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BOR - Papers in Press, published online ahead of print June 23, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031401
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BIOLOGY OF REPRODUCTION 71, 1508–1516 (2004)
DOI: 10.1095/biolreprod.104.031401
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Composition of Luminal Fluid Secreted by the Seminiferous Tubules and After Reabsorption by the Extratesticular Ducts of the Japanese Quail, Coturnix coturnix japonica1

J. Clulow, and R.C. Jones2

Discipline of Biological Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present report examines the composition of luminal fluid in the seminiferous tubule (STF), rete testis (RTF), and ductus epididymidis of the Japanese quail (Coturnix coturnix japonica). This subject is of particular interest, both because the reproductive ducts are intra-abdominal and because sperm production is more rapid in birds than in mammals. It was interpreted that micropuncture samples of STF contain varying amounts of contamination with intracellular solute, particularly K and protein. The concentration of solute in samples was correlated with packed cell volume (spermatocrit), and when the latter was used to assess estimates of solute concentration in STF, the magnitude of the estimates were much the same as determinations in RTF. Consequently, it is concluded that the fluid entering the rete testis of the quail is the primary secretion of the seminiferous tubules. The composition of RTF in the quail was determined to be 148 mM Na, 126 mM Cl, 9.8 mM K, 2.7 mM Mg, 1.4 mM Ca, 2.1 mM glutamate, 3.4 mM glutamine, 20.2 mM bicarbonate, 1.8 µg µl–1 of protein, pH 7.34, and 310 mmol kg–1, and it is significantly different from the composition of blood plasma. Estimates of solute output by the testis and reabsorption by the extratesticular ducts indicate, first, that most of the solutes secreted into the seminiferous tubules are subsequently reabsorbed from the extratesticular ducts and, second, that sufficient solute of testicular origin (except for protein) exists to account for the concentrations of solutes throughout the lumen of the duct system. Changes in the concentration of solute in the extratesticular ducts probably result from different reabsorption rates of solute and water. The composition of fluid from the distal end of the ductus epididymidis was 133 mM Na, 125 mM Cl, 25 mM K, 1.0 mM Mg, 0.3 mM Ca, 6.7 mM glutamate, 4.0 mM glutamine, 19.5 mM bicarbonate, 6.0 µg µl–1 of protein, pH 7.33, and 335 mmol kg–1, and it is significantly different from those of RTF and blood.

epididymis, male reproductive tract, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sperm production in birds is of particular interest, because it occurs at a much higher temperature than in mammals and is completed in extratesticular ducts that are less highly differentiated. Also, the higher rate of fluid output from the avian testis makes it an attractive model to examine the composition and modification of testicular secretions. Fluid output from the testis of the Japanese quail (Coturnix coturnix japonica) is 133 µl g–1 h–1 [1], compared with 4–50 µl g–1 h–1 for mammals [2–4], and fluid reabsorption by the ductuli efferentes is 100 µl cm–2 h–1 for the quail [1], compared with 17 µl cm–2 h–1 for the rat [4]. The present study compares the composition of luminal fluid from the seminiferous tubules and rete testis of the Japanese quail, and it provides estimates for the rates of transmural solute flux and modification of the composition of luminal fluid in the extratesticular ducts.

The luminal fluid in the seminiferous tubule (STF) is of particular interest, because its composition is determined by Sertoli cell secretions that provide the milieu for developing spermatozoa and the vehicle to transport them from the testis. However, studies with the rat (to our knowledge, the only species studied in this area) have not satisfactorily resolved the origin and composition of this fluid [3, 5, 6]. A number of problems exist in developing an integrated model that explains earlier findings: 1) The composition of fluid collected from the STF varies depending on the method of collection [6–8]; 2) substantial differences in composition of the STF and of the fluid collected from the rete testis (RTF) have been reported, especially in the concentration of K but also in those of Na, Cl, and protein [5, 7– 13]; and 3) substantial differences in spermatocrits (packed cell volume, presumed to consist mostly of spermatozoa) have been obtained for samples of STF and RTF [7, 9–11]. Tuck et al. [7] suggested a two-fluid theory to explain the findings. Those authors propose that fluid leaving the testis is a mixture of STF (high in K and HCO3–, low in Na and Cl) and a plasma-like fluid (high in Na and Cl, low in K) transported from blood into the rete testis. However, when differences in the concentration of Na and K were compared in STF (free-flow micropuncture samples) and in RTF (collected by cannulation), it was estimated that only 20%–30% of the latter would originate from the seminiferous tubules [5, 7]. Also, Setchell et al. [14] showed that direct counts of sperm concentrations (as opposed to spermatocrit determinations) and inositol concentrations were similar in rat STF and RTF. Consequently, the two-fluid theory was rejected, and it was interpreted that testicular fluid is derived from secretions of the seminiferous tubules but that the fluid must be modified substantially by considerable solute secretion and reabsorption in the rete testis [3, 5].

