Captivity alters neuroendocrine regulators of stress and reproduction in the hypothalamus in response to acute stress.

Wild animals are brought into captivity for many reasons. However, unlike laboratory-bred animals, wild caught animals often respond to the dramatic shift in their environment with physiological changes in the stress and reproductive pathways. Using wild-caught male and female house sparrows (Passer domesticus) we examined how time in captivity affects the expression of reproductive and stress-associated genes in the brain, specifically, the hypothalamus. We quantified relative mRNA expression of a neurohormone involved in the stress response (corticotropin releasing hormone [CRH]), a hypothalamic inhibitor of reproduction (gonadotropin inhibitory hormone [GnIH]), and the glucocorticoid receptor (GR), which is important in terminating the stress response. To understand potential shifts at the cellular level, we also examined the presence of hypothalamic GnIH (GnIH-ir) using immunohistochemistry. We hypothesized that expression of these genes and the abundance of cells immunoreactive for GnIH would change in response to time in captivity as compared to free-living individuals. We found that GR mRNA expression and GnIH-ir cell abundance increased after 24 and 45 days in captivity, as compared to wild-caught birds. At 66 days in captivity, GR expression and GnIH cell abundance did not differ from wild-caught birds, suggesting birds had acclimated to captivity. Evaluation of CRH and GnIH mRNA expression yielded similar trends, though they were not statistically significant. In addition, although neuroendocrine factors appeared to acclimate to captivity, a previous study indicated that corticosterone release and immune responses of these same birds did not acclimate to captivity, suggesting that neuroendocrine endpoints may adapt more rapidly to captivity than downstream physiological measures. These data expand our understanding of the physiological shifts occurring when wild animals are brought into captivity.

Cloning and expression characterization in hypothalamic Dio2/3 under a natural photoperiod in the domesticated Brandt's vole (Lasiopodomys brandtii).

The Dio2/3 gene is related to the photoperiodic response in mammals and plays an important role in the development of gonadal organs and seasonal breeding. Our previous studies have reported synchronous variations in the gonadal mass and photoperiodical transition around the summer solstice in a wild Brandt's vole population, a species with striking seasonal breeding. To investigate the role of the Dio2/3 gene in the control of seasonal breeding in this species, we cloned and characterized its expression levels by high-throughput Real-Time PCR during the period around the summer solstice. We selected a domesticated strain to ensure similar development of samples. The synchronous variation pattern between the Dio2/3 expression levels and gonadal mass around the summer solstice supports the prediction that the Dio2/3 gene plays an important role in the seasonal transition in this species. We suggest that the observed photoperiod response may be triggered by differences in the day length rather than the absolute daylength in this species. However, the similar Dio2/3 gene expression patterns but inconsistent gonadal mass patterns between the domesticated strain and the wild strain in the samples collected on Sep 8th, an absolute nonbreeding stage in the wild, lead us to speculate that the core function of the Dio2/3 gene should be restricted in response to the photoperiod rather than factors directly regulating gonadal development, and this laboratory strain could be used as an animal model to test the mechanism of environmental adaptation.

Intermittent Moderate Energy Restriction Improves Weight Loss Efficiency in Diet-Induced Obese Mice.

BACKGROUND: Intermittent severe energy restriction is popular for weight management. To investigate whether intermittent moderate energy restriction may improve this approach by enhancing weight loss efficiency, we conducted a study in mice, where energy intake can be controlled. METHODS: Male C57/Bl6 mice that had been rendered obese by an ad libitum diet high in fat and sugar for 22 weeks were then fed one of two energy-restricted normal chow diets for a 12-week weight loss phase. The continuous diet (CD) provided 82% of the energy intake of age-matched ad libitum chow-fed controls. The intermittent diet (ID) provided cycles of 82% of control intake for 5-6 consecutive days, and ad libitum intake for 1-3 days. Weight loss efficiency during this phase was calculated as (total weight change) ÷ [(total energy intake of mice on CD or ID)-(total average energy intake of controls)]. Subsets of mice then underwent a 3-week weight regain phase involving ad libitum re-feeding. RESULTS: Mice on the ID showed transient hyperphagia relative to controls during each 1-3-day ad libitum feeding period, and overall ate significantly more than CD mice (91.1±1.0 versus 82.2±0.5% of control intake respectively, n = 10, P<0.05). There were no significant differences between CD and ID groups at the end of the weight loss or weight regain phases with respect to body weight, fat mass, circulating glucose or insulin concentrations, or the insulin resistance index. Weight loss efficiency was significantly greater with ID than with CD (0.042±0.007 versus 0.018±0.001 g/kJ, n = 10, P<0.01). Mice on the CD exhibited significantly greater hypothalamic mRNA expression of proopiomelanocortin (POMC) relative to ID and control mice, with no differences in neuropeptide Y or agouti-related peptide mRNA expression between energy-restricted groups. CONCLUSION: Intermittent moderate energy restriction may offer an advantage over continuous moderate energy restriction, because it induces significantly greater weight loss relative to energy deficit in mice.

