Localization and identification of tyrosine phosphorylated proteins in adult Sprague-Dawley rat testis / Localización e identificación de proteínas de tirosina fosforilada en testículos de rata Sprague-Dawley adultos

SUMMARY: Spermatogenesis is a major process in testis occurring from puberty through life span of males. The tyrosine phosphorylation is assumed to play roles in spermatogenesis because this process is important for cell proliferations, divisions, and differentiations. However, the localizations and identifications of phosphorylated proteins in testicular tissue of adult male rats are still unclear. Therefore, this study attempted to immuno-localize and identify such proteins in testicular tissues of Sprague-Dawley rats. The monoclonal anti-phosphotyrosine (clone 4G10) was used to probe tyrosine phosphorylated proteins and also to examine the expression of such proteins using immuno-Western blotting in rat testis. The result showed that positive reactivity of tyrosine phosphorylated proteins was clearly observed in interstitial endocrine cells (Leydig cells), sustentocytes (Sertoli cells), spermatogonia, spermatocytes, and spermatids (round and elongated), respectively. The expressions of testicular tyrosine phosphorylated proteins were 200, 131, 93, 70, 60, and 48 kDas, respectively. In conclusion, testicular tyrosine phosphorylated proteins were localized in both germinal epithelium and interstitial endocrine cells of adult Sprague-Dawley rats.

RESUMEN: La espermatogénesis es un proceso importante en los testículos que ocurre desde la pubertad a lo largo de la vida de los machos. Se supone que la fosforilación de la tirosina desempeña papeles en la espermatogénesis, debido a que este proceso es importante para las proliferaciones, divisiones y diferenciaciones celulares. Sin embargo, las localizaciones e identificaciones de proteínas fosforiladas en el tejido testicular de ratas macho adultas todavía no están claras. Por lo tanto, este estudio intentó inmuno-localizar e identificar dichas proteínas en tejidos testiculares de ratas Sprague-Dawley. La anti-fosfotirosina monoclonal (clon 4G10) se usó para sondar proteínas tirosina fosforiladas y también para examinar la expresión de tales proteínas usando inmunotransferencia Western en testículo de rata. El resultado mostró que la actividad positiva de las proteínas tirosina fosforiladas se observó claramente en endocrinocitos intersticiales (células de Leydig), sustentocitos (células de Sertoli), espermatogonias, espermatocitos y espermátidas (redondas y alargadas), respectivamente. Las expresiones de las proteínas tirosina fosforiladas testiculares fueron de 200, 131, 93, 70, 60 y 48 kDas, respectivamente. En conclusión, las proteínas tirosina fosforiladas fueron localizadas en ambos epitelios germinales y endocrinocitos intersticiales de ratas adultas Sprague-Dawley.

Beneficial effect of pentoxifylline into the testis of rats in an experimental model of unilateral hindlimb ischemia/reperfusion injury

The objective of the present study was to investigate the role of pentoxifylline (PTX) on remote testicular injury caused by unilateral hind limb ischemia/reperfusion of rats.

Fenitrothion induced oxidative stress and morphological alterations of sperm and testes in male sprague-dawley rats

OBJECTIVE: Fenitrothion residue is found primarily in soil, water and food products and can lead to a variety of toxic effects on the immune, hepatobiliary and hematological systems. However, the effects of fenitrothion on the male reproductive system remain unclear. This study aimed to evaluate the effects of fenitrothion on the sperm and testes of male Sprague-Dawley rats. METHODS: A 20 mg/kg dose of fenitrothion was administered orally by gavages for 28 consecutive days. Blood sample was obtained by cardiac puncture and dissection of the testes and cauda epididymis was performed to obtain sperm. The effects of fenitrothion on the body and organ weight, biochemical and oxidative stress, sperm characteristics, histology and ultrastructural changes in the testes were evaluated. RESULTS: Fenitrothion significantly decreased the body weight gain and weight of the epididymis compared with the control group. Fenitrothion also decreased plasma cholinesterase activity compared with the control group. Fenitrothion altered the sperm characteristics, such as sperm concentration, sperm viability and normal sperm morphology, compared with the control group. Oxidative stress markers, such as malondialdehyde, protein carbonyl, total glutathione and glutathione S-transferase, were significantly increased and superoxide dismutase activity was significantly decreased in the fenitrothion-treated group compared with the control group. The histopathological and ultrastructural examination of the testes of the fenitrothion-treated group revealed alterations corresponding with the biochemical changes compared with the control group. CONCLUSION: A 20 mg/kg dose of fenitrothion caused deleterious effects on the sperm and testes of Sprague-Dawley rats.

