RESUMO
During the course of a study of glycoprotein processing mannosidases in the rat epididymis, we have made an intriguing discovery regarding the presence of a novel alpha-D-mannosidase on the rat sperm plasma membranes. Unlike the sperm acrosomal "acid" mannosidase which has a pH optimum of 4.4, the newly discovered alpha-D-mannosidase has a pH optimum of 6.2, and 6.5 when assayed in sperm plasma membranes and intact spermatozoa, respectively. In addition, the two enzymes show different substrate specificity. The acrosomal alpha-D-mannosidase is active mainly towards synthetic substrate, p-nitrophenyl alpha-D-mannopyranoside, whereas the sperm plasma membrane alpha-D-mannosidase shows activity mainly towards mannose-containing oligosaccharides. Evidence is presented which suggest that the sperm plasma membrane alpha-D-mannosidase is different from several processing mannosidases previously characterized from the rat liver. The newly discovered alpha-D-mannosidase appears to be an intrinsic plasma membrane component, since washing of the purified membranes with buffered 0.4 M NaCl did not release the enzyme in soluble form. The enzyme requires nonionic detergent (Triton X-100) for complete solubilization. The enzyme is activated by Co2+ and Mn2+. However, Cu2+ and Zn2+ are potent inhibitors of the sperm plasma membrane alpha-D-mannosidase. At a concentration of 0.1 mM, these divalent cations caused nearly complete inactivation of the sperm enzyme. In addition methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, mannose, 2-deoxy-D-glucose, and D-mannosamine are inhibitors of the sperm surface alpha-D-mannosidase. The physiological role of the newly discovered enzyme is not yet known. Several published reports in three species, including the rat, suggest that the sperm surface alpha-D-mannosidase may have a role in binding to mannose-containing saccharides presumably present on the zona pellucida.
Assuntos
Manosidases/metabolismo , Interações Espermatozoide-Óvulo , Espermatozoides/enzimologia , Animais , Fracionamento Celular , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Centrifugação com Gradiente de Concentração , Feminino , Cinética , Masculino , Manosidases/isolamento & purificação , Microscopia Eletrônica , Orquiectomia , Ratos , Valores de Referência , Especificidade por Substrato , Testículo/fisiologia , alfa-ManosidaseRESUMO
We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.
Assuntos
Marcação de Genes , Vetores Genéticos , Proteínas Proto-Oncogênicas/genética , Recombinação Genética , Animais , Células Cultivadas , Mapeamento Cromossômico , Cromossomos/ultraestrutura , Genes , Hidrolases/genética , Técnicas In Vitro , Camundongos , Proteínas Tirosina Quinases/genética , Mapeamento por Restrição , Quinases da Família srcRESUMO
We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.
Assuntos
Técnicas Genéticas , Recombinação Genética , Alelos , Sequência de Aminoácidos , Troca Genética , DNA Complementar/metabolismo , Biblioteca Gênica , Dados de Sequência Molecular , Mutagênese , Mutação , Nocardia/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Rhodococcus/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Analgesics given preoperatively have the potential to decrease the amount of inhalant anesthetics required intraoperatively (i.e., to decrease the minimum alveolar concentration, or MAC, for the inhalant). Tepoxalin is an NSAID approved for the treatment of arthritis in dogs in the United States and, hence, could be administered to patients undergoing anesthesia. In this study, administration of a single dose or a 10-day course of tepoxalin did not affect the MAC for isoflurane or sevoflurane.
