhancement facilitates the assessment of the signaler's resource holding potential, important for male-male interactions, while signal duration provides information about aggressive motivation to same-sex competitors and reproductive state to the opposite sex. Moreover, I found that female signals also change in accordance to the social environment. Females also increase the amplitude of their signal when population density increases and elongate the duration of their signal's second phase when the sex ratio becomes female-biased. Indicating that some degree of sexual selection operates in females. ^ I studied whether male B. gauderio use signal plasticity to reduce the cost of reproductive signaling when energy is limited. Surprisingly, I found that food limitation promotes the investment in reproduction manifested as signal enhancement and elevated androgen levels. The short lifespan and single breeding season of B. gauderio diminishes the advantage of energy savings and gives priority to sustaining reproduction. I conclude that the electric signal of B. gauderio provides reliable information about the signaler, the quality of this information is reinforced rather than degraded with signal enhancement.^

The effect of male -male competition and its underlying regulatory mechanisms on the electric signal of the gymnotiform fish Brachyhypopomus gauderio

Sexually-selected communication signals can be used by competing males to settle contests without incurring the costs of fighting. The ability to dynamically regulate the signal in a context-dependent manner can further minimize the costs of male aggressive interactions. Such is the case in the gymnotiform fish Brachyhypopomus gauderio, which, by coupling its electric organ discharge (EOD) waveform to endocrine systems with circadian, seasonal, and behavioral drivers, can regulate its signal to derive the greatest reproductive benefit. My dissertation research examined the functional role of the EOD plasticity observed in male B. gauderio and the physiological mechanisms that regulate the enhanced male EOD. To evaluate whether social competition drives the EOD changes observed during male-male interactions, I manipulated the number of males in breeding groups to create conditions that exemplified low and high competition and measured their EOD and steroid hormone levels. My results showed that social competition drives the enhancement of the EOD amplitude of male B. gauderio. In addition, changes in the EOD of males due to changes in their social environment were paralleled by changes in the levels of androgens and cortisol. I also examined the relationship between body size asymmetry, EOD waveform parameters, and aggressive physical behaviors during male-male interactions in B. gauderio, in order to understand more fully the role of EOD waveforms as reliable signals. While body size was the best determinant of dominance in male B. gauderio, EOD amplitude reliably predicted body condition, a composite of length and weight, for fish in good body condition. To further characterize the mechanisms underlying the relationship between male-male interactions and EOD plasticity, I identified the expression of the serotonin receptor 1A, a key player in the regulation of aggressive behavior, in the brains of B. gauderio. I also identified putative regulatory regions in this receptor in B. gauderio and other teleost fish, highlighting the presence of additional plasticity. In conclusion, male-male competition seems to be a strong selective driver in the evolution of the male EOD plasticity in B. gauderio via the regulatory control of steroid hormones and the serotonergic system.

Androgen Treatment Increases Female Aggression in the Electric Fish Brachyhypopomus Gauderio

Androgens regulate aggression in male vertebrates however the exact role they play in regulating aggression in females is not as well understood. Female aggression is commonplace in many vertebrate groups where it can provide various advantages to the aggressors. I explored whether androgens serve as important hormonal mediators of aggressive behavior in female electric fish. I paired adult females of the weakly-electric fish Brachyhypopomus gauderio in aggressive encounters and compared bloodtestosterone (T) levels of dominant and subordinate groups. Afterwards, I implanted a new set of females with the androgen 5a-dihydrotestosterone (DHT) and compared frequency of different aggressive behaviors to a blank-implanted group. I created dyads ofblank-blank (BB), blank-DHT (BD), and DHT-DHT (DD). I demonstrate that dominant females have higher T-levels than subordinates. I also show that the frequency of aggressive behaviors is dependent upon treatment type. Androgens increased both the intensity and level of female aggression, however the degree and type of aggressive behavior depended on the opponent being fought.

The Effect of male-male competition and its Underlying Regulatory Mechanisms on the Electric Signal of the Gymnotiform fish Brachyhypopomus gauderio

Sexually-selected communication signals can be used by competing males to settle contests without incurring the costs of fighting. The ability to dynamically regulate the signal in a context-dependent manner can further minimize the costs of male aggressive interactions. Such is the case in the gymnotiform fish Brachyhypopomus gauderio, which, by coupling its electric organ discharge (EOD) waveform to endocrine systems with circadian, seasonal, and behavioral drivers, can regulate its signal to derive the greatest reproductive benefit. My dissertation research examined the functional role of the EOD plasticity observed in male B. gauderio and the physiological mechanisms that regulate the enhanced male EOD. To evaluate whether social competition drives the EOD changes observed during male-male interactions, I manipulated the number of males in breeding groups to create conditions that exemplified low and high competition and measured their EOD and steroid hormone levels. My results showed that social competition drives the enhancement of the EOD amplitude of male B. gauderio. In addition, changes in the EOD of males due to changes in their social environment were paralleled by changes in the levels of androgens and cortisol. I also examined the relationship between body size asymmetry, EOD waveform parameters, and aggressive physical behaviors during male-male interactions in B. gauderio, in order to understand more fully the role of EOD waveforms as reliable signals. While body size was the best determinant of dominance in male B. gauderio, EOD amplitude reliably predicted body condition, a composite of length and weight, for fish in good body condition. To further characterize the mechanisms underlying the relationship between male-male interactions and EOD plasticity, I identified the expression of the serotonin receptor 1A, a key player in the regulation of aggressive behavior, in the brains of B. gauderio. I also identified putative regulatory regions in this receptor in B. gauderio and other teleost fish, highlighting the presence of additional plasticity. In conclusion, male-male competition seems to be a strong selective driver in the evolution of the male EOD plasticity in B. gauderio via the regulatory control of steroid hormones and the serotonergic system.