| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Research Article |
Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331
ABSTRACT
In previous work, variation in sperm mobility phenotype was attributed to the proportion of ejaculated fowl sperm containing dysfunctional mitochondria. In the present work, latent mitochondrial dysfunction was inferred from patterns of sperm egress from the oviduct's sperm-storage tubules. In addition, experiments were performed to help explain how mitochondrial function could be compromised in viable sperm cells. Confocal microscopy demonstrated that sperm Ca2+ content differed between low and high sperm-mobility phenotypes when sperm were stained with rhod-2 AM, a Ca2+-specific dye. Fluorescence was associated with the nuclear envelope, a variant of the endoplasmic reticulum, and greater fluorescence was observed in sperm from low sperm-mobility males. Fluorescence was reduced by 50% when motile sperm were rendered immotile by incubation with a Ca2+ chelator. Thus, a relationship was established between a dynamic intracellular Ca2+ pool and sperm motility. Sperm N-methy-D-aspartic acid (NMDA) receptors were inferred by the action of D-homocysteinesulfinic acid, a potent NMDA receptor agonist. Seminal plasma from low sperm mobility males was characterized by an elevated glutamate concentration. Thapsigargin, which inhibits the smooth endoplasmic reticulum Ca2+ pump and thereby promotes Ca2+ efflux, rendered sperm immotile. This effect was blocked by cyclosporin A, which prevents the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca2+ content. In summary, we propose that 1) glutamate enables Ca2+ uptake into sperm before ejaculation, 2) excessive Ca2+ uptake triggers formation of the PTP in a subpopulation of sperm, and 3) sperm mobility is decreased in proportion.
calcium, gamete biology, sperm, sperm maturation, sperm motility and transport
INTRODUCTION
Sperm mobility is a quantitative trait and a primary determinant of fertility in the domestic fowl [1, 2]. The term sperm mobility denotes the net movement of a sperm cell population against resistance at body temperature. The trait's discovery was based on the observation that sperm from different males penetrated an Accudenz solution at different rates even though the number of viable sperm in the overlying sperm suspension was held constant [3]. The number of mobile sperm is proportional to the absorbance of the Accudenz solution after a 5-min incubation. Thus, the sperm mobility assay provides a context for the separation of two sperm subpopulations. Mobile sperm segregate from immobile sperm by penetrating the Accudenz solution.
Previous work has shown that such segregation is the result of variation in straight-line velocity (VSL) among sperm within an ejaculate [4, 5]. Specifically, sperm mobility phenotype was a function of the size of the sperm subpopulation in a semen sample with a VSL > 30 µm/sec [5]. In other words, whereas all mobile sperm are motile, not all motile sperm are mobile. This distinction becomes evident when sperm populations are characterized by VSL distributions, for the size of the mobile subpopulation in any ejaculate is proportional to the area within the upper tail of the distribution [5–7]. Variation in VSL among sperm within an ejaculate was attributed to mitochondrial dysfunction in sperm within the immobile subpopulation [7].
The present work outlines a series of experiments based on the hypothesis of Froman and Kirby [7], who proposed that variation in sperm mobility phenotype was explicable in terms of excessive mitochondrial uptake of Ca2+ induced by seminal plasma glutamate before ejaculation. Males from chickens characterized by low or high sperm mobility were used as semen donors. Patterns of sperm egress from the oviduct's sperm storage tubules were compared to assess sperm cell function in vivo. Sperm cell Ca2+ content was estimated with a cell-permeant fluorescent Ca2+ indicator and confocal microscopy. Subsequent experiments addressed the interrelationship between seminal plasma properties, the Ca2+ store observed at the level of the nuclear envelope, mitochondrial integrity, and sperm motility. Based on our experimental outcomes, we propose a mechanism that explains variation in sperm-mobility phenotype.
MATERIALS AND METHODS
Experimental Animals
Experimental animal care was in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Birds were caged (30 x 46 x 63 cm) and maintained on a 14L:10D photoperiod. Sperm mobility phenotype was measured according to the method outlined by Froman et al. [2] using males from lines of chickens selected for either low or high sperm mobility. Unless specified, individual ejaculates were used to replicate observations within experiments.