Assuntos
Anestésicos Inalatórios/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Cães/fisiologia , Alvéolos Pulmonares/efeitos dos fármacos , Pirazóis/farmacologia , Administração por Inalação , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Interações Medicamentosas , Feminino , Isoflurano/administração & dosagem , Masculino , Éteres Metílicos/administração & dosagem , Medicação Pré-Anestésica/veterinária , Alvéolos Pulmonares/metabolismo , Pirazóis/uso terapêutico , Distribuição Aleatória , Sevoflurano , Resultado do TratamentoRESUMO
The expression and androgen regulation of egasyn, the endoplasmic reticulum-targeting protein of beta-D-glucuronidase, was examined in the mouse-epididymis. The proximal (caput) and distal (corpus & cauda) epididymal tissue extracts were prepared by homogenization and sonication in buffered Triton X-100 solution, and high speed centrifugation. The supernatant when resolved by 2D-PAGE under non-denaturing conditions and stained for esterase activity showed that the distal (but not proximal) epididymis of the normal mouse contain several specific forms of esterases. These forms include a series of four variants (pI 5.2-5.75) with high mobility (HM) and esterase activity, and three faintly staining variants (beginning at pI 6.0) with low mobility (LM). Several lines of evidence indicate that the specific esterases seen in the corpus/cauda epididymidis are egasyn-esterases. Firstly, these molecular forms were not seen in the distal epididymal extracts from the egasyn-deficient mouse. Secondly, the HM forms can be immunoprecipitated with anti-egasyn antibody, suggesting the presence of free egasyn. Finally, the LM forms disappeared after heat treatment (56 degrees C for 8 min), a condition known to dissociate egasyn:beta-D-glucuronidase complex. This result indicates that a small amount of egasyn is complexed with beta-D-glucuronidase. Immunoblotting (Western blot) studies (using anti-egasyn antibody) following resolution of egasyn released from the egasyn:beta-D-glucuronidase complex revealed a single band of an apparent molecular weight 64 kDa in the distal (but not proximal) epididymis, indicating that the mouse epididymal egasyn is identical or very similar to the liver egasyn. Castration of mice lead to the appearance of free and complexed egasyn forms in the proximal epididymis. Testosterone supplementation to the castrated mice resulted in the disappearance of the induced egasyn forms from the caput epididymidis. Taken together, these results indicate that the expression of egasyn in the epididymis is region-specific and is differentially regulated by androgens.
Assuntos
Androgênios/farmacologia , Hidrolases de Éster Carboxílico/análise , Hidrolases de Éster Carboxílico/efeitos dos fármacos , Epididimo/efeitos dos fármacos , Epididimo/enzimologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/efeitos dos fármacos , Animais , Antineoplásicos Hormonais/administração & dosagem , Hidrolases de Éster Carboxílico/deficiência , Castração , Esterases/química , Masculino , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Testes de Precipitina , Testosterona/administração & dosagemRESUMO
As a result of examining regional-specific gene expression in the mouse epididymis, a novel cystatin-related epididymal specific (CRES) gene was identified. Substantial homology between the CRES gene and members of the cystatin family of cysteine proteinase inhibitors was observed at the amino acid level. This homology included the presence of four highly conserved cysteine residues in exact alignment with the cystatins as well as other regions of sequence characteristic of the cystatins. However, unlike the cystatins, the CRES gene does not contain specific highly conserved sequence motifs thought to be necessary for cysteine proteinase inhibitory activity. Also, in contrast to the ubiquitous expression of the cystatin C gene, Northern blot analysis and in situ hybridization demonstrated that the CRES gene is very restricted in its expression. The 0.75-kilobase CRES transcript is dramatically restricted to the very proximal caput region of the epididymis with 15- to 20-fold less expression in the testis and no expression detected in any of the other 24 tissues examined. In addition, the CRES transcript disappears 2-3 weeks after castration, suggesting a dependence on androgens. However, its expression remained undetectable even after the administration of testosterone or dihydrotestosterone. Unilateral castration also resulted in the disappearance of the CRES mRNA from the castrate epididymis, but not from the intact epididymis, suggesting that testicular factors or hormones other than androgens may be involved in the regulation of CRES gene expression. Therefore, the unique sequence of the CRES gene as well as its highly restricted expression and unusual regulation by the testis suggests that it has a very specialized role in the epididymis.
