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Gamete Biology |
Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, female reproductive tract, glutamate, oviduct, sperm motility and transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Likewise, the means by which sperm emerge from the SST has remained unknown. In general, this phenomenon has been attributed to an action external to sperm, hence the term sperm release mechanism. As first reviewed by Zavaleta and Ogasawara [2] and most recently by Bakst et al. [1], different mechanisms have been proposed. These include cyclic secretion from epithelial cells and physical distortion of the oviduct. Neither smooth muscle cells nor myoepithelial cells are found in association with SST, and tubular epithelial cells are nonciliated [3]. Thus, expulsion could not be attributed to either tubular contraction or ciliary action. Van Krey et al. [4] proposed that sperm egress results from a failure of resident sperm to persist in an agglutinated, immobilized state. In this regard, reversible inhibition of sperm motility within the SST has been assumed [1]. Nonetheless, the term quiescent is used in reference to sperm within the SST [5].
The present study entails a synthesis of previous knowledge of SST structure with new knowledge gained from computer-assisted sperm motion analysis. This technique enabled an integrated understanding of how fowl sperm motility is maintained under physiological conditions. Likewise, insight was gained regarding the behavior of sperm populations through time, a dimension that is critical to the phenomenon of sperm storage. It is proposed that such knowledge, within the context of SST histology, affords a simple and reasonable explanation for sperm residence within and emergence from the SST. This synthesis of information represents the first coherent working model for sperm storage in birds.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Roosters (n = 10) were selected from a base population of 110 individuals based on sperm mobility phenotype as outlined by Froman et al. [6]. Only males with mobility scores greater than 1 SD from the population mean were used as semen donors. Roosters were housed and handled in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, First Edition, 1988. Unless specified, individual ejaculates were used to replicate observations within experiments.
Extracellular Milieu and Sperm Motility
Sperm concentration was measured spectrophotometrically [7]. Unless specified, reagents were purchased from Sigma-Aldrich (St. Louis, MO). Accudenz was purchased from Accurate Chemical & Scientific Corporation (Westbury, NY). Tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a Ca2+ chelator, was purchased from the Calbiochem-Novabiochem Corporation (San Diego, CA).
Sperm motility was inhibited in the first experiment as follows: sperm were diluted to 2.0 x 109 cell/ml with 128 mM sodium glutamate buffered with 50 mM N-tris[hydroxy-methyl]methyl-2-amino-ethanesulfonic (TES), pH 7.4. The TES-buffered sodium glutamate contained 5 mM BAPTA. A 2-ml volume of the sperm suspension was placed in a 12- x 75-mm borosilicate glass culture tube, and the tube was incubated at 41°C for 2 min. Then, sperm were washed by centrifugation through 12% (w/v) Accudenz [8] with three modifications. First, the Accudenz solution was prepared with TES-buffered sodium glutamate containing 5 mM BAPTA. Second, this Accudenz solution was prewarmed to 41°C before sperm suspension overlay. Third, centrifugation was performed at 20°C. Computer-assisted sperm motion analysis was performed [9] after washed sperm were resuspended in TES-buffered sodium glutamate containing 5 mM BAPTA (control sperm) or 2 mM CaCl2 (treated sperm). Motile concentration, measured at 1-min intervals, was plotted as a function of time. In a second experiment, computer-assisted sperm motion analysis was performed with nonwashed sperm. In this case, sperm were diluted with 128 mM NaCl buffered with 50 mM TES, pH 7.4 (TES-buffered saline). Control sperm were diluted with TES-buffered saline containing 2 mM CaCl2. Treated sperm were diluted with TES-buffered saline containing 2 mM CaCl2 and 1 mM N-methyl-D-aspartic acid (NMDA), a glutamate-receptor agonist. Average straight-line velocity (VSL) was analyzed in a paired comparison [10].
Maintenance of Sperm Motility
Computer-assisted sperm motion analysis was performed in two related experiments as described by Froman and Feltmann [9] with the exception that 10-µl samples of sperm suspension were withdrawn at 10-min intervals from a 300-µl volume of sperm suspension incubated at 41°C for 1 h. In the first experiment, sperm were incubated in TES-buffered saline containing 2 mM Ca2+. In the second experiment, sperm were incubated in TES-buffered saline containing 5 mM BAPTA. In this case, a 1-µl volume of excess Ca2+ was added to the residual sperm suspension after the measurement at 60 min.