Identification and expression of GnRH2 and GnRH3 in the black sea bass (Centropristis striata), a hermaphroditic teleost.

We cloned two cDNAs for two gonadotropin-releasing hormones, GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH), respectively, from the black sea bass (Centropristis striata). Black sea bass are protogynous hermaphroditic teleosts that change from females to males between 2 and 5 years of age. Similar to other GnRH precursors, the precursors of black sea bass GnRH2 and GnRH23 consisted of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide. Our analyses failed to identify GnRH1. GnRH3 precursor transcript was more widely distributed in a variety of tissues compared with GnRH2. Further examination of GnRH expression and gonadal histology was done in black sea bass from three different size groups: small (11.4-44.1 g), medium (179.4-352.2 g) and large (393.8-607.3 g). Interestingly, GnRH3 expression occurred only in the pituitaries of males in the small and medium groups compared with expression of GnRH2. Future functional studies of the sea bass GnRHs will be valuable in elucidating the potential underlying neuroendocrine mechanisms of black sea bass reproduction and may ultimately contribute to management advances in this commercially important fish.

Fecundidad de la medusa Stomolophus meleagris (Rhizostomeae: Stomolophidae) en el Golfo de California / Stomolophus meleagris fecundity (Rhizostomeae: Stomolophidae) in the Gulf of California

The cannonball jellyfish Stomolophus meleagris is regarded as a fishery resource with high commercial value, but with scarce biological information. With the aim to generate preliminary information on reproductive aspects, the present study analyzes its fecundity, based on the estimated number of vitellogenic oocytes in the gonad; in addition, we evaluated its relationship with the jellyfish body length, diameter and wet weight; and we established the relationship of the gonadosomatic index (IGS) with the jellyfish diameter and length. For this, a total of 30 specimens were collected, measured and weighed in a monthly basis, in Las Guásimas lagoon from January to May 2006. In order to estimate the gonad volume, 60 mature females were analyzed and their gonads were extracted and stained with hematoxylin-eosin. Gonad images from histological preparations were analyzed and the number of vitellogenic and previtellogenic oocytes obtained. We used catch data from The Guásimas for population recruitment analysis, that was estimated with FISAT II. Among results, we found that fecundity increased with jellyfish length, diameter and total wet weight. The lower fecundity rates occured on February and the highest during May (11 873 071 and 37 528 197 millions of vitellogenic oocytes per gonad, respectively). The IGS increased with length and diameter of the specimens, at its maximum value of 3.7% was observed in May. A continuous production of previtellogenic oocytes were observed during the full collection period. The maximum recruitment was observed in July and November (37.50% and 28.01% respectively). The high fecundity observed in S. meleagris, suggests a high population reproductive potential that may support the possibility of an important fishery consolidation on this jellyfish.

La medusa Stomolophus meleagris es considerada un recurso pesquero de alto valor comercial. Debido a la relevancia de conocer aspectos de importancia biológica actualmente desconocidos, el presente trabajo analiza la fecundidad con base en la estimación del número de ovocitos vitelogénicos en la gónada; su relación con la longitud, diámetro y peso húmedo; y la relación del índice gonadosomático (IGS) con su diámetro y longitud. De enero a mayo 2006 se recolectaron 30 ejemplares mensuales que fueron medidos y pesados, de los cuales se analizaron 60 hembras maduras para calcular el volumen de sus gónadas. Se extrajeron las gónadas, se tiñeron con hematoxilina-eosina y se seleccionaron las hembras con madurez gonadal. Mediante la digitalización de imágenes se estimó el número de ovocitos vitelogénicos y previtelogénicos. Se emplearon datos de captura en Las Guásimas para el análisis de reclutamiento poblacional, estimado con FISAT II. La fecundidad se incrementó con la longitud, el diámetro y el peso húmedo total. La menor fecundidad se observó en febrero y la mayor en mayo (11 873 071 y 37 528 197 millones de ovocitos vitelogénico por gónada, respectivamente). El IGS se incrementó con la longitud y diámetro de los ejemplares, con su máximo en mayo de 3.7%. Se observó una producción continua de ovocitos previtelogénicos en todo el periodo de recolecta. El máximo reclutamiento se observó en julio y noviembre (37.50% y 28.01% respectivamente). La alta fecundidad encontrada en S. meleagris sugiere un alto potencial reproductivo para la población y sustenta la posibilidad de la consolidación de una importante pesquería de esta medusa.