The avian extratesticular duct system differs significantly from the duct system in common laboratory and domestic mammals [15] in that it has more ductuli efferentes relative to testis mass and a relatively shorter and completely undifferentiated ductus epididymidis. The latter is joined to the ductuli efferentes by short, connecting ducts with an epithelium similar in structure to that of the ductus epididymidis. The role of the duct system in fluid reabsorption has been reported [1]. However, only a summary report on the composition of luminal fluid has appeared [16], and to our knowledge, nothing regarding the origins and rates of transepithelial solute transport in the avian genital ducts has been published in the literature.

In the present report, we interpret that luminal samples from the seminiferous tubules are often contaminated by K and protein from cells damaged by the collection process and that when this contamination is accounted for, the composition of STF and RTF is much the same. We also interpret that the composition of fluid leaving the testis is modified in the extratesticular ducts but that all the solute in the duct lumen could come from testicular secretions except (probably) for some of the protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Procedures were based on established methods for micropuncturing the seminiferous tubules and epididymis of mammals [7, 9, 10, 17, 18] and were accepted by the University on Newcastle's Animal Care and Ethics Committee.

Anesthesia

Quail were anesthetized with i.m. injections of 5-sec-butyl-5-ethyl-thiobarbituric acid (Inactin; Byk Gulden Pharmaceuticals, Konstanz, Germany) at an initial dose of 200 mg/kg, supplemented as necessary to maintain anesthesia. The animals were tracheotomized and maintained under anesthesia on a heated operating table (Institute of Physiology, Munich, Germany), with body temperature monitored by a rectal probe and maintained at 41–42°C. After sampling, quail were killed by an overdose of anesthetic. An initial study (n = 6 animals per group) comparing hematological parameters of samples collected immediately after anesthesia and after collection of micropuncture samples (1–2 h later) indicated that the collection procedures had little effect on the physiological state of the animals and no effect on the composition of the luminal milieu.

Collection of Fluids

A testis was exposed by removing a ventrodorsal section of the animal's most posterior rib. A cup and stabilizer manufactured with epoxy resin (Fig. 1) were used to support the testis and epididymal ducts and to immobilize them from rapid body movements caused by respiration [19]. The seminiferous tubules were exposed by an incision along the tunica albuginea and teasing the tunica apart with forceps. A hole in the stabilizer permitted access to the seminiferous tubules. Warmed saline rather than paraffin oil was used to maintain moist tissue surfaces, because oil may enter the lungs through air sacs that are (unavoidably) perforated during abdominal surgery. Testis temperature was maintained by its position within the body cavity. Seminiferous tubules were micropunctured (pipette tip diameter, 40–50 µm) under a Zeiss OPM1 (Carl Zeiss, Oberkochen, Germany). Samples from the seminiferous tubules were 0.1–0.5 µl except for that from one quail; its sample was 9.9 µl (presumably because of anastomoses between several seminiferous tubules in close proximity to the site of puncture). Samples that were obviously contaminated by collapse or rupture of the seminiferous tubules were not used (no samples contained erythrocytes). The RTF was collected by micropuncturing the proximal efferent ducts (pipette tip diameter, 50–60 µm), which are cavernous structures that are continuous with the rete testis (Fig. 2). Samples of 1–8 µl were collected over several minutes with the micropuncture pipette pointing upstream toward the rete testis. Although some movement of the testis and epididymis occurred during micropuncture, this did not affect sampling, because once penetrated, the duct wall sealed around the pipette tip and the ducts were flexible and did not tear. Samples of 0.5–3 µl of fluid were collected from the "proximal" ductus epididymidis, and samples of 1–8 µl were collected from both the "mid" and "distal" ductus epididymidis (Fig. 2) by micropuncture (pipette tip diameter, 50–80 µm) using a retractor (not shown) to hold the abdominal viscera away from the micropuncture site.