Sperm Egress from Sperm-Storage Tubules
Rate of sperm egress from sperm storage tubules (SST) was estimated as follows. Each of 30 hens was artificially inseminated with 30 x 106 sperm from the pooled ejaculates of roosters characterized by high sperm mobility (n = 5). Egg collection began on the second day following insemination and continued for 15 consecutive days. Density of perforations through the inner perivitelline layer overlying the germinal disk was estimated as outlined by Howarth and Donoghue [8]. Hens were inseminated a second time after a 3-wk interval. In this case, each hen was inseminated with 90 x 106 sperm from the pooled ejaculates of roosters characterized by low sperm mobility (n = 5). Egg collection and data collection were performed as above. Data sets approximated exponential functions. Therefore, parameters of y(x) = 
+ ße–
(x) were estimated by iterative least squares [9].
The equivalence of estimates for the decay constant, , was tested using the principle of conditional error. In brief, a conditioned sum of squared residual errors (CSSE) was estimated by imposing the hypothetical condition 1 = 2 on the model. An extra sums of squares F-test was performed as follows: fr (n–p) = (SSH/r)/[SSE/(n – p)], where SSE was the sum of squared residual errors obtained unconditionally; SSH was the difference between CSSE and SSE; r was the number of independent parametric statements implied by the condition (e.g., r = 1 for the hypothesis 1 = 2), n was the number of observations, and p the number of parameters within the observational model.
Confocal Microscopy
Males characterized by either low or high sperm mobility were used as semen donors (n = 10 per phenotype). The experimental design was a randomized complete block design [10] in which a sample from each phenotype was viewed on a common slide. Unless specified, reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Sperm mobility phenotype was confirmed [2] before incubation, and sperm incubated in 5 µM rhod-2 AM (Invitrogen, Carlsbad, CA) as follows. A 50-µl volume of semen was diluted to 5 x 108 sperm per ml in 50 mM N-tris[hydroxy-methyl]methyl-2-amino-ethanesulfonic acid (TES), pH 7.4, containing 128 mM NaCl and 2 mM Ca2+ (TES-buffered saline). An 80-µl volume of sperm suspension was combined with 415 µl TES-buffered saline and 5 µl of 500 µM rhod-2 AM in dimethyl sulfoxide (DMSO) within a polystyrene cuvette. Each cuvette was covered with a 2-cm2 piece of Parafilm (VWR International, Inc., Seattle, WA), and cuvettes were incubated at 41°C for 30 min.
Microscopy was performed as follows. A 250-µl volume of each sperm suspension was diluted with an equal volume of 3% (w/v) NaCl to dilute and immobilize sperm cells. Sperm cells were visualized under oil with a Zeiss 510-META inverted confocal microscope (OSU Center for Genome Research and Biocomputing) using a 63x objective. Incident light with a wavelength of 543 nm was emitted from an HeNe laser. Image analysis was performed with Zeiss Image Browser software. Relative fluorescence units were measured from each of approximately 200 sperm cells per male. A random sample of 180 sperm cells per male was used in the ANOVA [10].
Data within a sample were normalized by transforming each observation into a percentage of the maximal value observed. Normalized data were pooled and frequencies compiled to evaluate staining profiles.
A second experiment was performed as follows. Ejaculates were pooled from roosters characterized by high sperm mobility (n = 5). Sperm were immobilized by incubation with 5 mM tetrasodium 1,2 -bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) as outlined by Froman [6]. Control sperm were suspended in TES-buffered saline. Control and treated sperm were stained with rhod-2 AM as outlined above. Confocal microscopy was performed as outlined above. Data (n = 188 sperm cells per treatment) were analyzed by single-classification ANOVA [11].
Computer-Assisted Sperm-Motion Analysis
Computer-assisted sperm-motion analysis was performed in the first experiment according to methods outlined previously [6]. The effect of the N-methyl-D-aspartate (NMDA) receptor agonist D-homocysteinesulfinic acid was tested using a randomized complete block design [10]. Ejaculates from males characterized by high sperm mobility (n = 10) served as blocks. Thus, sperm from each male were exposed to TES-buffered saline or 500 µM D-homocysteinesulfinic acid in TES-buffered saline. Likewise, the effect of cyclosporin A was tested on the motility of thapsigargin-treated sperm using methods outlined by Froman and Feltmann [4] and a randomized complete block design [10]. In this case, sperm from each male (n = 10) were exposed to 1% (v/v) DMSO, 20 µM thapsigargin in 1% (v/v) DMSO, or 40 µM cyclosporin A and 20 µM thapsigargin in 1% (v/v) DMSO.
