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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.25 n.2 Temuco jun. 2007

http://dx.doi.org/10.4067/S0717-95022007000200003 

 

Int J. MorphoL, 25(2):259-264, 2007.

 

Localization of Total Proteins and Lactate Dehydrogenase in Hamster Epididymis

Localization de Proteínas Totales y LDH en el Epidídimo del Hámster

 

*Celia C. L. Beu; **,***Antonio M. Orsi; **Raquel F. Domeniconi & ****Ethel L. B. Novelli

* Departamento de Ciencias Morfofisiologicas/UNIOESTE, Campus de Cascavel, PR, Brasil.
** Departamento de Anatomia/UNESP, Campus de Botucatu, SP, Brasil.
*** Setor de Anatomía, Faculdade de Medicina e Enfermagem, UNIMAR, Campus de Marilia, SP, Brasil.
**** Departamento de Bioquimica/UNESP, Campus de Botucatu, SP, Brasil.

Dirección para correspondencia


SUMMARY: The concentration of total protein measured by photocolorimetric methodology and reported as units per 100 mg of tissue decreased from the initial segment to the cauda epididymidis of the Golden hamster, being significant the numeric difference observed between these two regions. This observation was related with an increased synthesis and secretion of proteins to the lumen in proximal segments of the epididymidis duct, mainly in initial segment, as proposed for other rodents. LDH activity was higher in initial segment and distal cauda than in the caput and corpus epididymidis, although no significant differences in mean values had been observed. The high LDH activity observed in initial segment and cauda epididymidis of hamster had been related to an expressive epithelium metabolic activity presented in these regions. This metabolic activity help to guarantee the survival of spermatozoa stored in cauda epididymidis. Furthermore, lower LDH activity noted in the caput and corpus epididymidis might be related with a progressive reduction of glycolysis in initial maturation step of spermatozoa mainly verified in corpus epididymidis.

KEY WORDS: Total protein; LDH, Tissue localization; Epididymis, Hamster.

RESUMEN: La concentración de proteínas totales medidas a través de la fotocolorimetría y reportada como unidades por 100 mg de tejido, decreció desde el segmento inicial hacia la cola del epidídimo del hámster dorado, siendo significativa la diferencia numérica observada entre estas dos regiones. Esta observación estaba relacionada con el aumento de la síntesis y secreción de proteínas por el epitelio del epidídimo, siendo exportadas después hacia el lumen tubular en los segmentos proximales del epidídimo, especialmente a nivel del segmento inicial, ya obsevada en otros roedores. La actividad del LDH fue mayor en el segmento inicial y en la cola distal que en la cabeza y cuerpo del epidídimo, no obstante no haya sido observada ninguna diferencia significativa en los valores promedios. La intensa reacción del LDH observada en el segmento inicial y cola del epidídimo había sido relacionada con la expresiva actividad metabólica del epitelio, especialmente observada en estas regiones del conducto. Esta actividad metabólica permite la sobrevivencia y el metabolismo de los espermatozoides almacenados en la cola del epidídimo. Además, la baja actividad del LDH evidenciada en la cabeza y el cuerpo del epidídimo podría estar relacionada con la reducción progresiva de la glucólisis observada en la etapa inicial de maduración de los espermatozoides, siendo especialmente notoria en el cuerpo del epidídimo.

PALABRAS CLAVE: Proteínas totales; LDH; Localización tisular; Epidídimo; Hamster.


INTRODUCTION

Among the several components of the epididymidis fluid had been characterized glycoproteins and proteins, including enzymes such as lactate dehydrogenase (LDH), (Setchell et al, 1994). Most proteins presented in the epididymal fluid were synthesized and secreted by the lining epithelium of the epididymal duct and were located in specific epithelium regions (Dacheux ei a/., 1989; Syntin et al, 1996). Moreover, there were proteins associated with the surface of intraluminal spermatozoa (Dacheux & Paquignon, 1980; Jones et al, 1983, Dacheux et al.). Epididymidis proteins had two major roles; one consisted of regulation of the structural and functional integrity of the epididymal epithelium, while the other was related to a direct action on spermatozoa in order to protect them and promote their maturation during epididymal transit (Jones et al; Robaire &Hermo, 1988, Syntin et al.). Perhaps, proteins interact with spermatozoa changing their membrane properties in different ways, and might be absorbed by spermatozoa surface or proteins modify preexisting compounds on the plasma membrane through specific action of proteases such as glucosaminidases or glycosyltransferases (Syntin et al).