[Stomolophus meleagris fecundity (Rhizostomeae: Stomolophidae) in the Gulf of California]. / Fecundidad de la medusa Stomolophus meleagris (Rhizostomeae: Stomolophidae) en el Golfo de California.

The cannonball jellyfish Stomolophus meleagris is regarded as a fishery resource with high commercial value, but with scarce biological information. With the aim to generate preliminary information on reproductive aspects, the present study analyzes its fecundity, based on the estimated number of vitellogenic oocytes in the gonad; in addition, we evaluated its relationship with the jellyfish body length, diameter and wet weight; and we established the relationship of the gonadosomatic index (IGS) with the jellyfish diameter and length. For this, a total of 30 specimens were collected, measured and weighed in a monthly basis, in Las Guásimas lagoon from January to May 2006. In order to estimate the gonad volume, 60 mature females were analyzed and their gonads were extracted and stained with hematoxylin-eosin. Gonad images from histological preparations were analyzed and the number of vitellogenic and previtellogenic oocytes obtained. We used catch data from The Guásimas for population recruitment analysis, that was estimated with FISAT II. Among results, we found that fecundity increased with jellyfish length, diameter and total wet weight. The lower fecundity rates occured on February and the highest during May (11 873 071 and 37 528 197 millions of vitellogenic oocytes per gonad, respectively). The IGS increased with length and diameter of the specimens, at its maximum value of 3.7% was observed in May. A continuous production of previtellogenic oocytes were observed during the full collection period. The maximum recruitment was observed in July and November (37.50% and 28.01% respectively). The high fecundity observed in S meleagris, suggests a high population reproductive potential that may support the possibility of an important fishery consolidation on this jellyfish.

Gene structure analysis of kisspeptin-2 (Kiss2) in the Senegalese sole (Solea senegalensis): characterization of two splice variants of Kiss2, and novel evidence for metabolic regulation of kisspeptin signaling in non-mammalian species.

Kisspeptin signaling in the brain is involved in the control of the onset of puberty in vertebrates. In this study, we present novel evidence indicating that kisspeptin may link energy balance and reproduction. For that purpose, we determined the complete gene structure of kisspeptin in a teleost fish, the Senegalese sole (Ss). In contrast to the situation evident in several fish, in this species only Kiss2 was found. Yet, two Ss Kiss2 isoforms generated by alternative splicing through intronic retention were detected: Ss Kiss2_v1, producing the functional protein, and Ss Kiss2_v2, coding for a truncated, non-functional protein. Specific qPCRs showed that the expression of these two isoforms varied differently in brain and gonads throughout maturation. In addition, and in contrast to what has been observed in mammals, fasting increased hypothalamic mRNA levels of Ss Kiss2_v1, which also caused a concomitant rise in pituitary Ss LH and Ss FSH mRNA. Together, these data indicate the impact of the nutritional status on Kiss mRNA expression as a potential regulatory mechanism for the metabolic control of reproduction in non-mammalian species, albeit with some significant differences with respect to the situation described in mammals.

Nitric oxide modulates gonadal and adrenal function in Japanese quail Coturnix coturnix japonica.

