Inductively coupled plasma emission spectroscopic and flame photometric analysis of goat epididymal fluid

Meenakshi Gaur, Vikas Pruthi,  Ramasare Prasad, Ben M.J. Pereira

Reproductive Biology Laboratory, Dept. of Bioscience and Biotechnology, University of Roorkee, Roorkee-247 667, U.P., India

Asian J Androl  2000 Dec; 2: 288-292

Keywords: goat epididymis; epididymal luminal fluid; elements;  spectroscopy; flame photometry

Abstract

1 Introduction

In most species it is generally recognized that the microenvironment provided by the proximal segments of the epididymis promotes sperm maturation while the distal segments preserve the sperm in a quiescent, yet viable state until ejaculation^[1-3]. The biochemical and physiological changes that occur in spermatozoa during passage through the epididymis, are to a great extent influenced by the luminal fluid in which they are bathed^[1,4]. The composition of this fluid is believed to fluctuate all along the epididymis due to the mixed absorptive and secretory nature of the epithelium lining the duct^[5,6]. Information on the composition of fluid collected from various sites of the epididymis would therefore help in understanding the interaction between the epididymal tissue, luminal fluid and sperm.

Due to the minuteness of the epididymal duct, investigators faced technical difficulties in the collection of luminal fluid free from contaminants. A satisfactory micropuncture technique described by Levine and Marsh^[7] has helped to circumvent this limitation. Unfortunately, the quantity of sample that could be drawn by this procedure is very small. Initially, the lack of microanalytical techniques necessitated pooling of samples, which complicated the interpretation of data. However, Jenkins et al^[8] showed how an ultra micro-electron probe elemental analysis could be carried out on rat testicular and luminal fluids with amazingly small quantities of samples. Such types of microanalytical techniques have not been used for elemental analysis of the epididymal luminal fluid (ELF) from other animal models.

2 Materials and methods

2.1 Sample collection

Six adult goats (Capra indica), aged 3-5 years, were used. The ELF was collected by the micropuncture technique described by Levine and Marsh^[7]. Based on the demarcation made by Besancon et al^[9], samples were drawn from each of the twelve serial segments of the epididymis. The elemental composition of the fluid samples was determined after appropriate dilution with triple distilled deionized water.

2.2 ICP analysis (refer to Table 1 for its sensitivity)

ICP has an edge over other flame spectroscopy methods in that it can generate higher temperatures and provide a chemically inert environment. The best advantage is the provision for simultaneous analysis of a number of elements using small amounts of sample without compromising on sensitivity. In the present study, the polychromatic system of Labtam (Plasma Lab 8440, Australia) was used, which permits simultaneous detection of up to 48 elements at wavelengths ranging 170-820 nm. However, only biologically relevant elements were analyzed.

Initially standard stock solutions of 1 mg/L (or 1000 ppm) were prepared. At the time of analysis, the instrument was calibrated using working standards of 1g/mL and 5 g/mL. The quantity of the elements detected in the luminal fluid samples was directly read as ppm. The dilution was taken into consideration while computing the results. The values obtained as ppm were then converted and expressed as millimolar concentrations.

2.3 Flame photometry (refer to Table 1  for its sensitivity)

The sodium and potassium concentrations were analyzed by flame photometry. The instrument used was produced by the Evans Electroselenium Ltd., England.

2.4 Statistical analysis

Data were represented as means. Statistical analyses were made using Student's t-test.

Table 1. Sensitivity of the analytical methods.

¹Percentage co-efficient of variation between duplicate estimations of same sample (chosen at random)
²Percentage co-efficient of variation between samples drawn from same segments of different animals (n=6)
Percentage co-efficient of variation=standard deviation/mean100

3 Results

The concentration profiles of various elements in ELF are shown in Figure 1. Copper, calcium, nickel, iron, magnesium, chromium, titanium and zinc were found to be present with levels fluctuating at different sites along the length of the epididymis. Cadmium, cobalt, lead and manganese were not detected. The sodium and potassium concentrations are depicted in Figure 2. In the proximal segments, the values for sodium were significantly higher as compared to that of potassium and the situation was reversed in the distal segments. It can be seen from Figure 3 that the Na⁺/K⁺ ratio was higher in ELF from the proximal segments and lower towards the distal end.

Figure 1. Elemental composition of ELF in male goats. means, n=6.
Figure 2. Concentration profile of sodium and potassium in ELF. means, n=6. 
Figure 3. Na⁺/K⁺ ratio in ELF in twelve epididymal segments (n=6).

4 Discussion

The present study is unique in the sense that no previous investigations have analyzed the sequential changes in the elemental composition of goat ELF by ICP. Although many investigators have analyzed the ELF of other species, they have restricted their studies to selected regions of the epididymis^[7,8]. This has been mainly due to micro, nano and sometimes pico quantity of luminal fluid available for analysis. This limitation has been overcome in the present study by using ICP analysis; the latter is particularly suitable for the simultaneous determination for many elements in minute volumes of sample.

Calcium, magnesium, zinc, copper and iron have been shown to be present in the ELF of several animal species^[4,8,10]. Some of these elements are so important that deficiencies are associated with fertility disorders^[11,12] that could be rectified by replacement therapy^[13]. Various roles have been ascribed to these elements in the epididymal environment, including the initiation of sperm motility^[14], the acquisition of fertilizing ability^[1], the stabilization of sperm structure^[15,16], and the regulation of metabolism^[17].

To the best of our knowledge, the present study is perhaps the first that reports the presence of nickel, chromium and titanium in the ELF of goat under normal physiological conditions. Heavy metals have been shown to be present in male reproductive tissues and bring about profound changes both in laboratory animals^[18-20] and in humans in occupational exposure^[21]. It may be relevant to point out that the pastures used for grazing our goats are located at the foothills of the Himalayas rich in mineral deposits. It is therefore likely that these elements were acquired through dietary sources and eventually found a berth in the male reproductive tract. Further investigations would be needed to establish if these elements in any way affect epididymal function.

