Nonsense-mediated Decay of mRNA for the Selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase Is Detectable in Cultured Cells but Masked or Inhibited in Rat Tissues

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Refractoriness to melatonin occurs independently at multiple brain sites in Siberian hamsters

The mid-winter development of refractoriness to melatonin (Mel) triggers recrudescence of the atrophied reproductive apparatus of rodents. As a consequence, over-wintering animals become reproductively competent just before the onset of spring conditions favorable for breeding. The neural target tissues that cease to respond to winter Mel signals have not been identified. We now report that the suprachiasmatic nucleus of the hypothalamus, which contains the principal circadian clock, and the reuniens and paraventricular nuclei of the thalamus, each independently becomes refractory to melatonin. Small implants of Mel that were left in place for 40 wk and that act locally on these brain nuclei, induced testicular regression within 6 wk in male Siberian hamsters; 12 wk later Mel implants no longer suppressed reproduction and gonadal recrudescence ensued. Hamsters that were then given a systemic Mel infusion s.c. immediately initiated a second gonadal regression, implying that neurons at each site become refractory to Mel without compromising responsiveness of other Mel target tissues. Refractoriness occurs locally and independently at each neural target tissue, rather than in a separate “refractoriness” substrate. Restricted, target-specific actions of Mel are consistent with the independent regulation by day length of the several behavioral and physiological traits that vary seasonally in mammals.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

The human CAS protein which is homologous to the CSE1 yeast chromosome segregation gene product is associated with microtubules and mitotic spindle.

Human CAS cDNA contains a 971-aa open reading frame that is homologous to the essential yeast gene CSE1. CSE1 is involved in chromosome segregation and is necessary for B-type cyclin degradation in mitosis. Using antibodies to CAS, it was shown that CAS levels are high in proliferating and low in nonproliferating cells. Here we describe the distribution of CAS in cells and tissues analyzed with antibodies against CAS. CAS is an approximately 100-kDa protein present in the cytoplasm of proliferating cells at levels between 2 x 10(5) and 1 x 10(6) molecules per cell. The intracellular distribution of CAS resembles that of tubulin. In interphase cells, anti-CAS antibody shows microtubule-like patterns and in mitotic cells it labels the mitotic spindle. CAS is removed from microtubules by mild detergent treatment (cytoskeleton preparations) and in vincristine- or taxol-treated cells. CAS is diffusely distributed in the cytoplasm with only traces present in tubulin paracrystals or bundles. Thus, CAS appears to be associated with but not to be an integral part of microtubules. Immunohistochemical staining of frozen tissues shows elevated amounts of CAS in proliferating cells such as testicular spermatogonia and cells in the basal layer cells of the colon. CAS was also concentrated in the respiratory epithelium of the trachea and in axons and Purkinje cells in the cerebellum. These cells contain many microtubules. The cellular location of CAS is consistent with an important role in cell division as well as in ciliary movement and vesicular transport.

Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules.

We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.

Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by approximately 35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than approximately 35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial, mutations. The most prominent feature of the ENU-induced base-pair mutations in testicular germ cells sampled 55 days after treatment is that 70% are induced in A.T base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.

Sazonalidade reprodutiva em machos de tartaruga (Kinosternon scorpioides) de vida livre no Nordeste brasileiro evidenciado por imunolocalização de enzimas esteroidogênicas no testículo e epidídimo

No Estado do Maranhão, na região da Baixada Maranhense, presenta na fauna silvestre o réptil Kinosternon scorpioides, um quelônio de água doce popularmente conhecido como jurará e que possui valor social, econômico e ambiental para os ribeirinhos da cidade de São Bento. Este estudo contempla suas características biológicas reprodutivas baseadas em seu ambiente natural, com o intuito de permitir a preservação e o estabelecimento de planos de manejo reprodutivo e de uso sustentável da espécie. Recentemente poucos estudos em todo o mundo tratam sobre os aspectos do ciclo reprodutivo de tartarugas em face das características endócrinas, na América do Sul estudos desse tipo são recentes e escassos, sendo assim este é o primeiro estudo, que se tem conhecimento, que elucida um padrão sazonal reprodutivo da espécie K. scorpioides, associando hormônios gonadais com aspectos comportamentais. Trinta e oito animais adultos tiveram seus órgãos reprodutivos caracterizados para as enzimas esteroidogênicas P450 aromatase, P450c17 e PNADPH redutase através de imunomarcação e blotting, além de índice gonadossomático, morfometria e concentração de testosterona, corticosterona e estradiol pela técnica de radioimunoensaio. As mudanças biométricas, morfometria celular e a esteroidogênese testicular entre os períodos chuvoso e seco sugerem que o estrógeno produzido pelas células de leydig podem afetar a produção e a apoptose de células germinais durante o processo de espermatogênese, e a presença das enzimas P450aromatase e P450c17 em células de leydig acompanhados com a recrudescência testicular também reforça a ideia, de que elas podem desempenhar um papel na quiescência testicular. Esse trabalho evidenciou que as enzimas citocromo P450, citocromo P450c17 e NADPH-citocromo P450-redutase estão presentes no testículo e epidídimo nos diferentes períodos climáticos e podem estar relacionados à síntese de testosterona em tartarugas concordando com os diferentes achados para biometria e espermiogênese nos períodos chuvoso e seco, o que, nos leva a sugerir um estado de quiescência durante o período seco e atividade espermatogênica no período chuvoso, semelhante ao que ocorre com as espécies que apresentam comportamento reprodutivo sazonal