Development of spermatozoa motility required changes in the metabolic energetic mechanism that supply energy for flagellum movement. One of the most specific enzyme that took place in this metabolic mechanism was the LDH (Vermouth et al, 1986). LDH isoenzyme as LDH-X, or C4, was specific for the testes and epididymis of various mammalian species (Blanco & Zinkhan, 1963), and showed unique catalytic properties (Blanco et al, 1976). Furthermore, LDH was a highly specialized enzyme and was related to metabolic energetic processes to supply motility and survival of spermatozoa (Gerez de Burgos et al, 1978). According to Storey & Kayne (1977), LDH played a role in the pyruvate-lactate shuttle system transferring reducing equivalents from cytosol to mitochondria.

Thus, in view of the importance of proteins and LDH to the process of maturation and metabolism of spermatozoa, the aim of this study was to determine the presence and concentration (localization) of total proteins and LDH in the epididymidis initial segment, caput, corpus and distal cauda in adult laboratory hamster.

MATERIAL AND METHOD

Biochemical analysis for determination of total proteins and LDH (E.C. 1.1.1.27) activity was performed on epididymidis tissue samples obtained from 14 adult laboratory hamsters (Mesocricetus auratus), of the Golden variety. The hamsters were maintained under a 12-h light/ dark cycle at controlled room temperature, with water and food available ad libitum. Afterwards, the rodents suffered euthanasia with an overdose of anesthetic formed by a mixture of ketamine hydrochloride (20 mg/kg) and xylazine hydrochloride (1.5 mg/kg). The bilateral epididymis were collected and divided into the initial segment, caput, corpus and cauda distal. Epididymidis tissues were weighed and frozen until the time for biochemical studies. Three samples of each epididymal region were used for biochemical analysis, with each sample weighing 135 mg of medium average. Tissue samples were homogenized in 5 ml sodium phosphate buffer, pH 7.4 with a Potter-Elvehjem Homogenizer (Curtin Matheson Scientific, Houston, TX, USA) using a Teflon® pestle. The homogenates obtained were centrifuged in a refrigerated centrifuge at 10,000 rpm during 15 min at -4°C, and the resulting supernatants were used for the biochemical determinations.

Total proteins were determined in 50 µl supernatant of each sample by Colorimeter .£ 14 -method using a CELM kit for total protein evaluation (Modern Laboratory Equipment 0 10 -Company, Sao Paulo, Brazil). Absorbance was j; g-read with a Spectrophotometer at 540 nm. read with a Spectrophotometer at 540 nm. Results were reported as mg of protein per 100 mg of tissue evaluated. LDH was determined in 15 µl supernatant for each sample using the same CELM kit (Modern Laboratory Equipment Company), and UV method optimized for determination of tissular LDH at 25°C. Absorbance at 340 nm was read after 1,2 and 3 minutes using an ELISA Microplate Reader (Bio-tech Instruments, Inc., Winooski, VT, USA). LDH activity was reported as units per 100 mg of tissue (U/100 mg tissue). Statistical analysis was made by a completely random design, having four treatments and three replicates. The data were submitted to analysis of variance (ANOVA) and mean values were compared at 5% level by the Tukey test.

Histochemical analyses of total proteins and LDH activities were performed in 10 mm cryostatic sections of the same epididymidis regions biochemistry analyzed, utilizing tissue samples obtained from 4 hamsters. The samples were previously frozen in n-hexane (-60°C), and the sections were used for histochemical localization of LDH (Pearse, 1985), and total proteins utilizing the Millon's reaction (Lillie, 1965). As convention, the specific tissular reactivity at light microscopy was signalized as strong, medium and low.

RESULTS

Analysis of the results showed a decline in total protein concentration along the epididymal segments. The highest concentration was observed in the initial segment (11.8 ± 4.12 mg protein/100 mg tissue) and the lowest concentration in the cauda epididymidis (5.1 ±0.48 mg protein/100 mg tissue), with the difference being significant. Intermediate and similar total protein concentrations were observed for the caput and corpus epididymidis (7.4 ±0.83 and 7.0 ± 2.39 mg protein/ 100 mg tissue, respectively), with no significant differences between these two regions or between the caput, corpus and distal cauda epididymidis or between initial segment, caput and corpus epididymidis (Fig. 1).


The highest LDH activity was observed in the cauda epididymidis (7.88 ± 0.60 U/100 mg tissue), while the lowest activity was found in the corpus epididymidis (4.70 ±2.28 U/l 00 mg tissue). Intermediate values were observed for the initial segment (7.38 ± 0.61 U/100 mg tissue) and the caput epididymidis (6.11 ± 1.27 U/100 mg tissue) (Fig. 2). Statistical analysis showed no significant differences in mean LDH activity between the epididymal segments studied.