Assuntos
Cistatinas/genética , Epididimo/fisiologia , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , DNA/genética , DNA/isolamento & purificação , Epididimo/citologia , Feminino , Expressão Gênica , Humanos , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA/genética , RNA/isolamento & purificação , RNA Antissenso , Homologia de Sequência de AminoácidosRESUMO
Using subtractive hybridization to identify genes that are androgen regulated in the mouse epididymis, a number of cDNAs were identified that represented mitochondrial genes including cytochrome oxidase c subunits I, II, and III, cytochrome b, NADH dehydrogenase subunit 5, a region of the displacement loop, and the 16S rRNA. Northern blot analysis of RNA from intact, castrate, or testosterone-replaced epididymides confirmed that these mitochondrial mRNAs as well as the rRNA were androgen regulated with a 2- to 5-fold reduction in expression observed after 4 weeks castration with partial to full recovery to precastrate levels upon 4 weeks of testosterone replacement. In contrast to the mitochondrial genes, the expression of the RNA component of the mitochondrial RNA-processing endoribonuclease (RNAase MRP), a nuclear factor which is thought to be involved in the regulation of mitochondrial DNA synthesis, increased in the epididymis upon castration and then returned to precastrate levels after testosterone replacement. An examination of other androgen-responsive tissues showed that mitochondrial gene expression was also regulated by androgens in the kidney. The RNAase MRP RNA levels, however, showed an increase after castration only in the reproductive tissues (epididymis, vas deferens, and seminal vesicle) and not in the kidney. No correlative increase in mitochondrial DNA levels was observed for any of the tissues. Finally, an analysis of various mouse tissues as well as the different regions of the epididymis revealed large differences in mitochondrial mRNA levels. While for most tissues the mRNA levels correlated with the mitochondrial DNA content, the levels of the RNAase MRP RNA did not. Taken together, these findings not only show the large variations in mitochondrial gene expression between tissues but also demonstrate that the expression of mitochondrial genes and ultimately mitochondrial function are androgen regulated in the epididymis and kidney.
Assuntos
DNA Mitocondrial/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Testosterona/farmacologia , Animais , Sequência de Bases , Epididimo/efeitos dos fármacos , Epididimo/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL/genética , Dados de Sequência Molecular , Orquiectomia , Especificidade de ÓrgãosRESUMO
Infertile spermatozoa from the proximal corpus epididymidis will become fertile when this epididymal segment is cultured 24 h in vitro with 5alpha-dihydrotestosterone (5alpha-DHT). The effect of antiandrogens and inhibitors of RNA and protein synthesis upon this 5alpha-DHT-induced sperm maturation was investigated. The response to 5alpha-DHT was abolished by cyproterone acetate, SKF 7690, actinomycin D, cycloheximide, puromycin, and the aminonucleoside analog of puromycin. Addition of cyproterone acetate or actinomycin D to a suspension of distal corpus spermatozoa did not change their fertilizing ability. Culture of segments of the distal corpus for 24 h with the same antiandrogens and RNA and protein synthesis inhibitors did not change the fertilizing ability of spermatozoa. These results indicate that the development of the sperm-fertilizing ability observed in the proximal corpus epididymidis in vitro in response to 5alpha-DHT is dependent upon binding of 5alpha-DHT and synthesis of new RNA and protein molecules by the target cell. They also indicate that antiandrogen and inhibitors of RNA and protein synthesis do not have a nonspecific toxic effect on spermatozoa during the time period studied. It is likely that the target cells are the epididymal epithelial cells rather than the spermatozoa themselves.
Assuntos
Antagonistas de Androgênios/farmacologia , Ciproterona/farmacologia , Di-Hidrotestosterona/farmacologia , Epididimo/fisiologia , Metiltestosterona/análogos & derivados , Biossíntese de Proteínas , RNA/biossíntese , Espermatozoides/fisiologia , Animais , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Masculino , Metiltestosterona/farmacologia , Técnicas de Cultura de Órgãos , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , Ratos , Espermatozoides/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
The hypothesized relationship between androgen-binding protein (ABP) and sperm maturation was investigated using a mutant rodent: the restricted rat. The seminiferous epithelium of these animals undergoes a spontaneous degeneration, but changes are progressive. Restricted rats in the transition to infertility were used to determine if changes in ABP were related to the decreased fertility found in these animals. Fertilizing ability was determined by insemination of cauda epididymal spermatozoa into hormonally primed female rats and examination of ova for evidence of fertilization 48 h later. Epididymal and testicular tissues were analyzed for ABP using a charcoal assay. Androgen levels were determined by RIA. Testicular weights were significantly reduced compared to those of normal littermates in restricted rats at all ages; epididymal weights were significantly reduced in rats 140 days and older. Among restricted rats, sperm fertilizing ability was variable, but was significantly lower than that in normal littermates; it was consistently highest at 90 days of age. Epididymal ABP content (picomoles per organ) was significantly reduced in restricted rats at all ages; peak values occurred at 90 days. Testicular ABP content was significantly reduced only in the youngest and oldest animals. Plasma testosterone levels were not statistically lower than those found in normal littermates, and ventral prostate weights were maintained at normal levels in all four groups of animals. A significant positive correlation existed between sperm fertilizing ability and epididymal ABP, but not between sperm fertilizing ability and plasma testosterone. Since ABP is an index of Sertoli cell function, these data indicate that sperm fertilizing ability is closely related to Sertoli cell function and/or ABP.