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  		FIG. 1. Testis cup (a) and testis stabilizer (b) used to support the testis and epididymal region (c) during micropuncture of the seminiferous tubules (e). Testis cup only (d) was used during micropuncture of the ductuli efferentes and ductus epididymidis



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  		FIG. 2. Diagram of the reproductive tract of the Japanese quail showing the sites used to collect samples of luminal fluid from the extratesticular ducts (using the terminology proposed by Jones [15]). CD, Connecting ducts; d, distal ductus epididymidis; ED, ductuli efferentes; m, mid-ductus epididymidis, just caudal to region overlying the testis; p, proximal ductus epididymidis, approximately in the middle of the region lying over the testis; RT, rete testis

Immediately after collection, samples of luminal fluid were transferred under water-saturated paraffin oil into 0.5- to 2.0-µl microcaps (Drummond Scientific, Broomall, Pennsylvania), sealed with Sealease (Clay Adams, Parsippany, NJ), and centrifuged immediately at 12 000 x g for 15 min in a Hawksley microhematocrit centrifuge. Spermatocrits were measured, and the supernatants were stored at –20°C between paraffin oil columns in microcaps. Samples of blood were collected by cardiac puncture after collecting the micropuncture samples.

Assays

Osmotic pressure was determined using a Wescor vapor-pressure osmometer (Wescor, Inc., Logan, UT) adapted for 0.5-µl samples by reducing the size of the paper sample-holding discs to 0.125x standard size. Standard curves were linear using the microsystem from 200 to 1000 mOsm kg–1. Concentrations of seven elements (Na, Mg, P, S, Cl, K, and Ca) were determined by z-ray microanalysis of microdroplets [4, 20, 21].

The pH of samples of blood (>1 ml), RTF (5–8 µl), and luminal fluid from the distal ductus epididymidis (5–10 µl) was determined using an MI-410 microcombination pH electrode (Microelectrodes, Inc., Londonderry, NH). Bicarbonate concentrations were calculated using the Henderson-Hasselbalch relationship [9, 22, 23].

Amino acids were identified and quantified against 22 amino acid standards using thin-layer chromatography with single- and double-dimension solvent systems [24–26] and ninhydrin staining. Glutamate and glutamine were identified as the two major amino acids that were present. Their concentrations were determined after separation using a single-dimension solvent system consisting of n-butanol:acetone:glacial acetic acid:water (35:35:10:20, v/v) in which the Rf values for glutamine and glutamate were 0.16 and 0.25, respectively (double-dimension systems confirmed they were not running with other amino acids). Glutamate and glutamine concentrations were determined photometrically [27] using a microassay system by removal of ninhydrin-stained spots (samples and standards) from the plates and measurement of absorbance at 570 nm in 0.2-ml microcuvettes. Protein concentrations were determined by the method described by Bradford [28].

Transmural Electrical Potentials and Estimation of Electrochemical Gradients

Transmural potential differences were measured using glass electrodes filled with 3 M KCl connected via silver-silver chloride half-cells to a marconi TF 2655 high-impedance voltmeter [7, 9, 29]. Glass electrodes were made from either single-filament glass capillary tubes (outer diameter, 2.0 mm; inner diameter, 1.2 mm; Clarke Electromedical Instruments) or from standard micropuncture capillary tubing pulled on a Narashige PN-3 electrode puller (Narishige Scientific Laboratories, Tokyo, Japan). The difference between the measured and equilibrium potentials was used to determine whether solute transport was against the electrochemical gradient (nominally designated as "active" transport) or down the electrochemical gradient (nominally designated as "passive" transport).