Histological and histochemical analysis of the gonadal development of males and females of Armases rubripes (Rathbun 1897) (Crustacea, Brachyura, Sesarmidae) / Análise histológica e histoquímica do desenvolvimento gonadal de machos e fêmeas de Armases rubripes (Rathbun, 1897) (Crustacea, Brachyura, Sesarmidae)

The objective of this study was to provide information on the histological characteristics of the gonads of male and female Armases rubripes crabs, and to try to establish a relationship between the microscopic and macroscopic stages previously identified. Thirty-six crabs were collected by hand between February 2003 and January 2004 in banks of Spartina alterniflora on Sahy Beach in Mangaratiba, Rio de Janeiro state, Brazil. The histological analysis of the ovaries of A. rubripes demonstrated a gradual process of development of the oocytes. According to their cellular characteristics, five types of cells were distinguished: oogonia, oocyte I, oocyte II, oocyte III and oocyte IV. The ovaries showed four stages during gonadal activity: stage I (rudimentary), stage II (developing or maturing), stage III (developed or mature) and stage IV (resting). The results of the histochemical analyses showed that the ovaries vary according to the gonad development stage. The histological aspect of one section of the male gonad was always the same in all of the seminiferous tubules, where the lumen of these tubules always contained spermatozoa and/or spermatids. It was not possible to characterize the three stages of gonad development in the males. This agrees with previous reports in the literature. However, in the females there was a relationship between the gonad stages distinguished macroscopically and the results obtained through the histological and histochemical analysis, due to the presence of different cell types, as well as the lysis process and reabsorption of the oocytes in spent females.

O objetivo deste estudo foi fornecer informações sobre as características histológicas das gônadas de machos e de fêmeas de Armases rubripes, tentando estabelecer uma relação entre os estágios microscópicos e os macroscópicos anteriormente identificados. Foram coletados manualmente 36 caranguejos, durante o período de fevereiro de 2003 a janeiro de 2004, em bancos de Spartina alterniflora na praia do Sahy Mangaratiba, Estado do Rio de Janeiro. A análise histológica dos ovários de A. rubripes demonstrou um processo gradual de desenvolvimento dos oócitos. De acordo com sua característica celular, cinco tipos de células foram distinguidos: ovogônias, oócito I, oócito II, oócito III, oócito IV. Os ovários revelaram quatro estágios de atividade gonadal: estágio I (rudimentar), estágio II (em desenvolvimento ou em maturação), estágio III (desenvolvido ou maduro), estágio IV (desovada). Os resultados das análises histoquímicas permitem afirmar que os ovários variam de acordo com o estágio de desenvolvimento gonadal. O aspecto histológico de uma sessão de gônada masculina é sempre o mesmo em todos os túbulos seminíferos, onde o lúmen deste túbulo sempre contém espermatozóides e/ou espermátides. Não foi possível a caracterização de três estágios de desenvolvimento gonadal em machos, conforme descrito previamente na literatura. Entretanto, em fêmeas, houve uma relação entre os estágios gonadais distinguidos macroscopicamente e os resultados obtidos através da análise histológica e histoquímica, devido à presença de diferentes tipos celulares, assim como processo de lise e reabsorção dos oócitos em fêmeas desovadas.

Purification of protein from a crude mixture through SDS-PAGE transfer method.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie bluc/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.

Tissue mRNA expression in rat of newly described matrix metalloproteinases

Matrix metalloproteinases (MMPs) comprise a large group of endoproteinases that degrade all protein components of the extracellular matrix. Functionally, MMPs contribute to several different physiological as well as pathological conditions. The number of newly described MMPs has increased in recent years, although current knowledge about their expression pattern in various tissues remains incomplete. Here we analyzed the relative mRNA expression of the most recently described MMPs _ MT5-MMP (MMP-24), MT6-MMP (MMP-25), MMP-27 and epilysin (MMP-28) _ in a broad selection of rat tissues using real time-PCR. MMP-24 mRNA was found to be widely expressed with predominance in the central nervous system. MMP-25 mRNA, in contrast, exhibited peak expression levels in testis, kidney and skeletal muscle, differing from previously described distribution patterns in humans. mRNAs for MMP-27 and MMP-28 were generally expressed at a lower level. All four MMPs studied were detected at higher mRNA levels in bone and kidney, suggesting a possible role of these MMPs in physiological processes within these two organs. The present study highlights the differential distribution pattern of newly described MMPs among different tissues and underlines differences in the mRNA expression between different species.