It is noteworthy that the Na⁺/K⁺ ratio in the luminal fluid decreased from the proximal to the distal segments of the epididymis. Similar observations have been made in other animal species^[1,8]. The secretory and absorptive nature of the epididymal epithelium is perhaps responsible for the change in the ionic composition, which may ultimately be of functional significance^[6]. Eearlier reports also underlined the importance of sodium and potassium concentrations in the male reproductive tract^[12] and abnormality would lead to epididymal dysfunction. Moreover, in separate experiments, it has been convincingly shown that the levels of sodium and potassium delicately control the sperm motility, and quiescence and viability^[1,12]. The importance of K on the quiescent phenomenal was elaborated as early as 1986^[1]. We are of the opinion that the influence of a single ion is quite different from that of ions when present together. Thus, it is concluded that the unique pattern in Na⁺/K⁺ ratio observed in the luminal fluid may be related to sperm maturation in the proximal segments of the epididymis and sperm storage in the distal segments.

Acknowledgements

References

[1] Cooper TG. The epididymis, sperm maturation and fertilization. Berlin: Springer-verlag, 1986. p 18 & 281.
[2] Setchell BP, Sanchez-Partida LG, Chairussyuhur A. Epididymal constituents and related substances in the storage of spermatozoa: a review. Reprod Fertil Dev 1993; 5: 601-12.
[3] Hinton BT, Palladino MA, Rudolph D, Lan ZJ, Labus JC. The role of the epididymis in the protection of spermatozoa. Current Topics Devel Biol 1996; 33: 61-102.
[4] Howards SS, Lechene CP, Vigersky R. The fluid environment of the maturing spermatozoa. In: Fawcett DW, Bedford JM, editors. The spermatozoon. Baltimore: Urban-Schwarenberg, 1979. p 35-41.
[5] Hinton BT, Palladino MA. Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment. Microsc Res Tech 1995; 30: 67-81.
[6] Setchell BP, Maddocks S, Brooks DE. Anatomy, vasculature, innervation and fluids of the male reproductive tract. In: Knobil E, Neill JD, editors. The Physiology of Reproduction, Volume I, Second edition. New York: Raven Press Ltd; 1994. p 1063-175.
[7] Levine N, Marsh DJ. Micropuncture studies of the electrochemical aspects of fluid and electrolyte transport in individual seminiferous tubules, the epididymis and the vas deferens in rats. J Physiol 1971; 213: 557-70.
[8] Jenkins AD, Lechene CP, Howards SS. Concentration of seven elements in the intraluminal fluids of the rat seminiferous tubules, rete testis and epididymis. Biol Reprod 1980; 23: 981-7.
[9] Besancon J, Dacheux JL, Paquin R, Tremblay RL. Major contribution of epididymis to -glucosidase content of ram seminal plasma. Biol Reprod 1985; 33: 296-301.
[10] Stoltenberg M, Ernst E, Andreason A, Danscher G. Histochemical localization of zinc ions in the epididymis of the rat. Histochem J 1996; 28: 173-85.
[11] Hartoma TR, Nahoul K, Netter A. Zinc, plasma androgens and male sterility. Lancet 1977; 11: 1125-6.
[12] Wong PYD, Lee WM. Ionic mechanisms of sperm motility initiation. In: Lobl TJ, Hafez ESE, Editors. Male fertility and its regulation. Lancaster: MTP Press; 1985. p 411-6.
[13] Antoniou LD, Shalhoub RJ, Telechery S, Smith JC Jr. Reversal of uremic impotence by zinc. Lancet 1977; 11: 895-8.
[14] Serres C, Feneux D, Bernon B. Decrease of internal free calcium and human sperm movement. Cell Motil Cytoskeleton 1991; 18: 228-40.
[15] Silvestroni L, Menditto A, Modesti A, Scarpa S. Zinc uptake in human seminal spermatozoa: Characterization and effects on cell membranes. Arch Androl 1989;23: 97-103.
[16] Jochenhovel F, Bols-Pratsch M, Bertam HP, Nieschlag E. Seminal lead and copper in fertile and infertile men. Andrologia 1990; 22: 503-11.
[17] Eliasson R, Johnsen O, Lindholmer C. Effect of zinc on human sperm respiration. Life Sci 1971; 10: 1317-20.
[18] Barratt CLR, DaviesAG, Bansal MR, William ME. The effect of lead on the male reproductive system. Andrologia 1989; 21: 161-6.
[19] Calflisch CR, DuBose TD Jr. Cadmium induced changes in the luminal fluid pH in the testis and epididymis of the rat in vivo. J Toxicol Environ Health 1991; 32: 49-57.
[20] Hess RA. Effects of environmental toxicants on the efferent ductules, epididymis and fertility. J Reprod Fertil Suppl 1998; 53: 247-59.
[21] Wildt K, Eliasson R, Berlin M. Effects of occupational exposure to lead on sperm and semen. In: Clarkson TW, Nordberg GF, Sager PR, Editors. Reproductive and developmental toxicology of metals. New York: Plenum Press; 1985; p 279-99.

Correspondence to: Dr. Ben M.J. Pereira,  Reproductive Biology Laboratory, Department of Bioscience and Biotechnology,University of Roorkee, Roorkee-247 667, U.P., India.
Tel: +91-133-285 216, Fax: +91-133-273 560
e-mail: benmjfbs@rurkiu.ernet.in
Received 2000-07-10     Accepted 2000-11-07