Estimation of Testicular Output and Net Fluxes

Estimates of testicular output of solute and net reabsorption by the extratesticular ducts were made using our published estimates of luminal plasma output from the testis and stereological determinations of spermatocrits along the genital ducts [1]. The expected concentration of solute at each sampling site (CE2) was calculated, assuming that no transport of the solute occurred either into or out of the duct between the site under consideration and the adjacent proximal site, as

where CE2 = expected concentration at the distal site (with no fluid transport), CM1 = mean (measured) concentration of solute at the proximal site, S1 = spermatocrit at the proximal site, and S2 = spermatocrit at the distal site.

It was interpreted that significant net transport of a solute (reabsorption or secretion) occurred if CE2 was outside the range of CM2 ± 1.96 (mean ± SEM), where CM2 = measured mean concentration of solute at the distal site.

The net rate of transport (reabsorption or secretion) of solutes between two regions in the genital ducts (Rsol) was estimated as

where F1 = fluid flow rate through the genital ducts at the proximal site, F2 = fluid flow rate through the genital ducts at the distal site, CM1 = measured mean concentration of solute at the proximal site, and CM2 = measured mean concentration of solute at the distal site.

A positive value indicates net reabsorption, and a negative value indicates net secretion. Transport rates were expressed as a function of epithelial surface area by dividing transport rates by A, the luminal surface area of duct epithelium between a proximal site and a distal site.

The concentration of solutes in the fluid that was reabsorbed (CR) between proximal and distal sites were estimated as

where Rsol = solute reabsorbed per unit time and RF = fluid volume reabsorbed per unit time.

It was interpreted that reabsorption of a solute was isosmolar relative to the luminal concentration if CR was within the range of CM1 ± 1.96, where CM1 = measured mean concentration of solute at the proximal site.

Statistics

Values for pH and solute concentrations are presented as the mean ± SEM. The SEMs were estimated from the variance between animals. F-tests were used to determine significant differences between means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Composition of STF and RTF

Spermatocrits of STF samples varied widely, from 1%– 2% to 70%, whereas spermatocrits for RTF samples were less than 7%. Even after extended centrifugation of STF samples with a high spermatocrit, an indistinct boundary was observed between the cellular and plasma phases. This was interpreted as the compacted material in STF containing matter in addition to spermatozoa that, under the light microscope, appeared to be cellular material from germinal epithelium disrupted during micropuncture (see Discussion). The evidence for this interpretation is as follows: 1) The dense cellular pellet that formed in spermatocrits of STF did not form in spermatocrits of RTF, 2) our determinations of sperm concentration were not significantly different for STF and RTF [1], 3) we did identify similar material under the electron microscope in micropuncture samples from the initial segment of the epididymis with long stereocilia extending into the lumen of the duct [18], and 4) analyses of the plasma component of the STF samples indicated correlations between spermatocrit values and the concentration of solutes that are normally higher in intracellular than in extracellular fluid. Figure 3 shows that a statistically significant, positive regression of osmolality and the concentrations of K, P, S, and protein against spermatocrit and a statistically significant, negative regression of the concentration of Cl against spermatocrit.



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  		FIG. 3. Linear regression lines for concentration of solutes versus spermatocrits (packed cell volumes) for centrifuged micropuncture samples from the seminiferous tubules. Spermatocrit values expressed as percentages were transformed to angles before calculation of correlation (r) and regression (b) coefficients. Asterisks indicate statistical significance of regression coefficients: **P < 0.01, ***P < 0.001

Two interpretations of the data were made to estimate the composition of uncontaminated STF. Because estimates of spermatocrits using stereological methods are 0.2% or less [1], the intercepts of the regression lines in Figure 3, with the ordinate for a spermatocrit of 0%, were used as an estimate of the concentration of luminal solute (intercept method). The second method (low-spermatocrit method) used only data with spermatocrits lower than the values shown in Table 1. Because the regression coefficients were not statistically significant for concentrations of glutamate and glutamine, all the data were used to determine the concentrations of these compounds in STF.


