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Prolactin PRL in fish is considered to be an osmoregulatory hormone, c-mario some studies suggest that it may influence the production of steroid hormones in the gonads.

The objective of the present study was to establish if PRL is involved in reproduction of the gilthead seabream—a protandrous hermaphrodite. Northern blot analysis revealed a single pituitary PRL transcript, the expression of which was significantly reduced by E 2 treatment in adults but significantly increased in juvenile fish.

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In adult gonads, four sbPRLR transcripts of 1. A competitive reverse transcription-polymerase chain reaction was developed and used to determine how E 2 treatment alters expression of the gonadal sbPRLR gene.

Seabream PRLR was detectable in all samples analyzed by this assay. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected the receptor in spermatogonia and oocytes. Taken together, the preceding results suggest that in the seabream, PRL may act on both testis and ovary via its receptor and that the stage of maturity influences this process.

The full characterization and relative importance of the different transcripts of sbPRLR in eliciting the action of PRL in the gonads remain to be elucidated. Prolactin PRL is a kDa polypeptidic hormone secreted by the anterior pituitary in all vertebrates [ 12 ]. One of the well-known actions of PRL is in the reproductive physiology of mammals. For example, PRL acts on the mammary gland and specifically stimulates DNA synthesis, epithelial cell proliferation, and synthesis of milk proteins casein and lactalbuminfree fatty acids, and lactose [ 5 ].

Prolactin has also been shown to regulate luteal functions, follicular steroidogenesis, and follicular growth in the ovary during reproduction [ 67 ], and homozygous female PRLR knockout mice are completely infertile and lack normal mammary gland development [ 8 ]. In male animals, PRL plays an important role in maturation of the gonads, and PRL supplementation to immature, hypophysectomized rats stimulates the multiplication and differentiation of Leydig cells and germ cells in a dose-dependent manner [ 9 ].

These observations suggest that in the testis, PRL may be a trophic hormone that acts directly on testicular tissue. In fish, PRL is considered to be primarily an osmoregulatory hormone [ 12 ], although some studies suggest PRL may be associated with production of steroid hormones in the gonads, the onset of gonadal development, and reproductive behavior [ 13 ]. Mammalian PRL stimulated testosterone production in the goby Gobius niger [ 14 ] suggesting a possible role for PRL in the regulation of testicular function.

Furthermore, specific PRL-binding sites have been detected in seminal vesicle cells of the same species [ 15 ] and in tilapia Oreochromis mossambicus testis [ 16 ]. In mammals, estrogens have a positive effect on PRL secretion. In teleosts, the way in which E 2 influences PRLR expression in the gonads and the cellular localization of the receptor in fish have, to our knowledge, never been described.

Thus, the primary aim of the present study was to determine the effects of E 2 on the expression of PRL in the pituitary gland and on the gonadal expression of sbPRLR in the gilthead seabream. The gonads of this protandrous hermaphrodite are characterized by their bisexuality and consist of a mediodorsal ovarian area and a lateroventral testicular zone, separated by connective tissue [ 20 ]. The hermaphroditic nature and the poorly understood mechanisms underlying sex reversal of this species make the seabream an interesting comparative model for studying the involvement of PRL and its receptor in reproduction.

The generally low level of receptor expression means that methods such as mRNA blots, image analysis of in situ hybridization, and immunohistochemistry may be too insensitive for quantifying receptor gene expression. The cellular localization of sbPRLR in the gonads was determined by immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR.

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All procedures involving experimental animals were conducted in accordance with Portuguese national regulations. The length of the control and E 2 fish were, respectively, One week after implantation, fish were anesthetized in 0. Fish were weighed and measured, and blood samples were collected in 2-ml, heparinized syringes from the messangr vasculature.

The gonads were removed and weighed, and the ratio between testicular tissue white color and ovarian tissue yellow color was scored. The antiserum against the extracellular domain of sbPRLR has previously been characterized for immunohistochemistry [ 18 ]. Total RNA was extracted from whole pituitaries, from mg of the gonads adultsand from all gonads juveniles using TRI Reagent Sigma-Aldrich according to the manufacturer’s protocol.

The expected product was base pairs bp.

Primers were then prepared for competitor template DNA. Sense and antisense primers were composed of a sequence specific for the competitor template and were flanked by the sequence of sense and antisense primers of target DNA, respectively. In addition, the sense primer for the competitor template was flanked messanget the sequence of the SP6 promoter region. The size of the DNA competitor was bp.

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In vitro transcription was performed according to a standard protocol from Promega Madison, WI with a few modifications. The aqueous phase was transferred to a fresh tube, and one volume of chloroform: The aqueous phase was then transferred to a fresh tube, and the jessanger was precipitated by addition of 2. To confirm that the amplification c-nrio for competitor and target templates were similar, equal amounts of competitor cRNA and target RNA were introduced into the RT reaction, and synthesis was subsequently analyzed.

As a negative control, sterile water substituted for cDNA, and as positive controls, cDNA synthesized from competitor or intestine sample alone was used. The PCR cycle was as follows: Band intensities were quantified using Image Master Amersham-Pharmacia and plotting of optical density as a function of cycle number.