Nitric oxide (NO) a gaseous neurotransmitter is reported to play an important role in controlling the release of luteinizing hormone releasing hormone (LHRH) in mammalian system. But its role has not been investigated in birds, where photoperiod plays an important role in regulating gonadal development. To investigate the effect of NO on gonadal and adrenal function of Japanese quail, in the first experiment, 3-weeks-old sexually immature quail received NO donor sodium nitroprusside (SNP, 5mg/100g body weight) orally or nitric oxide synthase (NOS) inhibitor N-nitro L-arginine methyl ester (L-NAME, 25 microg/100g body weight) intraperitoneally for 13 days in continuous condition of light (LL). Thereafter treated quail along with their respective controls were shifted to long day length (LD 16:8) for 21 days when the study was terminated. Results indicate that SNP treatment stimulated and L-NAME suppressed body weight, cloacal gland volume (an androgen dependent sex accessory organ), testes (gonado-somatic index, spermatogenesis), epididymis (histology) and adrenal (weight, histology, cortico-medullary ratio) function as well as total nitrite and nitrate concentration in plasma, hypothalamus and testes. In the second experiment, two groups of 3-weeks-old birds were maintained in short day length (LD 6:18) or long day length (LD 16:8) for 5 weeks to induce gonadal suppression and stimulation, respectively. Thereafter birds of both the photoperiod were divided into two subgroups, the short day quail receiving normal saline (SD Con) or SNP (SD+SNP) while long day quail received normal saline (LD Con) or L-NAME (LD+L-NAME) for 13 days and were maintained in their respective photoperiods, until the termination of study (21 days post treatment period). SNP stimulated all the parameters even in short day condition and L-NAME suppressed in long day quail compared to their respective controls. These findings indicate positive control of NO on the gonad and adrenal function of Japanese quail which exhibits parallel adrenal-gonad relationship. Further, NO donor induces long day effects while NOS inhibitor mimics short day effects. It is concluded that NO may not only regulate hypothalamo-hypophyseal-gonadal and -adrenal axis of Japanese quail but may also modulate its photosexual responses.

Health effects in fish of long-term exposure to effluents from wastewater treatment works.

Concern has been raised in recent years that exposure to wastewater treatment effluents containing estrogenic chemicals can disrupt the endocrine functioning of riverine fish and cause permanent alterations in the structure and function of the reproductive system. Reproductive disorders may not necessarily arise as a result of estrogenic effects alone, and there is a need for a better understanding of the relative importance of endocrine disruption in relation to other forms of toxicity. Here, the integrated health effects of long-term effluent exposure are reported (reproductive, endocrine, immune, genotoxic, nephrotoxic) . Early life-stage roach, Rutilus rutilus, were exposed for 300 days to treated wastewater effluent at concentrations of 0, 15.2, 34.8, and 78.7% (with dechlorinated tap water as diluent). Concentrations of treated effluents that induced feminization of male roach, measured as vitellogenin induction and histological alteration to gonads, also caused statistically significant alterations in kidney development (tubule diameter), modulated immune function (differential cell count, total number of thrombocytes), and caused genotoxic damage (micronucleus induction and single-strand breaks in gill and blood cells). Genotoxic and immunotoxic effects occurred at concentrations of wastewater effluent lower than those required to induce recognizable changes in the structure and function of the reproductive endocrine system. These findings emphasize the need for multiple biological end points in tests that assess the potential health effects of wastewater effluents. They also suggest that for some effluents, genotoxic and immune end points may be more sensitive than estrogenic (endocrine-mediated) end points as indicators of exposure in fish.

The anatomy of the nervous system of the hydrozoan jellyfish, Polyorchis penicillatus, as revealed by a monoclonal antibody.

Dissociated cells from the margin and tentacles of the hydromedusa Polyorchis penicillatus were centrifuged in a Percoll gradient to remove cnidocytes. The resulting formaldehyde-fixed cells were used to inoculate mice to produce monoclonal antibodies. One of the hybridomas, which secreted antibodies against all neurons, was cloned and designated as mAb 5C6. Immunohistochemical labelling with mAb 5C6 of whole-mount preparations and paraffin sections provided a far more complete picture of the organisation of the hydromedusan nervous system than was previously available when using neuronal labelling techniques that restrict labelling to certain neuronal types. Besides confirming anatomical features described in earlier studies these techniques allowed us to discover a number of new structures and to determine connections that were only suspected. Such findings included:1. The discovery of an arch-like connection between the swimming motor neuron network at the apices of the subumbrellar muscle sheets 2. An orthogonal network connecting each pair of radial nerves in each radius 3. Continuity of a central branch of the radial nerve with the radial innervation of the manubrium 4. Details of the sensory neuronal contribution to the microanatomy of the ocelli and cnidocyte batteries 5. Presence of specialised receptor cells in the margin at the bases of tentacles 6. Neurons apparently innervating the radial muscles of the velum 7. Isolated neurons in the peduncle and gonads

Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus).