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  		TABLE 1. Composition of STF, RTF, and blood from Japanese quail.*

Table 1 shows that for STF, the two estimates of solute concentrations and osmolality are in fairly close agreement with one another. They are also much the same as the composition of the one large sample (9.9 µl) of STF that was collected (see Materials and Methods): 170 mM Na, 10.0 mM K, 171 mM Cl and 1.7 µg µl–1 of protein. The estimate of protein concentration in STF is negative by the intercept method, but the estimate by the low-spermatocrit method is similar to the concentration in RTF. The estimates of elemental concentrations in STF are also in general agreement with determinations for RTF, except that concentrations of Na, Cl, and S in STF were a little higher than those in RTF. The concentrations of glutamate and glutamine in STF were also higher than those in RTF.

It is noteworthy that both STF and RTF are isosmotic with blood. For the STF samples, the sum of the mean concentrations of Na, K, and Cl (low-spermatocrit method) account for 104% of the measured mean osmolality (316 mmol kg–1) when an osmotic coefficient of 0.91 is applied [9], indicating that these solutes generate most of the osmolality in STF but also that the means in Table 1 may be overestimates of the actual concentrations. In this respect, the equivalent Na, K, and Cl values in RTF were 17 to 26% lower and, together, accounted for 83% of the measured osmolality (89% when bicarbonate, as shown in Table 5, was included).


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  		TABLE 5. pH and bicarbonate concentration of blood and luminal fluid from the rete testis and distal ductus epididymidis of the Japanese quail.a

Composition and Reabsorption of Fluid and Solute in Extratesticular Ducts

Tables 2 and 3 show the concentrations of the major solutes that contribute to the osmolality of the extratesticular fluids, and Table 4 shows their contribution to the total osmolality of the fluids. Luminal fluids from the proximal ductus epididymidis were isosmotic with samples from the rete testis, but samples from the mid (P < 0.001) and the distal (P < 0.001) ductus epididymidis were hyperosmotic relative to these samples and blood, with the increase of 19–32 mmol kg–1 along the ductus epididymidis being associated with a similar increase in osmotic difference (Table 4). Most of the osmotic pressure of the fluids from the ductus epididymidis was accounted for by Na and Cl, and their concentrations were much the same as in RTF (Table 2). However, whereas the concentration of Cl was much the same (and higher than in blood, P < 0.01) along the ductus epididymidis, the concentration of Na decreased distally along the duct (P < 0.05 for comparison of proximal vs. distal samples). Consequently, the concentration of Na in the distal ductus was lower than in blood (P < 0.01). The concentration of K was 2.5-fold greater along the ductus epididymidis than in RTF (P < 0.001) and 5.5-fold greater than in blood (P < 0.001). The changes in concentrations of Na and K along the extratesticular ducts were caused by their different rates of reabsorption (see below). The changes resulted in the ratio of Na to K reducing from 15:1 in RTF to 5:1 in fluid from the distal ductus epididymidis (this ratio was 35:1 in blood plasma).


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  		TABLE 2. Concentration of elements (mM) in blood plasma and luminal fluids from the extratesticular ducts of the Japanese quail.a


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  		TABLE 3. Concentrations of elements in protein-free ultrafiltrates of plasma from blood and luminal fluids from the extratesticular ducts of the Japanese quail.a


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  		TABLE 4. Osmotic pressure, electrical potential difference, and concentrations of glutamate, glutamine, and protein in blood plasma and luminal fluids from the extratesticular ducts of the Japanese qual.a

The concentrations of Mg and Ca along the ductus epididymidis were lower than in RTF (P < 0.01). The concentration of Mg increased distally along the ductus (P < 0.05 for comparison of mid vs. distal samples). The concentration of Ca was low (at the limits of detection by energy-dispersive x-ray microanalysis) along the ductus epididymidis, and it was much lower than in blood (P < 0.001).

The concentration of P was 5.5-fold greater in luminal fluids from the ductus epididymidis than in RTF (P < 0.001) but lower than in blood (P < 0.01). Table 3 shows that much of the P was associated with protein in blood (unfiltered fraction, P < 0.001) and the luminal fluids from the extratesticular ducts (P < 0.01 for samples from the distal ductus epididymidis). A greater concentration of filterable P in fluid from the ductus epididymidis than in RTF (P < 0.001) suggests that the concentration of inorganic P increased between the regions. The concentration of S was 1.6-fold greater in luminal fluids from the ductus epididymidis than in RTF (P < 0.001) but lower than in blood (P < 0.01). However, the concentration of S increased along the ductus epididymidis (P < 0.05) in a manner that closely paralleled the increase in the concentration of protein. Consistent with this interpretation, the concentration of S in ultrafiltrates was significantly less than that in unfiltered samples (P < 0.01), being approximately 2.0 mM throughout the extratesticular ducts.