The target RNA was quantified by densitometry plotting of the ratio of competitor to target band intensity as a function of the initial amounts of the competitor cRNA added fold dilutions ranging from 0.

A ratio of 1 equivalent concentrations was taken to indicate that equal amounts of competitor and target RNA were present and was used to determine the amount of target RNA in the reaction. Levels of E 2testosterone, and ketotestosterone are in nanograms per milliliter of plasma. Data from the kinetics of amplification are expressed as optical density units. In adults implanted with E 2circulating E 2 steroid levels at the end of the experimental period were twice as high as in controls control, 0.

No significant differences in the GSI control, 0. In juveniles, circulating E 2 levels in E 2 -treated fish increased significantly control, 0. No mortality occurred during both experiments. Treatment with E 2 did not alter transcript size or number. In contrast, in juveniles, E 2 treatment caused a significant 1. The blot was hybridized with the full-length sbPRL probe and c-nnario for 1 h. In the adult gonads, four PRLR transcripts of 1.

No significant differences were observed between control and E 2 -treated messnger regarding the number, size, and abundance of transcripts. The smallest transcript 1. The blot was hybridized with a sbPRLR probe and exposed for 1 wk. The c-narii indicate the sizes of the transcripts. The linear portion of the amplification curves had very similar slopes Fig.

The plot of log ratio of sbPRLR: However, sbPRLR expression was significantly increased by fold, 0. A clear difference in control values of the receptor was registered between adult and juvenile animals. Kinetics of amplification of the sbPRLR target and competitor.

The upper band corresponds to the competitor. Products corresponding to target gene sbPRLR and competitor are indicated. Following gel electrophoresis, the bands intensity was determined. Data are plotted as units of optical density versus cycle number. Note that the competitor was loaded in decreasing concentrations from left to right on the gel. For immunohistochemistry, only gonads from control animals were analyzed.

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The mature gonads were composed principally of testicular tissue, with abundant spermatozoa in the sperm duct Fig. The ovarian region of the gonads was filled with oogonia and perinucleolar oocytes Fig. Immunoreactivity in testis was messwnger intense in the spermatogonia, particularly in the region surrounding the nucleus, although the heads of spermatozoa also stained less intensely.

In the oocytes of the ovarian tissue, the staining pattern was cytoplasmic, and a strong reaction was found in the region nearest to the perinuclear membrane Fig. A positive immunoreaction is present in spermatogonia SG and the heads of some spermatozoa SZ. A strong signal is observed in some oocytes in the perinuclear region Po. Despite the well-known roles of PRL in the reproductive physiology of higher vertebrates, most of the identified hormonal functions in fish have been in hydromineral balance e.

The present study provides strong evidence for a role of PRL in fish reproduction. Treatment with E 2 resulted in a significant increase in pituitary PRL mRNA in juvenile gilthead seabream, and this agrees with a previously observed, dose-dependent increase in pituitary synthesis of two forms of PRL in E 2 -treated tilapia in vivo and in vitro [ 2425 ]. This was taken to suggest that PRL cells are not regulated by estrogens in this species [ 26 ].

The dosage and duration of the experiments may nevertheless explain the conflicting results. A direct action on PRL cells is supported by the presence of estrogen-response elements in the upstream regulatory regions of the rat PRL gene [ 27 — 29 ], and mammalian lactotrophs and gonadotrophs have been shown to express estrogen receptor ER [ 30 — 33 ].

Furthermore, E 2 strongly increases GnRH fibers and pituitary innervation in immature African catfish Clarias gariepinusand an estrogen-response element has been found in the human GnRH gene [ 39 ].

In addition, a homologue of mammalian PRL-releasing peptide PRP has been isolated from tilapia brain, characterized, and shown to significantly increase circulating PRL levels [ 41 ].

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Whether the neurons producing PRP could be a target for E 2 needs investigation. In contrast to juvenile fish and mammals, in which E 2 has a stimulatory effect on PRL gene transcription [ 19 ], in adult seabream it caused a drastic reduction in PRL gene expression.

A range of factors may explain this difference, although it is not possible from the present results to determine the underlying cause s. However, the differing hormonal status of the mature adult and immature juvenile seabream e. In fact, one of the characteristic features of juvenile vertebrates is the quiescent state of activity of the brain-pituitary-gonad axis as a functional unit [ 4243 ].

The difference in the status of the gonads may also be a contributing factor: Further studies will be required to elucidate the answers to this question. The difference between fish species may be explained again by the developmental status of the brain-pituitary-gonad axis, which in African catfish is more sensitive to positive feedback of sex steroids during puberty [ 4243 ].

The presence of numerous PRLR transcripts is in direct contrast to previous observations in tilapia Oreochromis niloticus3. The pattern of PRLR expression in seabream is more like that in other vertebrate groups, in which more than one PRLR transcript is present in the gonads e. Both in chicken and seabream, the 1. In addition, adult chickens produced three distinct, truncated, testis-specific cPRLR transcripts, of which two lacked regions coding for the extracellular and transmembrane domains [ 51 ].

The functional significance of this is unclear. In mammals, the PRLR gene consists of 11 exons [ 5253 ], which generate alternative splicing long and short forms of the receptor, which differ only in the intracellular domains [ 7 ].

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