Assuntos
Proteína de Ligação a Androgênios/metabolismo , Proteínas de Transporte/metabolismo , Fertilização , Maturação do Esperma , Espermatozoides/fisiologia , Animais , Peso Corporal , Epididimo/anatomia & histologia , Feminino , Masculino , Tamanho do Órgão , Ratos , Ratos Mutantes , Testículo/anatomia & histologia , Testosterona/sangueRESUMO
Hypophysectomized rats maintained with 2 mg pregnenolone have low plasma testosterone levels, but rete testis levels are normal. This model system was used to examine the importance of androgen-binding protein (ABP) in maintaining high luminal androgen concentrations for the development and maintenance of sperm fertilizing ability in the epididymis. Hypophysectomized rats were injected with pregnenolone (1, 0.5, 0.2, or 0.02 mg/100 g BW) for 14 days, starting 1 day after surgery. Sham-operated rats and a group of hypophysectomized rats were injected with oils as controls. At the end of the experimental period, sperm fertilizing ability, tissue weights, ABP, and testosterone levels were determined. The 1- and 0.5-mg doses of pregnenolone resulted in rete testis testosterone levels that were 67% and 29%, respectively, of the levels found in sham-operated animals. Plasma testosterone levels were not different from levels found in hypophysectomized, oil-injected controls with any of the pregnenolone doses. The three highest doses of pregnenolone resulted in levels of testicular ABP that were not statistically different from sham-operated levels (2 pmol/organ). Epididymal ABP content was maintained at sham-operated control levels (30 pmol/organ) with the 1-mg dose; with the 0.5- and 0.2-mg doses, ABP levels were 76% and 73% of sham-operated levels, respectively. Epididymal ABP specific content (picomoles per 100 mg tissue) was maintained at sham-operated control levels with the 1-, 0.5-, and 0.2-mg doses. Sperm fertilizing ability was maintained at sham-operated control levels with the three highest doses of pregnenolone. The 0.02-mg dose of pregnenolone resulted in values that were not statistically different from hypophysectomized, oil-injected control values for all parameters tested. A significant positive correlation existed between ABP and sperm fertilizing ability. These data demonstrate that testicular and epididymal ABP levels can be maintained in hypophysectomized rats with pregnenolone treatment alone, and that sperm fertilizing ability can be maintained when intraluminal androgen levels are low and ABP levels are near normal. This suggests that ABP may be important in the maintenance of normal sperm fertilizing ability.
Assuntos
Proteína de Ligação a Androgênios/metabolismo , Proteínas de Transporte/metabolismo , Fertilidade/efeitos dos fármacos , Pregnenolona/farmacologia , Espermatozoides/fisiologia , Testosterona/metabolismo , Animais , Relação Dose-Resposta a Droga , Epididimo/efeitos dos fármacos , Hipofisectomia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/fisiologiaRESUMO
The effects of estradiol benzoate (EB), testosterone propionate (TP), and Tamoxifen, alone or in combination, on the weight and morphology of the male accessory sex glands were studied in intact and in castrated immature rabbits. TP treatments (2 mg/kg) exerted a stimulatory effect on the glands, resulting in a significant increase in the weight of the epididymis, the proprostate, and the prostate of castrated rabbits, and of the bulbourethral glands of both intact and castrated rabbits, and in marked morphological changes in all the glands. the epithelium was stimulated but retained its pseudostratified columnar appearance and resembled more the epithelium of normal mature males than that of age matched controls. The bulbourethral glands were the most responsive and the vesicular gland the least responsive to androgen treatment. The response of the glands to EB (25 micrograms/kg) was characterized by significant weight increases in all the glands of both castrated and intact rabbits, hypertrophy of the musculo-fibrous components and proliferation of the basal layer of the epithelium leading to squamous metaplasia and leukocytic infiltration. Hyperplasia of the fibromuscular stroma was most evident in the cauda epididymidis and in the vesicular gland. Squamous metaplasia and leukocytic infiltration were most evident in the ejaculatory duct and in the structures adjacent to it. The antiestrogen, Tamoxifen (250 micrograms/kg), and TP (2 mg/kg) given in conjunction with EB (25 micrograms/kg) tended to reduce the weight increase caused by estrogen, but the decrease was significant in only a few instances. Tamoxifen (250 micrograms/kg) administered alone stimulated the epithelium of the accessory sex glands and induced squamous metaplasia, but did not induce hyperplasia of the fibromuscular stroma. The study demonstrates that accessory sex glands display a consistent pattern of differential sensitivity to both androgens and estrogens and that these hormones exert their action on different cell types within the organ.