Computer-assisted sperm motion analysis was performed in a third experiment in which sperm suspensions were preincubated at 41°C for 10 min in 30, 60, or 120 µM arachidonyltrifluoromethyl ketone (Calbiochem), a specific inhibitor of phospholipase A2. The buffer control was TES-buffered saline containing 2 mM Ca2+ and 5% (v/v) dimethyl sulfoxide. Data were analyzed with a randomized complete block design [10]. The Student-Newman-Keuls test was used for a posteriori comparison among means [11].
Acylcarnitine analysis was performed in a fourth experiment in which replicate observations (n = 5) were made with pooled semen. In each case, sperm were washed by centrifugation through 12% (w/v) Accudenz as outlined above. However, duplicate tubes were used for each iteration. Washed sperm were resuspended to a concentration of 3 x 109 sperm/ml in one of two media: TES-buffered saline containing 5 mM BAPTA, or TES-buffered saline containing 2 mM Ca2+. Sperm suspensions, approximately 0.3–0.5 ml in volume, were placed in 12- x 75-mm culture tubes, which were incubated at 41°C for 3 min. Motility was assessed at 41°C by light microscopy. A 10-µl volume of sperm suspension was placed on a coverslip. The coverslip was inverted and placed above the concavity of a hanging drop slide. Immediately thereafter, sperm suspensions were frozen at -80°C. Acylcarnitine analyses were performed by the Center for Inherited Disorders of Energy Metabolism (Case Western Reserve University, VA Medical Center, Cleveland, OH). Concentrations of long-chain acylcarnitine species were summed for each sample. Data were analyzed by paired comparison [10].
VSL Distributions
The effect of temperature on the shape of the VSL distribution was tested by performing computer-assisted sperm motion analysis at 20, 30, and 40°C. In each case, data were accrued by incubating sperm suspensions as outlined above at the experimental temperature and then sampling at 5-min intervals over the course of 30 min. Linear regression [12] demonstrated that motile concentration and average VSL were independent of time for each treatment. Thus, frequency distributions were generated for each temperature using pooled data. The VSL of each observed sperm cell was assigned to 1 of 12 categories ranging from 0 to 120 µm/sec in increments of 10 µm/sec. Each category was plotted as a proportion of the total motile concentration (i.e., the y-intercept estimated by linear regression).
A retrospective comparison was made among VSL distributions using data from experiments in which motile concentration was independent of time (see Fig. 5; n = 6668 tracks), when motile concentration decreased as a function of time because of BAPTA (see Fig. 3; n = 3660 tracks), and when motile concentration was independent of time but average VSL was enhanced by glutamate (see Fig. 1; n = 3039 tracks). Frequency distributions were generated as above with the exception that data were normalized (i.e., each category within a distribution was plotted as a percentage of the maximal value within that distribution).
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VSL and Trajectory
The database generated with TES-buffered sodium glutamate containing 2 mM Ca2+ (see Fig. 1, upper line; n = 3039 tracks) was used as a data source. Straightness, a measure of sperm cell trajectory [9], was plotted as a function of VSL from 10 to 100 µm/sec in increments of 10 µm/sec. Data approximated an exponential function in which straightness increased abruptly between 10 and 30 µm/sec toward an asymptote. Therefore, the data set was augmented with observations corresponding to velocities of 12, 15, and 25 µm/sec.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Washed sperm were essentially immotile when incubated at body temperature in the presence of BAPTA (Fig. 1, lower line). In contrast, sperm rendered immotile by washing became motile en masse when resuspended in a medium containing Ca2+ and Na+ (Fig. 1, upper line). Extracellular Ca2+ did not maintain sperm motility in buffered, isotonic media prepared with sucrose, choline chloride, lithium chloride, potassium chloride, or potassium glutamate (data not shown). Average VSL (mean ± SEM) was 52 ± 3.1 µm/sec versus 70 ± 4.1 µm/sec (P < 0.0001) for nonwashed sperm suspended in TES-buffered saline containing 2 mM Ca2+ versus TES-buffered saline containing 2 mM Ca2+ and 1 mM NMDA.