Protein detection in spermatids and spermatozoa of the butterfly Euptoieta hegesia (Lepidoptera)

This study was undertaken to detect protein components in both sperm types of the butterfly Euptoieta hegesia. These spermatozoa possess complex extracellular structures for which the composition and functional significance are still unclear. In the apyrene sperm head, the proteic cap presented an external ring and an internal dense content; basic proteins were detected only in external portions. In the tail, the paracrystalline core of mitochondrial derivatives and the axoneme are rich in proteins. The extratesticular spermatozoa are covered by a proteic coat, which presented two distinct layers. In eupyrene spermatozoa, acrosome and nucleus were negatively stained, probably because of their high compaction. In the tail, there is no paracrystalline core and the axoneme presented a very specific reaction for basic proteins. The lacinate and reticular appendages are composed of cylindrical sub-units and presented a light reaction to E-PTA and a strong reaction to tannic acid. A complex proteic coat also covers the extratesticular spermatozoa. We found similarities between both extratesticular coats, indicating a possible common origin. Both spermatozoon types are rich in proteins, especially the eupyrene appendages and the extratesticular coats. We believe that both coats are related to the sperm maturation and capacitation processes.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Impact of feeding ethanolic extracts of Achyranthes aspera Linn. on reproductive functions in male rats.

Feeding 50% ethanolic extract of A. aspera to male rats resulted in reduced sperm counts, weight of epididymis, serum level of testosterone and testicular activity of 3beta-hydroxysteroid dehydrogenase, while motility of the sperm and activity of the HMG CoA reductase were not affected. Cholesterol level in the testis, incorporation of labelled acetate into cholesterol, 17-ketosteroids in urine and hepatic and fecal bile acids were increased. The results suggest that ethanolic extract of A. aspera caused reproductive toxicity in male rats and the action may be by suppressing the synthesis of androgen.

Flow cytometric evaluation on the age-dependent changes of testicular DNA contents in rats

An age-dependent cellular change of DNA contents in the testis of Sprague-Dawley rats was investigated by flow-cytometric method. Testicular cell suspensions at the age of 4, 5, 6, 7, 8, 10, 12, 16 and 26 weeks were prepared and stained with propidium iodide. The relative proportions in the number of mature and immature haploid (1n), diploid (2n), S-phase and tetraploid (4n) cells were calculated. The proportion in the number of mature haploid cells was sharply increased to the age of 10 weeks (about 38%), thereafter increased slightly to the level of 42% at the age of 26 weeks. The proportion of immature haploid cells was dramatically increased to the age of 6 weeks, then maintained at the level of 20 to 30% thereafter. The proportion of diploid cells was 64% at the age of 4 weeks, then decreased gradually through the age of 26 weeks. The proportion of S-phase cells was increased to the age of 4 weeks, then maintained at a plateau level to the age of 26 weeks. The proportion of tetraploid cells were about 26% at the age of 4 weeks, then decreased gradually to the age of 26 weeks. These results suggest that the proportions of testicular cells may depend on the age of the rat and that the flow cytometric method may be useful in the evaluation of the spermatogenic status with regard to accuracy and sensitivity.

Early lipid alterations in spontaneously diabetic rats

Human and experimental diabetes mellitus extensively alters lipid metabolism. The eSS is a rat strain that develops a spontaneous diabetes of slow evolution, resembling the non-insulin-dependent diabetes mellitus of young people. We report here disturbances in lipid metabolism of 5-month old eSS rats compared to age-matched alpha-controls. Normal plasmatic glucose levels were found in the fasted state, whereas a diabetic curve was evident for eSS rats after glucose load. Triglyceride content was elevated in plasma and in liver microsomal preparations of eSS animals, when compared to the controls. The diabetic strain revealed a significant fall in the amount of linoleic acid in liver and kidney microsomes and in erythrocyte membranes. In liver, an increase in 22:6 (n-3) was also noted. A depression in the content of linoleic acid as well as an enhancement of docosahexaenoic acid were detected in phosphatidylcholine and phosphatidylethanolamine fractions from liver microsomes of eSS rats. The fatty acid pattern of eSS rat testis showed a raise in the relative percentage of arachidonic and a decrease in 22:5 (n-6), 22:5 (n-3) and 22:6 (n-3) acids compared to their controls. Diabetic rats exhibited a significant increase in microsomal cholesterol content and cholesterol/phospholipid ratio in liver and testis. In the latter tissue, higher values of fluorescence anisotropy were also observed. The current observations indicate that in early stages of the diabetes onset, when eSS rats are still normoglycemic, severe alterations of lipid metabolism may contribute to the establishment and progression of the diabetic syndrome.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods us for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll(). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll( gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ñ 8.2 per cent, mean ñ SEM) and spermatids (84 ñ 2.6 per cent) using centrifugal elutriation followed by continuous Percoll( gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll( gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis.