Moreover, protein content was seen to be high in the epididymal plasma obtained from the caput epididymidis of bulls and even was higher in the proximal corpus (Crabo, 1965). Data obtained with studies on rabbit and sheep epididymidis (Riar et al.) and also in bull (Crabo), suggested that the area of resorption extended next to the regional limit of the proximal corpus, and perhaps exceeded this level (Riar et al). In golden hamster, the highest total protein concentration was observed in the initial segment which represented an area of intense seminal fluid absorption in rodents (Robaire & Hermo; Hermo et al., 1994; Setchell et al.).

Histochemistry analysis of the epididymidis tissues showed similar results as the biochemistry tests, occurring a strong reactivity for LDH in samples of the cauda epididymidis mainly at the lining epithelium level. Corpus epididymidis tissues showed low activity of LDH, as well as a low reactivity was also observed along the connective tissues of epidydimal support. Medium intermediate patterns of epithelium reactivities for LDH were verified in initial segment and corpus epididymidis comparatively to the cauda epididymidis in which a major reaction was noticed for LDH. Total protein patterns were also similar to the biochemical results, occurring a strong reactivity in the initial segment; medium reactivities in the caput and corpus and minor (low) protein reaction in the cauda epididymidis of the Golden hamster.

DISCUSSION

Epithelium epididymidis synthesized and secreted numerous proteins which synthesis and secretion had been variable in accordance with the epididymal region focused (Syntin et al). These regional variations were confirmed in the present study showing differences in total protein expression in the epididymal duct of the Golden hamster. Previous investigation comparing epididymidis histochemistry of rabbit, rat, dog, sheep, monkey and humans (Riar et al, 1973) had demonstrated that in only two of these species, i.e. dog and man, were lower total protein concentration in caput than in cauda epididymidis. On the other hand, in rat, rabbit and sheep the highest total protein concentration was observed mainly in proximal and distal corpus epididymidis and the lowest levels occurred in cauda epididymidis (Riar et al). These results indicated that absorption of fluid deriving from the testis was higher in the caput epididymidis and also that testicular fluid contributed directly to the high protein content found in this region (Riar et al).

Moreover, protein content was seen to be high in the epididymal plasma obtaines from the caput epididyms of bulls and even was higher in the proximal corpus (Crabo, 1965). Data obtained with studies on rabbit and sheep epididymidis (Riar et al.) and also in bull (Crabo), suggested that the area of resorption extended next to the regional limit of the proximal corpus, and perhaps exceeded this level (Riar et al.), In golden hamster, the highest total protein concentration was observed in the initial segment wich represented an area of intense seminal fluid absorption in rodents (Robaire & Hermo; Hermo et al., 1994: Setchell et al.).

Number and concentration of proteins presented in the epididymal luminal fluid changed along the ductus (Dacheux & Paquignon). Electrophoretic analysis detected few common proteins secreted in several points along the epididymis, indicating that epididymal epithelium secreted selectively regionalized proteins inside the tubular lumen (Syntin et al). Epididymidis proximal portion had been the most active region concerning to protein secretion, in agreement with our results. In swine, the high protein synthesis activity of the proximal epididymidis was related to secretion of only two main components which account for 34.5% and 31.4% of total proteins secreted by this organ (Syntin et al). In contrast in rodents, which perhaps included the hamster, an elevated number of proteins was secreted into the luminal fluid (Robaire & Hermo). Previous base for protein secretion was proposed concerning to epididymal micromerocrine secretion, presented at the level of the initial segment of some mammals (Nicander & Malmqvist, 1977).

Autoradiographic studies on protein synthesis in mice had showed a higher activity in epididymidis initial segment, having a progressive decline of synthesis in the middle and terminal epididymal segments (Kopecny & Pech, 1977), as was also observed here for the hamster. In bulls (Lavonet al., 1971) and rats (Jones et al), a decrease in spermatozoon protein content was observed during epididymal maturation, in which total protein levels resulted from the balance between synthesis and degradation of proteins. Previous study regarding to presence of proteins in rat spermatozoa during epididymal transit (Vermouth et al.), confirmed the evident predominance of protein degradation in spermatozoa during their progression through the epididymal duct and we agree with this affirmation based in our findings.