Both RTF and fluid from the ductus epididymidis were alkaline but also more acidic than blood plasma (P < 0.001) by nearly 0.2 pH units (Table 5). The concentrations of bicarbonate in these fluids were much the same and approximately 70% of the concentration in blood.

Glutamate and glutamine were the major free amino acids in luminal fluids from the extratesticular ducts (Table 4). The concentration of both amino acids was greater in samples from the proximal ductus epididymidis than in RTF (P < 0.001 for glutamate, P < 0.05 for glutamine). However, the concentration of glutamine varied along the ductus (P < 0.001), whereas little change was found in the concentration of glutamate along the ductus.

The concentration of protein was substantially lower in the extratesticular ducts than in blood (P < 0.01). The concentration doubled between the rete testis and proximal ductus epididymidis (P < 0.001), but the increase along the ductus was not statistically significant.

Solute Output by Testis and Reabsorption by Extratesticular Ducts

Table 6 summarizes the estimates of solute output by the testis and reabsorption (or secretion) by the extratesticular ducts and compares them to published estimates of fluid output and reabsorption [1]. Table 6 also shows when transepithelial transport of solute in the extratesticular ducts is against or down an electrochemical gradient.


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  		TABLE 6. Testicular output and subsequent rates of reabsorption of fluid and solute by the ductuli efferentes and ductus epididymidis (DE) of the Japanese quail.a

Both Na and Cl leave the testis at a rate (40 000 nmol h–1) approximately one order of magnitude greater than that for K (2800 nmol h–1) and two orders of magnitude greater than those for Mg, Ca, P, and S (<1000 nmol h–1). The output of Mg was threefold greater than that of Ca, and the rate for glutamine was approximately twofold greater than that for glutamate.

Significant amounts of all the substances examined were reabsorbed in the ductuli efferentes (P < 0.001). However, when expressed relative to testicular output, a greater proportion of fluid (99%) than that for any of the solutes, except for Ca and protein, was reabsorbed by the ductuli. The rates of reabsorption of Na and Cl were much greater than the rate for K, both in absolute amounts (30 000, 26 000, and 200 nmol h–1, respectively) and as a proportion of testicular output (75%, 68%, and 7%, respectively). It was interpreted that reabsorption of Na and Cl from the efferent ducts was isosmolar. This interpretation is based on their concentrations being much the same in samples from the proximal and distal end of the ductuli (Table 2) and there being no significant difference in their concentrations in RTF and estimates of their concentrations in reabsorbates from the ductuli efferentes (data not shown). However, it is interpreted that fluid reabsorption of the other solutes shown in Table 6 deviated slightly from isomolar. This is because their concentrations in samples from the rete testis and proximal ductus epididymidis were significantly different.

The rates of reabsorption by the ductuli efferentes were several orders of magnitude greater than those in the ductus epididymidis. The estimates indicate that Na, Cl, K, and P (P < 0.001) as well as glutamate (P < 0.05) were transported from the ductus epididymidis. They also indicate that glutamine was secreted into the proximal part of the ductus epididymidis (P < 0.01), that Mg was secreted into the distal ductus epididymidis, and that the rates of secretion were very low (5.1 and 0.9 nmol h–1, respectively). Estimates of fluid reabsorption from the ductus epididymidis generally deviated significantly from isosmolar for all the solutes in Table 6 except for Mg and Ca. This indicates a difference in the net rates of reabsorption of water and solutes in the ductus and the ductuli efferentes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Composition of STF and RTF

