RIA Determination and immunofluorescence localization of cyclic nucleotides in rainbow trout (Salmo gairdneri) testes

Schulz, Rüdiger; Blüm, Volker

doi:10.1016/0016-6480(85)90275-8

Cited by 6 publications

(3 citation statements)

References 25 publications

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“…Cyclic AMP has been localized in the germ cells by immunofluorescence staining [28,29], but production of cAMP by germ cells has not been shown. Cyclic AMP can cross gap junctions [30]; therefore the cAMP detected in germ cells could be derived from the Sertoli cell.…”

Section: Discussionmentioning

confidence: 97%

Differential Regulation of Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Response Element-Binding Protein and cAMP Response Element Modulator Messenger Ribonucleic Acid Transcripts by Follicle-Stimulating Hormone and Androgen in the Adult Rat Testis

West

Sharpe²,

Saunders

1994

Biology of Reproduction

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Hormonal regulation of the expression of mRNA transcripts for cAMP response element-binding protein (CREB) and cAMP response element modulator (CREM) during spermatogenesis was studied in the adult rat testis. Northern analysis of CREB and CREM identified two mRNA transcripts for CREM (2.4 and 1.6 kb) and one transcript for CREB (2.0 kb). Analysis of mRNAs from isolated testicular cells by reverse transcriptase polymerase chain reaction (RT/PCR) showed that CREM mRNAs were expressed by the germ cells but not the Sertoli or interstitial cells, whereas CREB mRNA was located in germ cells, Sertoli cells, and interstitial cells. RNA was isolated and analyzed from the testes of 1) rats treated for 24 h with FSH, 2) rats in which androgen withdrawal had been induced by ethane dimethane sulphonate (EDS) treatment 6 days earlier (EDS-treated), 3) EDS-treated rats supplemented with testosterone (EDS + T), or 4) intratesticular administration or dibutyryl cAMP (dbcAMP) in the preceding 24 h. CREM mRNA transcript expression was found to be decreased after all of these treatments in samples from intact testis and from isolated cells. Expression of the CREB transcript was also decreased by EDS-induced androgen withdrawal, but not by FSH or EDS + T. In situ hybridization of paraffin-embedded testis sections probed with digoxigenin-labeled riboprobes confirmed the localization of CREB and CREM mRNA to the same cell types as found with RT/PCR. No stage-dependent expression of CREM mRNA transcripts could be observed. Hybridization of the CREB probe was highest around the base of stage VII-VIII tubules, and this was shown to be androgen-dependent. The data presented suggest that regulation of the expression of CRE-binding protein mRNAs in Sertoli and germ cells during spermatogenesis is dependent on both androgen and FSH. However, the effects of androgen or FSH on the regulation of CRE-binding protein mRNAs are different.

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Section: Discussionmentioning

confidence: 97%

Differential Regulation of Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Response Element-Binding Protein and cAMP Response Element Modulator Messenger Ribonucleic Acid Transcripts by Follicle-Stimulating Hormone and Androgen in the Adult Rat Testis

West

Sharpe²,

Saunders

1994

Biology of Reproduction

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“…Various testicular somatic cells and germ cells were present in our cultures and no information enables the determination of which cell type re¯ects altered cAMP level induced by AR activation. Using radioimmunoassay and indirect immuno¯uorescence microscopy, Schulz and Blu È m (1985) have shown that spermatogenetic trout testes contain high levels of cAMP, and that in these testes, this cyclic nucleotide is present in somatic cells and germ cells. However, regulation of cAMP levels may differ in the two cell types, especially if they differentially express A 1 and A 2 ARs.…”

Section: Discussionmentioning

confidence: 99%

Adenosine receptor-adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

Loir¹

2001

Mol. Reprod. Dev.

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To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H‐thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5′‐N‐ethylcarboxamidoadenosine (NECA), and by 2‐p(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS‐21680), an adenosine A2A receptor‐selective agonist. The increase in cAMP induced by the adenylate cyclase activator L‐858051 was inhibited by the adenosine A1 receptor‐selective agonists R‐N6‐(2‐phenylisopropyl)adenosine (R‐PIA) and N6‐cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2 receptor antagonists 3,7‐dimethyl‐1‐propargylxanthine (DMPX) and 8‐(3‐chlorostyryl)caffeine (CSC), and by the adenosine A1 receptor‐selective antagonist 8‐cyclopentyl‐1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2′,5′‐dideoxyadenosine. These data suggest that A1 and A2 adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H‐thymidine incorporation decreased in the presence of the adenylate cyclase activator L‐858051 and of the cAMP analogs 8‐CPT cAMP and Sp‐5,6‐DCI‐cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20‐1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short‐term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP‐dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation. Mol. Reprod. Dev. 58:307–317, 2001. © 2001 Wiley‐Liss, Inc.

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“…The measurement of adenylate cyclase activity can be accomplished using radiometric assays that follow the incorporation of a radioactive precursor into cAMP (Salomon, 1979; Schulz and Blum, 1985). More commonly, however, a variety of methods that quantify cAMP have been used both for assessment of adenylate cyclase activity, as well as for measuring tissue content of cAMP or breakdown of this second messenger.…”

Section: Introductionmentioning

confidence: 99%

Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: Application in GPCR-mediated adenylate cyclase assays

Brown¹,

Kant²,

Mailman³

2009

Journal of Neuroscience Methods

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Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high-throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

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RIA Determination and immunofluorescence localization of cyclic nucleotides in rainbow trout (Salmo gairdneri) testes

Cited by 6 publications

References 25 publications

Differential Regulation of Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Response Element-Binding Protein and cAMP Response Element Modulator Messenger Ribonucleic Acid Transcripts by Follicle-Stimulating Hormone and Androgen in the Adult Rat Testis

Differential Regulation of Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Response Element-Binding Protein and cAMP Response Element Modulator Messenger Ribonucleic Acid Transcripts by Follicle-Stimulating Hormone and Androgen in the Adult Rat Testis

Adenosine receptor-adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: Application in GPCR-mediated adenylate cyclase assays

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