So, our results were in agreement with those reported by other authors, such as Kopecny & Pech, showing a higher total protein concentration in the initial segment and a progressive reduction along the more distal tubular segments. The results obtained showed intense epithelial activity of protein synthesis in the proximal regions of the epididymal duct. Moreover, different patterns of protein degradation or resorption could be suggested, in which most proteins secreted by the epididymal epithelium were rapidly degraded or reabsorbed after secretion. In swine, most proteins found in epididymal fluid in vivo probably did not result from abundant secretion by the different regions, but rather corresponded to less degraded or less reabsorbed proteins (Syntin et al). Rodents as the hamster here studied, the rat (Olson; Hinton, 1985) and the mouse (Robaire & Hermo), apparently secreted a larger number of proteins through the epididymal epithelium, which were incorporated into the seminal fluid.

Studies realized on the activity and function of dehydrogenases in the epididymal duct suggested that they acted on the process of glycolysis, which played an important role in epididymal metabolism (Delhon & Lawzewitsch, 1994). LDH had been related to the fertility of mature human spermatozoa as a consequence of its role in energetic metabolism (Blanco, 1980), since low LDH levels were associated with a partial or total reduction on sperm concentration and motility (O 'Flaherty et al, 2002). Furthermore, LDH besides to be related to the Krebs cycle on the epididymal level (Orsi et al, 1993), played a relevant role on the concentration of lactate into the cauda epididymidis fluid, which aimed to regulate the spermatozoa motility (Acott & Carr, 1984; Carr & Acott, 1984; Turner & Reich, 1985; Orsi et al.).

Histochemical studies of epididymal LDH demonstrated species-specific variations in the epithelial reactivity observed in the different epididymal regions. Intense sperm LDH activity was detected in the cauda epididymidis of most species (Allen & Slater, 1961; Blackshaw & Samisoni, 1967; Tingari & Moniem, 1979). It was an observation consistent with the sperm storage function attributed to cauda epididymidis region (Delhon & Lawzewitsch), which might explain the marked LDH activity observed here in the distal cauda of the Golden hamster epididymis.

A study made on rat epididymidis (Vermouth et al.), showed that the transit of spermatozoa through the ductus was accompanied by a reduction of LDH activity from the caput to the corpus, and was not significant compared to the cauda epididymidis. The changes in LDH reported for the rat (Vermouth et al.), were suggestive of a reduced capacity of spermatozoa for glucose oxidation while spermatozoa matured along the luminal epididymidis transit. Similarly, a decline in tissue activity could be noted in epididymal homogenates of the Golden hamster from the initial segment to the corpus, being the corpus considered the most representative region from initial activities of sperm maturation in several mammals (Robaire & Hermo; Hermo et al.).

A reduction in the oxidative utilization of glucose in mature spermatozoa compared to glucose testicular utilization had been demonstrated in sheep and bull (Voglmayr, 1975; Dacheux & Paquignon). The profile of LDH reduction resembled that observed for glyceraldehyde-3-phosphate dehydrogenase (Ford; Harrison, 1983). Since both the enzymes were integrated in the Embden-Meyerhoff pathway, a decrease in their activities was correlated with lower glycolysis in the spermatozoa (Ford & Harrison).

The last observations and our results in Golden hamster epididymidis showed that LDH activity gradually decreased from the initial segment to the corpus epididymidis, and afterwards increased in the cauda epididymidis. These findings were in agreement with those reported for other species (Zinkham et al., 1964). Furthermore, comparatively, LDH was found to be the most active dehydrogenase in the camel epididymidis when compared to succinate dehydrogenase, glutamate dehydrogenase and glucose-6-phosphate dehydrogenase, in which a higher LDH activity had been observed in distal epididymal segments (Tingari & Moniem). Also, these findings made suggested the great importance of glycolytic pathways in mammalian epididymis. The higher LDH activity observed in the terminal segment (cauda) of the hamster epididymis indicated a high metabolic activity of the lining epithelium to provide energy for sperm survival. This thesis had been consistent with the main functional role attributed to the cauda epididymidis, as the prior spermatozoa storage site (Tingari; & Moniem; Robaire & Hermo; Hermo et al.).

ACKNOWLEDGMENTS

The authors thank a Grant from FAPESP, Sao Paulo Brazil (Proc. 04/05578-1), which provided partial resources to made this paper. It was a part of the Doctoral Thesis of the first author presented in 2005, next to the Instituto de Biologia da UNICAMP, Campinas, SP, Brazil. We also thank very much the technical assistance received in the Departamento de Bioquímica da UNESP, Botucatu, SP, Brazil.

 

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Correspondence to:

Prof. Dr. Antonio Marcos Orsi
Universidade Estadual Paulista
«Julio Mesquita Filho
Campus Botucatu
CP 18618-000
Botucatu- SP
BRASIL
Email: amorsi@ibb.unesp.br

Received: 10-01-2007 Accepted: 05-03-2007

 

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