An important interpretation in the present study was that the composition of quail STF collected by micropuncture was significantly affected by the presence of nonspermatozoal material in samples. Calculations of the potential amount of contamination in micropuncture samples justify this interpretation. Assuming (conservatively) that the micropuncture pipette was a cylinder of diameter 40 µm and of length 50 µm, it would displace (and possibly damage) 63 nl of epithelium, which is 13%–63% of the volume of the micropuncture samples collected (100–500 nl, except for the sample of 9.9 µl). Assuming that [K+]i = 140 mM, the volume of intracellular fluid that would be required to increase the [K+] in a sample of STF from 10 to 70 mM (the highest concentration determined) would be 43 nl, which is a volume smaller than the amount of epithelium that may be damaged. The main problem in collecting uncontaminated micropuncture samples from the lumen of the seminiferous tubules is that these tubules have a very tall epithelium and a narrow lumen (see Fig. 1 in [30]), like the initial segment of the epididymis, which cannot be micropunctured without some contamination of samples [18]. It is noteworthy that our interpretation of the composition of STF differs from that of Fisher [6], who recognized that perfusion may damage the seminiferous epithelium of the rat but who also accepted (without determining the composition) that samples of free-flow fluid are indicative of fluid secreted by the seminiferous tubules. Our determinations indicate that even this fluid is contaminated. Consequently, it is suggested that the most suitable approach for determining the composition of the secretions of the seminiferous epithelium is to examine frozen sections by x-ray microanalysis.

This report shows that the composition of avian RTF is similar in composition to that of the eutherian [4] and marsupial [21] mammals that have been studied. The interpretation that avian STF is similar in composition to RTF indicates that further work is required on the rat to determine the composition of its STF. As in the quail, micropuncture samples of STF from the rat have high spermatocrit values (e.g., 6%–20% [7], 20% [9], and 41% [10]). Hinton and Setchell [31] obtained spermatocrit values of 10%–15% when samples were centrifuged in tubing with an inner diameter of 80 µm [17]) but only 3.6% when centrifuged in tubing with an inner diameter of approximately 500 µm. However, it is suggested that the wider-bore tubing may have improved the packing among sperm of the contaminating cellular material, because the concentrations of K and protein in the STF were three- to fivefold greater than those in the RTF [7, 17]. Furthermore, Hinton [17] found that the [K+] in STF sampled from prenatally x-irradiated rats (Sertoli cell-only seminiferous epithelium) was much lower (6.8 ± 0.24 mM) than from rats that were not x-irradiated (46.4 ± 1.69 mM).

Composition and Reabsorption of Fluid and Solute in Extratesticular Ducts

To our knowledge, this present study is the first to report the composition of luminal fluids all along the avian male genital ducts. Our results indicate that the seminiferous tubules secrete a fluid that is rich in Na and Cl, that the concentration of K is at least twice that of blood, and that this fluid is the main—and possibly the only—source of the luminal solute in the extratesticular ducts. The concentrations of solute shown in Tables 1, 2, and 4 account for 93% and 89%, respectively, of the osmotic pressure of fluid from the rete testis and distal ductus epididymidis, indicating that most of the solute in quail luminal fluids was identified. Consequently, the common solutes secreted into the epididymis of eutherian mammals, such as inositol, phosphocholines, and carnitine, are not secreted into the quail ducts in significant amounts. The literature contains a number of reports on the composition of seminal plasma from the fowl and turkey collected without contamination by secretions from the cloacal glands [32–34], and these samples would be comparable to the luminal fluids from the distal ductus epididymidis in the present study. The fluids are similar to the quail samples regarding concentrations of Na (136–145 mM) and K (13–18 mM). However, they differ in that very high levels of glutamate (75–88 mM) replace Cl (23–46 mM) as the principal anion, and they are also isosmotic with blood. It is concluded that the concentrations of Na are higher, and that those of P are lower, in the birds that have been studied than in fluid from the cauda epididymidis of the mammals reported by Jones [35] (17–41 mM Na, 18–49 mM K). Consequently, a significant difference is found in the ratio of the concentrations of Na to K at the distal end of the extratesticular ducts of these birds (5.3– 11.2) and mammals (0.4–1.3). Although no acidification of the luminal fluid is found in the extratesticular ducts of the quail, as in the fowl and turkey, this may not be physiologically significant, because the pH of fluid from the cauda epididymidis of mammals varies from 6.7 in the dog to 7.0 in the rat, guinea pig, and rhesus monkey [35].