Assuntos
Epididimo/efeitos dos fármacos , Estradiol/farmacologia , Genitália Masculina/efeitos dos fármacos , Tamoxifeno/farmacologia , Testosterona/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Epididimo/patologia , Genitália Masculina/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Coelhos , Maturidade SexualRESUMO
An interstitial target site in the rat testis for vitamin A has been detected using radioactively labeled rretinol-binding protein (RBP) in autoradiography at the light microscope level. RBP purified from rat serum was iodinated by the lactoperoxidase procedure and was also complexes with [3H]-retinol. The radioactively labeled RBP was administered to rat testis tissue by a direct intratesticular injection, an iv injection, and through an in vitro incubation of decapsulated tissue in medium containing the labeled protein. Localization on the autoradiograms of both [125I]RBP and [3H]retinol-RBP was interstitial; there was no interaction of RBP with cells in or on the seminiferous tubules. The patterns of localization were identical in normal rats and vitamin A-deficient rats. [3H]etinol uncomplexed with RBP (administered with rat serum albumin) was distributed evenly throughout testicular tissue, with no specific localization. Although the labeling of the cells on the autoradiograms was not visible diminished by the presence of excess unlabeled RBP, an in vitro binding assay of a crude interstitial cell preparation using [125I]RBP displayed saturable binding, indicative of receptors for RBP. An in vitro binding assay of Sertoli cells in culture with [125I]RBP demonstrated no specific binding, nor did [125I]RBP in autoradiography of Sertoli cell monolayers display interaction with these cells. Results from this investigation show that the plasma transport protein for vitamin A, RBP, interacts initially with cells in the interstitium of the testis. (Endocrinology 108: 658, 1981)
Assuntos
Proteínas de Ligação ao Retinol/metabolismo , Testículo/metabolismo , Vitamina A/metabolismo , Animais , Autorradiografia , Histocitoquímica , Radioisótopos do Iodo , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Proteínas Plasmáticas de Ligação ao RetinolRESUMO
Using transgenic mice, we have recently shown that 5 kb of the 5'-flanking region of the mouse epididymal retinoic acid-binding protein (mE-RABP) gene contains all of the information required for spatial and temporal gene expression in the epididymis. To identify the important cis-DNA regulatory element(s) involved in the tissue-, region-, and cell-specific expression of the mE-RABP gene, the 5-kb DNA fragment was sequenced. A computer analysis of the nucleotide sequence showed the presence of a new gene located 1.7 kb upstream from the mE-RABP gene transcription initiation site. The analysis of the open reading frame showed that the new gene encoded a putative 17-kDa lipocalin (named mEP17) related to mE-RABP. A 600-bp complementary DNA encoding mEP17 was cloned by rapid amplification of 3'-cDNA ends from epididymal total RNA. Two mEP17 RNA species (1 and 3.1 kb in size) were detected by Northern blot in the epididymis, but not in other tissues tested. In situ hybridization analyses showed that, unlike mE-RABP messenger RNA (mRNA), which is expressed in the distal caput epididymidis, mEP17 mRNA was detected only in the principal cells of the initial segment. The spatial expression and homology with mE-RABP suggest that mEP17 may act as a retinoid carrier protein within the epididymis. mEP17 mRNA expression disappeared 5 days postcastration. Four days after unilateral castration, mEP17 mRNA had nearly disappeared in the epididymis from the castrated side, but not from the intact side. In addition, testosterone replacement to bilaterally castrated mice failed to restore gene expression. We conclude that mEP17 gene expression is dependent on testicular factors circulating in the luminal fluid. Together our results suggest that mE-RABP and mEP17 genes were generated by duplication and that evolution led to a different region-specific gene expression and regulation in the epididymis.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Epididimo/metabolismo , Duplicação Gênica , Receptores do Ácido Retinoico/genética , Testículo/metabolismo , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Sequência Conservada/genética , Regulação da Expressão Gênica , Genoma , Hormônios/fisiologia , Lipocalinas , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Orquiectomia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismoRESUMO
A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.