In order to study the physiological implication of sex steroid hormones in gonadal sex differentiation in fish, we first investigated the potential role of estrogens using two fish models: the rainbow trout (Oncorhynchus mykiss) and a tilapia species (Oreochromis niloticus). All experiments were carried out on genetically all-male (XY) and all-female (XX) populations. In vivo treatments with an aromatase inhibitor (ATD, 1,4,6- androstatriene-3-17-dione) result in 100% masculinization of an all-female population in rainbow trout (dosage 50 mg/kg of food) and 75.3% in tilapia (dosage 150 mg/kg of food). In tilapia, the effectiveness of the aromatase inhibition by ATD is demonstrated by the marked decrease of the gonadal aromatase activity in treated animals versus control. No masculinization is obtained following treatment with an estrogen receptor antagonist (tamoxifen) in both species. Aromatase and estrogen receptor gene expression was studied in rainbow trout by semi-quantitative RT-PCR in gonads sampled before, during and after sex-differentiation. Aromatase mRNA is specifically detected in female gonads, 3 weeks before the first sign of histological sex-differentiation, i.e., first female meiosis. Aromatase expression in male gonads is at least a few hundred times less than in female gonads. Estrogen receptor gene is expressed in both male and female gonads at all stages with no dimorphic expression between sexes. Specific aromatase gene expression before ovarian differentiation was also demonstrated using virtual Northern blot, with no expression detected in male differentiating gonads. From these results it can be concluded that estrogen synthesis is crucial for ovarian differentiation, and transcription of the aromatase gene can be proposed as a key step in that process in fish.

Morphofunctional study of the effects of fetal exposure to cyproterone acetate on the hypothalamo-pituitary-gonadal axis of adult rats.

Fetal exposure to cyproterone acetate (CPA), while allowing, normal sexual morphogenesis, has previously been shown to lead to functional endocrine abnormalities in adult rats of both sexes. Because of this, we examined morphologically and morphometrically the hypothalamic nuclei involved in sexual dimorphism as well as the pituitary lactotropes of rats exposed in utero from day 15 to 20 of gestation to CPA. Male and female offspring was studied at the age of 70-80 days. In both sexes the brain weight was lower (p less than 0.05) in CPA-treated than in control rats. Morphometrical investigations showed that the surface density (Sv) and the volume density (Vv) of the ventromedial nucleus were higher (p less than 0.05) in CPA-treated male than in control rats. By comparing sexes the Sv and Vv of the ventromedial nucleus were higher (p less than 0.01) in CPA-treated male than in corresponding female rats. Also the nuclear surface of the tyrosine hydroxylase-immunoreactive neurons of the arcuate nucleus was higher (p less than 0.05) in CPA-treated male than in female rats. In lactotropes of the pituitary gland the immunoreactive prolactin (PRL) was densitometrically increased (p less than 0.05) in CPA-treated female compared with control rats. By electron microscopy, PRL granules and autophagocytosis appeared to be more abundant in CPA-treated rats of both sexes. These data show that fetal exposure to CPA results in long-term anatomical and physiological alterations of hypothalamic and preoptic nuclei as well as of the pituitary lactotropes. These permanent changes support the functional endocrine abnormalities observed in adult rats.

Developmental sex differences in brain aromatase activity are related to androgen level.

Sex differences in the metabolism of testosterone (T) in the developing brain of quail were examined using an in vitro microassay. During each developmental stage (day before hatching, hatching and 2 days after hatching) aromatase activity was higher in hypothalamic areas than in a control neostriatal area. There was no sex difference in oestradiol-17 beta (E2) formation in the late embryonic brain or at hatching. But aromatase activity in the male preoptic-anterior hypothalamic area was 50% higher than in females by day 2. No regional differences in brain 5 beta-reductase activity were detected at any of the developmental stages sampled. There was a sex difference in production of catabolic 5 beta-reduced metabolites. Male 5 beta-reductase activity declined continuously from high embryonic levels in all areas, whereas female enzyme activity showed an increase at hatching. In contrast to plasma progesterone, levels of T were higher in the male than in the female by day 1 after hatching. We suggest that elevated circulating T in the male after hatching may account for the sexual dimorphism in brain aromatase activity.