Maintenance of Sperm Motility
Motile concentration was independent of time when sperm were incubated at body temperature for 1 h without an exogenous substrate (Fig. 2). As shown in Figure 3, BAPTA induced an exponential decay in motile concentration. Sperm rendered immotile by incubation with BAPTA became fully motile when the chelator was overcome with excess Ca2+. Motility was inhibited by arachidonyltrifluoromethyl ketone in a dose-dependent manner (Fig. 4). Washed sperm diluted to 3.0 x 109 sperm/ml in TES-buffered saline containing 5 mM BAPTA were immotile at 41°C, as evidenced by hanging drop preparation. In contrast, sperm rendered immotile by washing became motile when resuspended in TES-buffered saline containing 2 mM Ca2+. The long-chain acylcarnitine content of motile sperm was 1.7-fold greater than that of immotile sperm (P < 0.01). The relative proportions of long-chain acylcarnitine species in motile sperm are shown in Table 1.
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VSL Distributions
The VSL distributions were skewed at 20, 30, and 40°C (Fig. 5). Whereas temperature had little effect on motile concentration, temperature had a profound effect on the shape of the distribution: as temperature increased, the height of the distribution decreased as the area and length of the upper tail increased. The VSL distribution at physiological temperature was altered by glutamate, which acted as a motility agonist (Fig. 6).
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VSL and Trajectory
An exponential relationship was observed when trajectory, as measured by straightness, was plotted as a function of VSL (Fig. 7). Average straightness increased abruptly toward an asymptotic value as velocity increased from 10 to 30 µm/sec. However, this response was not uniform within a population of sperm, as evidenced by the magnitude of the SDs. In contrast, straightness was uniformly high at velocities of 30 µm/sec or greater.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Males with high sperm mobility were used as semen donors in the present study. Such males produce semen with a maximal concentration of highly motile sperm cells [9]. These sperm cell populations are most useful in the study of factors affecting either the maintenance or loss of motility. Based on the experimental outcomes presented in Results along with the histology of the SST [3], the SST epithelial cell ultrastructure [17], the phenomenon of sperm cell rheotaxis [18], and the pattern of sperm egress from the SST [19, 20], it is proposed that sperm cell motility is pivotal to sperm storage within the SST. Specifically, it is proposed that calcium acts as a motility agonist by activating phospholipase A2, which releases endogenous fatty acids and thereby enables sperm to ascend the vagina and enter the SST. It is proposed that resident sperm maintain their position against a fluid current generated by SST epithelial cells and that such sperm metabolize exogenous fatty acids released from surrounding lipid-laden epithelial cells. It is proposed that motile sperm emerge from the SST once their velocity decreases to a point at which position within the current cannot be maintained. Finally, it is proposed that the shape of the VSL distribution accounts for the exponential pattern of sperm emergence from the SST.
The first step in sperm storage is ascension through the vagina, followed by entrance into the SST. Whereas extracellular Ca2+ is a known motility agonist, a precise role for Ca2+ has remained elusive [5]. Computer-assisted sperm motion analysis confirmed that maintenance of motility at body temperature is a Ca2+-dependent phenomenon, but with the constraint that motility was dependent on the oxidation of endogenous substrates (Figs. 1 and 2). In preliminary work, cessation of motion was not immediate when freshly ejaculated sperm were diluted with media prepared without Ca2+. Such media contained micromolar amounts of Ca2+, as evidenced by atomic absorption spectrophotometry. Therefore, a high-affinity Ca2+ chelator (i.e., BAPTA) was used to control extracellular Ca2+ and thereby render sperm immotile, either via washing (Fig. 1) or incubation (Fig. 3). In other words, time was required for BAPTA to exert its effect on each cell within a sperm cell population.
Transient motility in the presence of BAPTA was attributed to an efflux of Ca2+ from the mitochondrial matrix into the incubation medium. This conclusion is consistent with that of Thomson and Wishart [21], who demonstrated that fowl sperm extrude Ca2+ at body temperature, and with the fact that mitochondria can store Ca2+ under physiological conditions [22]. The ability of extracellular Ca2+ to initiate motility was consistent with data presented by Ashizawa et al. [5]. However, in this case, the initiation of motility in the absence of an exogenous substrate (Fig. 3) was attributed to the activation of phospholipase A2, a Ca2+-dependent enzyme [23].