Assuntos
Androgênios/fisiologia , Clonagem Molecular , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Sequência Conservada , DNA Complementar/genética , Masculino , Camundongos , Dados de Sequência Molecular , Família Multigênica/genética , Orquiectomia , Receptores do Ácido Retinoico/metabolismo , Proteínas Plasmáticas de Ligação ao RetinolRESUMO
We previously demonstrated that the caput epididymis of intact sexually mature rabbits contains a specific high-affinity binding protein for 5alpha-dihydrotestosterone (5alphaDHT). The other anatomical segments (corpus and cauda) of the epididymes of these animals had no detectable 5alphaDHT-binding activity. We have further shown that this binding was due to an androgen-binding protein of testicular origin. In the present study we have investigated 5alphaDHT binding to epididymal cytosol from sexually immature rabbits (20-104 days old). Using sucrose gradient ultracentrifugation, we have detected a unique pattern of binding. The pattern correlated well with testicular and epididymal maturation, but there was little correlation with chronological age or body weight. In the most immature animals (Group I) the seminiferous tubules appeared as solid cords and the epithelium of the ductus epididymis detectable 5alphaDHT-binding activity. In the second group (Group II), there was 5alphaDHT-binding to all three segments. The seminiferous tubules of these rabbits exhibited spermatogenic activity and lumen formation. The height of the epididymal epithelium had increased uniformly throughout the duct. The third group (Group III) had 5alphaDHT-binding only in caput cytosol. Spermatogenesis had progressed to the formation of elongated spermatids in the most immature animals of this group to the release of spermatozoa in the most mature ones. The caput epithelium of this last group of rabbits was fully differentiated. Unilateral orchidectomy of Group II rabbits resulted in a decrease in [3H]5alphaDHT-binding activity on the operated side as compared to the contralateral non-operated control side, suggesting the testicular origin of the binding protein. The failure of cyproterone or cyproterone acetate to inhibit [3H]5alphaDHT-binding to the protein, the lack of effect of N-ethylmaleimide on binding, and the rapid dissociation rate of the [3H]5alphaDHT-binding protein complex suggested that the binding moiety was testicular androgen-binding protein (ABP).
Assuntos
Di-Hidrotestosterona/metabolismo , Epididimo/metabolismo , Maturidade Sexual , Envelhecimento , Animais , Sítios de Ligação , Ligação Competitiva , Peso Corporal , Citosol/metabolismo , Epididimo/anatomia & histologia , Epididimo/crescimento & desenvolvimento , Etilmaleimida/farmacologia , Masculino , Ligação Proteica , Proteínas/metabolismo , Coelhos , Receptores de Superfície Celular/efeitos dos fármacos , Testículo/anatomia & histologia , Testículo/efeitos dos fármacos , Testículo/ultraestruturaRESUMO
A highly specific, high affinity binding protein for estradiol-17beta (E2) is present in cytosol prepared from the epididymides of immature (21-53 day old) rabbits. This binding moiety sediments on sucrose gradients as an 8S species under low ionic strength conditions and as a 4S species under conditions of high ionic strength (0.3 M KCL). The relative binding affinities of estrogens for the binding protein was E2 is greater than estrone is greater than estriol. Neither 5alpha-dihydrostestosterone (5alphaKHT), progesterone, nor cortisol were able to inhibit binding of [3H]E2 to epididymal binding sites. An 8S binding moiety for E2 was present in testicular cytosol but not in muscle. An apparently non-specific binding component for E2 was present in plasma which sedimented in the 4S region of low ionic strength gradients. The epididymal E2 binding moiety was distinct from a 4S androen binding protein of testicular origin which is detectable in cytosol prepared from epididymides of rabbits at certain stages development. We were unalbe to detect a specific E2 binding protein in epididymal cytosol from mature intact or 4-day castrated rabbits. The E2 binding component in the cytosol of immature rabbits had an Kd congruent to 2-10 X 10-10 M and the concentration of binding sites was in the order of 1-4 X 10-13 mmoles/mg of protein. The binding component was thermo-labele and pronase, but not nuclease, sensitive.