The rat thymus contains a heparin-binding factor that modulates steroidogenesis in the testis

Thymus development and function are under the influence of hormones secreted by the gonads and pituitary. On the other hand, thymurus is crucial for the development of reproductive capacitities in female and male rats and we have shown that a factor derived from the prepubertal rat thy mus has antigonadotropic effect in ovarian and testis cells in vitro. In the present paper we show that the rat thymic factor which modulates gonadotropin action in the gonads is an heparin-binding factor. This capacity was also used as a useful tool to obtain this activity from semipure extracts. An acetone extract was prepared from 15 day old male rats and subjected to molecular filtration chromatography. The activity, of those fractions was investigated in a testis cells biossay, by measuring testosterone secretion under basal and hCG-stimulation. Active fraction were processed in a heparin-Sepharose affinity column. We found that fractions that eluted with 0.6 and 2M NaCl/10mM Tris had biological specific activity. The electrophoretic procedure showed that the apparent molecular weight of the Heparin Sephadex binding factor is 60 kDa. Since this factor was obtained from a protein peak that eluted in the volume of carbonic anhidrase a dimerization process could be involved. Present results show that the rat thymus has an heparin-binding factor that interacts with hCG in testis cells. This factor could play an interesting role in the mutual influence between thymus and gonads.

Immunohistochemical localization of riboflavin carrier protein in testicular cells of mammals.

Using specific polyclonal antibodies against chicken riboflavin carrier protein (cRCP), immunocytochemical localization of riboflavin carrier protein was carried out in testicular sections and isolated cells of mammals. A positive reaction was observed in the developing germ cells of rat testis, especially in meiotic and post-meiotic germ cells such as pachytene spermatocytes, round spermatids and spermatozoa. In addition both the somatic cells of the testis, viz. Leydig and Sertoli cells with vital function in germ cell proliferation and differentiation, displayed a moderate to strong staining reaction. This was further confirmed using in utero X-irradiated rat testis devoid of germ cells. Different types of cells isolated from testis when subjected to immunostaining showed similar patterns of reaction as in the intact tissue. Mature spermatozoa from different mammals (rat, bull and monkey) exhibited strong staining reaction in their head regions localized mainly in acrosomal caps. It is suggested that the testicular riboflavin carrier protein has a role in cell to cell communication and may be crucial during development of germ cells especially at the meiotic and post-meiotic stages.

Cytochemical changes in spinal cord and testis of mice during starvation

The effect of starvation on the content and distribution of RNA, proteins and glycogen of spinal cord and testis of mice was studied. Spinal cord and testis were taken from mice, non-starved and after one, two and three days of total starvation. In the neurons of the spinal cord, starvation decreased RNA and proteins early in the fast, decreases in glycogen occurred more slowly, late in the fast. In the testis there was no obvious decrease in RNA and proteins content and a very slight decrease of the glycogen granules, i.e. the mice conserve testis RNA, proteins and glycogen during prolonged starvation. It has been found that the effect of starvation on RNA, protein and glycogen of the testis and spinal cord was variable

Effect of low-intensity pulsed ultrasound on prepubertal rat testis

The testes of prepubertal male rats (N -12) aged 21 days were stimulated with low-intensity pulsed ultrasound (1.5-MHz frequency, 1-KHz repetion pulse rate, 200-*s pulse width, 30-V peak-to-peak amplitude and 20-mW/cm* intensity) applied to the skin for 20 min/day for 7 days. Control rats (N-8) were manipulated in the same manner but not submitted to ultrasound. Ultrasound stimulation promoted a significant increase in plasma testosterone (62%) leading to a significant increase in seminal vesicle relative weight (35%) as well as an increase in the fructose (92%) and DNA (200%) contents of the gland. No differences were detected between ultrasound-treated and control animals, in terms of body weight and the relative weights of testis, cauda epididymidis, testis DNA and mitosis