The present report suggests that the major electrolytes and organic compounds in the quail genital ducts originate as secretions from the testis rather than from the epithelium lining the extratesticular ducts. This is primarily a consequence of a large testicular output and subsequent reabsorption of fluid by the extratesticular ducts. All solute transport from the ductuli efferentes was shown to be absorptive, and most of the solute transport in the ductus epididymidis was absorptive. Even when estimates of solute secretion in the ductus epididymidis were statistically significant, the rates were very low. Consequently, the suggestion [32] that glutamate may be derived from epithelial secretion by the ductus epididymidis of the fowl should be revised. On the other hand, further studies are necessary to resolve whether any solute is secreted into the ductus epididymidis. For example, the testis may not be the only source of proteins in the extratesticular luminal fluids, because the estimated rate of testicular output (355 µg h–1) was slightly less than the rate of reabsorption from the ductuli efferentes (366 µg h–1), an increase in the concentration along the ductus epididymidis was found, and a low rate of protein secretion into the ductus epididymidis was estimated (although neither was statistically significant). However, a statistically significant increase was observed in the concentration of total (but not filterable) sulfur in the ductus epididymidis. Furthermore, polyacrylamide gels of luminal fluids from the ducts indicate that one protein is secreted into the extratesticular ducts that is not present in the RTF [16]. However, further studies are required to resolve whether this protein is secreted by the ductuli efferentes [36] or more distally.

In the seminiferous tubules of the quail, the major ions (Na, K, and Cl) were secreted into the lumen against an electrochemical gradient (transmural potential difference, –3.7 mV), and none of the elements studied was in electrochemical equilibrium along the extratesticular ducts of the quail. Because the reabsorption of Na was against its electrochemical gradient in all regions of the extratesticular ducts, and because it was reabsorbed isosmolar or hypermolar to its luminal concentrations, its transport may be the primary driving force for fluid reabsorption. Both Cl and K were reabsorbed down electrochemical gradients, so direct, active transport is not necessary to remove them from the ducts. It is possible that the epithelium of the ductus epididymidis is relatively impermeable to Cl and K ions, because these ions are maintained at concentrations greater than their electrochemical equilibrium for a period of time (22 h) sufficient for equilibrium to be reached [1]. Both Mg and Ca were transported out of the extratesticular ducts against their electrochemical gradient (except distally, where there may be some passive secretion). However, it is considered that these ions may not be actively transported if the primary source of active transport is the Na pump, and transport of both Ca and Mg is secondary to transport of Na.

The interpretation of isosmolar reabsorption of Na and Cl by the ductuli efferentes of the quail is in agreement with work involving the rat [4, 37]. Consequently, the reabsorption of water by the ductuli may be dependent on the transport of both Na and Cl in the quail, as it is in the rat [37]. However, there appear to be differences between the quail and rat in the mechanism of reabsorption of K, Mg, and Ca (but not of P and S). Whereas, between the rete testis and proximal ductus epididymidis in the quail, an increase occurs in the concentration of K and a decrease in the concentrations of Mg and Ca, no changes occur in the concentrations of these solutes in samples from the proximal and distal ends of the ductuli efferentes in the rat.

It is concluded that the extratesticular ducts of the Japanese quail play an important role in regulating the milieu of sperm while they develop the capacity to fertilize ova and are stored and available for mating. The finding that the milieu is not modified to the same extent as in the mammalian epididymis is consistent with the relative importance of posttesticular sperm maturation and storage in birds and mammals [15, 38].


   ACKNOWLEDGMENTS
 
We appreciate correspondence with B.T. Hinton and B.P. Setchell during the preparation of this manuscript.


   FOOTNOTES
 
1 Supported by grants from the Australian Research Council, The Research Management Committee, University of Newcastle, and a Commonwealth Postgraduate scholarship to J.C. Back

2 Correspondence. FAX: 61 2 4921 6923; bircj{at}cc.newcastle.edu.au Back

Received: 29 April 2004.

First decision: 2 June 2004.

Accepted: 18 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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