Consequently, the following argument was made: if the long-chain acylcarnitine content of sperm increases in response to Ca2+, then Ca2+ acts as a second messenger to modulate phospholipase A2 activity. The addition of Ca2+ to immotile sperm increased their long-chain acylcarnitine content by 1.7-fold (P < 0.01) as they regained motility. The action of phospholipase A2 was confirmed with a specific inhibitor (Fig. 4). Therefore, it was concluded that sperm ascend the vagina and enter the SST by oxidizing endogenous fatty acids, predominantly palmitic and stearic acid (Table 1), released by phospholipase A2 activity. Maintenance of motility in the absence of an exogenous substrate (Fig. 2) most likely depends on a Ca2+ circuit that is, in part, dependent on extracellular Na+, because Ca2+ did not maintain motility unless Na+ was present. In this regard, it is noteworthy that Ca2+ efflux from the mitochondrial matrix is mediated by a sodium/calcium exchanger, whereas Ca2+ uptake is mediated by a uniporter [22].
The effect of sodium glutamate on motility was unexpected. At first, this reagent was used in an attempt to reversibly inhibit sperm motility. Fowl sperm are essentially immotile at body temperature before ejaculation [24]. Deferent duct fluid contains 80–90 mM glutamate, and Na+ is the predominant cation [25]. Thus, buffered 128 mM sodium glutamate was used for simplicity. However, glutamate acted as a motility agonist when sperm were coincubated with Ca2+ under aerobic conditions, as evidenced by the VSL distribution shown in Figure 6. This effect was so pronounced that the experimental sperm concentration had to be reduced by half from that used in previous work with the Hobson SpermTracker [9] to avoid warning messages for overload conditions. The possibility of glutamate acting on a receptor was tested using 1 mM NMDA, which increased average VSL by 18 µm/sec (P < 0.0001). Therefore, the effect of glutamate was attributed to NMDA-receptor channels, which are permeable to Ca2+ in addition to Na+ [26]. Thus, glutamate in deferent duct fluid may facilitate the initiation of motility at ejaculation, particularly if oxygen tension is low within the deferent duct. This conclusion is consonant with the following observations: Whereas deferent duct fluid contains 1 mM Ca2+ [25], its glucose content is negligible [27]. Additionally, ATP content is marginal when sperm are incubated without glucose in buffered 100 mM sodium glutamate, pH 7.4, under anaerobic conditions at body temperature [28].
The assumption that sperm become quiescent once they enter the SST [1, 5] is problematic, because it leaves key questions unanswered. For example, how do sperm emerge from the SST? How is motility initiated? The SST are blind-end tubules [3]; thus, fluid secreted from SST epithelial cells would generate a current. Sperm with ample velocity could move against the current and remain within the tubule. In contrast, motile sperm would emerge from the tubule once velocity decreased to a point at which position could not be maintained against the current. The motility of resident sperm could be maintained by free fatty acids released from triglyceride stored within the pronounced supranuclear lipid droplet within SST epithelial cells [3]. These postulates afford a reasonable and simple explanation for sperm residence within the SST over a period of days.
The contention that sperm egress and VSL are interrelated is supported by the following experimental evidence: the Hobson SpermTracker reports VSL as an average for all cells observed within an operator-defined interval as well as for each cell observed therein. Velocities of individual cells can be compiled into frequency distributions. The VSL distribution was skewed at body temperature (Fig. 5). Motile concentration was independent of time during a 30-min interval. The y-intercept was 1.01 x 109 sperm/ml, and sperm had been diluted to 1.2 x 109 sperm/ml. Therefore, sperm motility was stable at 84%. Average VSL (data not shown) was also independent of time (P > 0.05). The y-intercept for this variable was 42 µm/sec. Thus, the data set represented a sperm cell population in a steady state. The VSL distribution was comparable to one generated during an interval in which motile concentration decreased as a function of time (Fig. 6). However, the shape of the VSL distribution was subject to change, as evidenced by the independent effects of temperature (Fig. 5) and glutamate (Fig. 6). Thus, it was concluded that the pronounced, skewed distribution observed at body temperature was characteristic of motile sperm cell populations in vivo.