Assuntos
Epididimo/metabolismo , Estradiol/metabolismo , Maturidade Sexual , Animais , Ligação Competitiva , Proteínas de Transporte/metabolismo , Centrifugação com Gradiente de Concentração , Citosol/efeitos dos fármacos , Citosol/metabolismo , Di-Hidrotestosterona/farmacologia , Epididimo/efeitos dos fármacos , Epididimo/ultraestrutura , Estriol/farmacologia , Estrona/farmacologia , Masculino , Músculos/metabolismo , Concentração Osmolar , CoelhosRESUMO
In previous studies we reported the synthesis, secretion, and immunolocalization at the light microscopic level of two mouse epididymal proteins, MEP 7 and MEP 10 [Rankin et al. (1992b), Biol. Reprod., 46:747-766]. MEP 7 is the mouse homologue of the rat metalloproteins, AEG/D and E, and MEP 10 is the mouse homologue of the rat retinoic acid binding proteins, B and C. We now describe the immunolocalization of MEP 7 and MEP 10 in the mouse epididymis at the electron microscopic level. MEP 7 was localized in the Golgi apparatus, in small electron-lucent secretory vesicles, and on microvilli of the principal cells from the distal caput epididymidis to the cauda. The luminal contents were also immunoreactive in these regions of the epididymis. Although some gold particles were associated with the sperm surface, there was no selective concentration of these particles. In addition, MEP 7 was localized in large (600 nm) supranuclear endocytic vesicles and in infranuclear lysosomes. MEP 10 immunoreactivity was also seen on the microvilli of the principal cells of the distal caput and corpus and the luminal contents from the distal caput to the cauda epididymidis. There was no association of gold particles with the sperm surface. In contrast to MEP 7, there was no detectable MEP 10 immunoreactivity on the organelles of the principal cells involved in protein secretion or endocytosis. Clear cells also demonstrated immunoreactivity to MEP 7 and MEP 10. However, the intensity of immunolabeling, and the number of clear cells labeled, was greater with MEP 10 than MEP 7. In the case of MEP 7, the gold particles were located on the large supranuclear endocytic vesicles and on some infranuclear lysosomes, from the proximal corpus to the middle cauda, while in the case of MEP 10, gold particles were predominantly present in infranuclear lysosomes from the distal caput to the middle cauda. These results suggest that the principal cells are involved in both the secretion and endocytosis of MEP 7. The MEP 10 and MEP 7 proteins present in the lumen of the mouse epididymis are endocytosed from the lumen and degraded in the clear cells. However, the process of endocytosis by the clear cells of these two proteins appears to be different.
Assuntos
Epididimo/metabolismo , Metaloproteínas/metabolismo , Microscopia Imunoeletrônica , Organelas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Animais , Endocitose/fisiologia , Epididimo/ultraestrutura , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organelas/ultraestrutura , Glândulas Seminais/metabolismo , Glândulas Seminais/ultraestruturaRESUMO
The acrosome is a unique organelle containing acid hydrolases common to lysosomes as well as unique enzymes. Its ultimate exocytosis, as well as the absence of several lysosomal markers, has led to the speculation that it should be considered a secretory or zymogen vesicle rather than a specialized lysosome. The basic targeting machinery for eukaryotic lysosomal acid glycosidases are the two mannose 6-phosphate receptors. Mouse testicular germ cells are known to express both the cation-independent (CI-MPR) and cation-dependent (CD-MPR) forms of the mannose 6-phosphate receptors, but the CD-MPR is predominant. In this report, we utilized the recent targeted disruption of the CD-MPR and CI-MPR genes to determine whether these mutations affect targeting of acid glycosidases to the acrosome. Antibody to luminal fluid beta-D-galactosidase was used to examine the targeting of immunoreactive product within the acrosome of permeabilized spermatozoa and testicular spermatids. No obvious changes in acrosomal immunoreactivity in either MPR homozygous mutant were observed when compared with the case of wild-type littermates. In addition, targeted disruption of either MPR did not result in decreased levels of beta-D-galactosidase, alpha-D-mannosidase, or N-acetylglucosaminidase activities in spermatozoa from either MPR-homozygous mutant. These results suggest that the targeted disruption of either MPR does not result in decreased acrosomal targeting efficiency.