Espermatogênese xenogênica pós-transplante de células tronco caninas no testículo de camundongos

As células tronco espermatogoniais (SSCs) são caracterizadas pela capacidade de autorrenovação, proliferação e transmissão das informações genéticas. Em caninos a primeira tentativa de xenotransplante não obteve o sucesso da produção de espermatozoides, no entanto, há evidências de que as células testiculares xenogênicas podem ser transplantadas no testículo do animal hospedeiro, e gerar espermatozoides viáveis do doador. Portanto, este estudo tem como objetivo realizar o xenotransplante das células germinativas caninas em camundongos imunosuprimidos, e com isto promover à produção de espermatozoides caninos viáveis, geneticamente modificados. E por meio desta técnica, analisar a eficiência da espermatogênese pós-transplante. Células germinativas testiculares foram caracterizadas, isoladas e cultivadas de cães pré-púberes, por meio de sistemas de cultura de enriquecimento e fatores de crescimento. As células foram transduzidas com um gene repórter GFP e LacZ, e por um vetor lentiviral para indentificar as SSCs nos testículos receptores. As SSCs transduzidas foram transplantadas nos testículos de camundongos (C57BL/6) tratados com Busulfan, após diferentes períodos os animais receptores foram eutanasiados e analisados. Aos 10 dias de cultivo as células germinativas adultas foram positivas para CD49f, CD117, e com 5 dias uma expressão semelhante de GFRA1 e DAZL, demonstrando a presença de SSCs e algumas células em meiose. Transplantamos 105 células e 20-43% das células transplantadas foram identificadas na membrana basal dos túbulos seminíferos do animal receptor. Portanto, o transplante das células germinativas caninas, mostrou que a purificação e o cultivo realizados são possíveis para obter SSCs caninas, as quais colonizaram os túbulos seminíferos dos camundongos imunodeficientes e mantiveram-se vivas na membrana basal por 90 dias após transplante, mesmo que estes animais tenham distância filogenética

Tumores de células germinativas do testículo : revisão da casuística do Serviço de Urologia do Hospital de Santa Maria (CHLN, EPE)

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

Organotypic Modeling Platform for Adverse Outcome Pathways of Male Reproductive and Developmental Processes

Thesis (Master's)--University of Washington, 2016-06

Haemodynamic assessment following inferior vena cava resection without replacement

Background: Treatment of bulky retroperitoneal malignancy may require en bloc resection of the infrarenal inferior vena cava. A number of reconstructive options are available to the surgeon but objective haemodynamic assessment of the peripheral venous system following resection without replacement is lacking. The aim of the present paper was thus to determine the symptomatic and haemodynamic effects of not reconstructing the resected infrarenal inferior vena cava. Methods: A retrospective descriptive study was carried out at Princess Alexandra Hospital in Queensland. Five patients underwent resection of the thrombosed infrarenal inferior vena cava as part of retroperitoneal lymph node dissection for testicular cancer (n = 3), radical nephrectomy for renal cell carcinoma (n = 1) and thrombosed inferior vena cava aneurysm (n = 1). Clinical effects were determined via the modified venous clinical severity score and venous disability score. Haemodynamic data were obtained postoperatively using venous duplex ultrasound and air plethysmography. Results: None of the present patients scored >2 (out of 30) on the modified venous clinical severity score or >1 (out of 3) on the venous disability score. Haemodynamic studies showed only minor abnormalities. Conclusions: Not reconstructing the resected thrombosed infrarenal inferior vena cava results in minor signs and symptoms of peripheral venous hypertension and only minor abnormalities on haemodynamic assessment.

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus)

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 mu g IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans. (c) 2005 Elsevier B.V. All rights reserved.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.