The association between the shape of the VSL distribution and the emergence of sperm from the SST stemmed from the following concepts. First, sperm move against resistance in the sperm mobility assay, and sperm mobility was directly proportional to the size of the subpopulation of sperm with a VSL of greater than 30 µm/sec [16]. Second, sperm emerge from the SST in a pattern that approximates an exponential decay [19, 20]. Consequently, any proposed mechanism of sperm egress must be consistent with this pattern. Third, if a motile sperm cell can hold its position in a current with fixed velocity, then the cell will experience retrograde movement should its velocity decrease. In this regard, it is noteworthy that sperm within SST do not adhere to epithelial cells and that they align with acrosomes pointed toward the blind end of the tubule [3]. Likewise, sperm manifest rheotaxis [18]. Fourth, the dense microvilli on the lumenal surface of SST epithelial cells constitute a pertinent feature, because they are comparable to those on the epithelial cells of the choroid plexus, a structure that exudes cerebrospinal fluid over its outer surface [29]. Fifth, when viewed head-on, a fowl sperm moves in a clockwise helix around its progression axis [30]. If the space through which a cell moves widens at low velocity, then the cell might experience greater drag if it were in a current. Thus, a cell's egress might be facilitated by its own digressive movement once VSL becomes less than 25 µm/sec (Fig. 7). This view is consistent with the observation that sperm from high-mobility males emerged from the SST at a slower rate than sperm from low-mobility males [15], because average VSL differed between these phenotypes [9]. In summary, the shape of the VSL distribution would predict that low-velocity sperm have the greatest potential for egress at any given moment if resident sperm move against a current. Thus, if the population of resident sperm declines exponentially over the course of days, then the size of the emergent motile subpopulation would decline similarly. This explanation accounts for the exponential decay in the number of sperm associated with oocytes following a single insemination [19, 20].
In conclusion, sperm storage in birds may be most readily explained in terms of sperm cell behavior. The proposed model is coherent and parsimonious. More importantly, this model can be tested. For example, if SST epithelial cells express genes for lipase and an array of channel proteins, then secretion of fluid containing free fatty acids would be certain in view of SST ultrastructure [17].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: David Froman, Department of Animal Sciences, 112 Withycombe Hall, Oregon State University, Corvallis, OR 97331-6702. FAX: 541 737 4174; david.froman{at}orst.edu
Received: 15 November 2002.
First decision: 26 December 2002.
Accepted: 4 March 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RV. Epithelial tissue. In: General Histology of the Mammal. Heidelberg: Springer-Verlag; 1984:15–97 This article has been cited by other articles:
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  M. R. Bakst and V. Akuffo Morphology of the turkey vagina with and without an egg mass in the uterus Poult. Sci., March 1, 2009; 88(3): 631 - 635. [Abstract] [Full Text] [PDF]
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 S. C. Das, N. Isobe, and Y. Yoshimura Changes in the expression of interleukin-1{beta} and lipopolysaccharide-induced TNF factor in the oviduct of laying hens in response to artificial insemination Reproduction, March 1, 2009; 137(3): 527 - 536. [Abstract] [Full Text] [PDF]
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 D.P. Froman, J.C. Wardell, and A.J. Feltmann Sperm Mobility: Deduction of a Model Explaining Phenotypic Variation in Roosters (Gallus domesticus) Biol Reprod, March 1, 2006; 74(3): 487 - 491. [Abstract] [Full Text] [PDF]
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 D. P. Froman Application of the Sperm Mobility Assay to Primary Broiler Breeder Stock J. Appl. Poult. Res., January 1, 2006; 15(2): 280 - 286. [Abstract] [Full Text] [PDF]
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D. P. Froman and J. D. Kirby Sperm Mobility: Phenotype in Roosters (Gallus domesticus) Determined by Mitochondrial Function Biol Reprod, March 1, 2005; 72(3): 562 - 567. [Abstract] [Full Text] [PDF]
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D.P. Froman and A.J. Feltmann Fowl (Gallus domesticus) Sperm Motility Depends Upon Mitochondrial Calcium Cycling Driven by Extracellular Sodium Biol Reprod, January 1, 2005; 72(1): 97 - 101. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
+ ße-
(x). After all sperm were immotile, supplemental Ca2+ induced motility equivalent to that of a preincubated control