Assuntos
Acrossomo/enzimologia , Receptor IGF Tipo 2/fisiologia , Espermatozoides/fisiologia , beta-Galactosidase/metabolismo , Animais , Cátions , Cruzamentos Genéticos , Epididimo/enzimologia , Epididimo/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor IGF Tipo 2/deficiência , Receptor IGF Tipo 2/genética , Espermatozoides/enzimologia , Testículo/fisiologiaRESUMO
The effects of different androgen and testicular fluid levels on the protein synthesis and secretion of the epididymis of mice and rats were examined. In mice, the in vitro protein synthesis and secretion of five epididymal segments were measured from 3 days up to 8 weeks after efferent duct ligation. During the entire period, alterations in the rate of protein secretion per milligram tissue were small. At 8 weeks, the mean rate of protein synthesis per milligram ligated epididymal tissue was 80% of that of the control side. As a consequence of the weight loss of the ligated epididymis, however, the protein secretion per organ can be estimated to be reduced by 50%. Changes in the protein profile were only found in the proximal segment, where a 40-kd protein appeared and a 29-kd protein disappeared. In rats, the effects of efferent duct ligation were studied in vivo for up to 8 months. Structural changes were present both in the proximal and in the distal epididymis. The most conspicuous change in the protein profile of secretory proteins was the disappearance of a 27-kd protein from the proximal segment. In the distal epididymis, a 32-kd protein was no longer secreted. In mice, the effects of castration on the profile of secreted proteins demonstrated that, without androgen stimulation, some proteins are still secreted 6 weeks after castration. Administering low or high doses of testosterone propionate to castrated mice resulted in almost similar profiles of secretory proteins. However one protein secreted in the proximal epididymis was preferentially stimulated by the high dose of testosterone propionate.
Assuntos
Epididimo/metabolismo , Orquiectomia , Biossíntese de Proteínas , Testosterona/farmacologia , Ducto Deferente/cirurgia , Animais , Epididimo/efeitos dos fármacos , Ligadura , Masculino , Camundongos , Proteínas/efeitos dos fármacos , Ratos , Ratos EndogâmicosRESUMO
Acrosin, an acrosomal serine protease, is believed to have a role in fertilization. The enzyme is synthesized in an enzymatically inactive precursor form, proacrosin, and is processed to enzymatically active form(s). In the studies presented here, maturation-associated changes in the proacrosin-acrosin system of rat spermatozoa are reported. Acid-solubilized components of spermatozoa from caput, corpus, and cauda epididymidis were resolved on gelatin-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and the proteolytic bands visualized by enzymography. These studies reveal the presence of one form (52 kDa), two forms (52 and 41 kDa), and four forms (52, 41, 34, and 31 kDa) in the spermatozoa from caput, corpus, and cauda, respectively. The findings suggest that the enzymatically inactive high molecular weight component (proacrosin) present in the caput spermatozoa is partially converted to the low molecular weight components (acrosin) during epididymal transit. The sensitivity of these molecular forms to an inhibitor of acrosin, p-nitrophenyl p'-guanidino benzoate (NPGB), and the fact that all four forms cross-reacted with the antibody against guinea pig testis proacrosin, suggest that these molecular forms are proacrosin-acrosin components. To understand the mechanism of the changes in molecular forms, spermatozoa from caput, corpus, and cauda regions were subjected to in vitro activation, and the acid-solubilized components resolved on gelatin-SDS-polyacrylamide gel. A smaller component of 34 kDa was generated from both the caput and corpus spermatozoa. No changes in the molecular form(s) of cauda spermatozoa were observed, even after in vitro activation for 4 hours. Inclusion of NPGB during in vitro activation blocked generation of the new molecular form from the caput spermatozoa. These studies indicate that intra-acrosomal events during epididymal transit may be important in the production of functionally mature spermatozoa.