Seminal Plasma Glutamate and Ca2+
Roosters characterized by low or high sperm mobility (n = 10 per phenotype) were ejaculated weekly over an interval of 10 wk. Each ejaculate was microcentrifuged for 7 min at 15600 x g. Seminal plasma supernatants were placed in screw-cap plastic vials and stored at –20°C. Samples were pooled by bird. Thereafter, each vial was thawed and seminal plasma filtered through a 0.2-µm Acrodisc filter (VWR). Each sample of filtered seminal plasma was subdivided for amino acid analysis (Molecular Analysis Facility, University of Iowa, Ames, IA) and electrolyte analysis (College of Veterinary Science's Diagnostic Laboratory, Oregon State University, Corvallis, OR). Each data set was analyzed using a single-classification ANOVA [11].
RESULTS
The pattern of sperm egress from the oviduct's sperm storage tubules differed between phenotypes, as evidenced by density of perforations within the perivitelline layer (Fig. 1). Whereas perforation density decreased exponentially in each case, decay constants differed between phenotypes (P < 0.01). Estimates of were 0.1953 and 0.0800 for low and high sperm-mobility phenotypes, respectively. Thus, ejaculates from males characterized by low sperm mobility contained a mixture of sperm that were immobile in vitro and sperm that were mobile in vivo, as evidenced by an ability to ascend the vagina and enter sperm-storage tubules. However, such sperm did not persist within the sperm-storage tubules to the extent that control sperm did, i.e., sperm from males characterized by high sperm mobility in vitro.
|
Sperm Ca2+ content differed between phenotypes (P < 0.0001), as evidenced by staining with the fluorescent Ca2+ indicator rhod-2 AM. A representative photomicrograph is shown in Figure 2. Fluorescence was observed primarily in association with the nucleus. The instrument range for relative fluorescence ranged from 0 to 255 U. Observed values ranged from 2 to 254 U. Means ± SEM were 166 ± 6.1 and 122 ± 8.9 U for roosters characterized by low and high sperm mobility, respectively. A significant block effect also was observed (P < 0.0001). Nonetheless, distinct patterns of staining intensity were evident from normalized frequency distributions (Fig. 3). Fluorescence decreased by 50% (P < 0.0001) when sperm were rendered immotile by incubation with BAPTA (Fig. 4).
|
|
|
D-homocysteinesulfinic acid enhanced sperm motility as evidenced by an increase (P < 0.001) in both motile concentration and VSL (Table 1). In contrast, a specific inhibitor of the smooth endoplasmic reticulum calcium (SERCA) pump, thapsigargin, inhibited sperm motility (Table 2). This effect was counteracted (Table 2) by cyclosporin A, which inhibits the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca2+ content. As shown in Table 3, differences in seminal plasma glutamate, Ca2+, and Mg2+ concentrations were observed between phenotypes (P < 0.05). Seminal plasma from roosters characterized by low sperm mobility contained 1.5-fold, 1.3-fold, and 2.5-fold more glutamate, Ca2+, and Mg2+, respectively, than seminal plasma from high sperm-mobility roosters.
|
|
|
DISCUSSION
Froman and Kirby [7] proposed that sperm mobility phenotype was determined by the extent to which glutamate induces excessive mitochondrial Ca2+ uptake in sperm before ejaculation. This hypothesis was based on the following facts. First, mitochondrial degeneration was observed in sperm from roosters characterized by low sperm mobility [7, 12]. Second, fowl sperm motility depends on extracellular Ca2+ cycling through the sperm cell [13]. Third, glutamate and NMDA increased sperm VSL [6]. Fourth, the NMDA receptor is one member of the glutamate receptor family that is permeable to extracellular Ca2+ [14]. Fifth, extracellular Ca2+ plays a critical role in the demise of neuronal mitochondria arising from glutamate-mediated toxicity [15, 16]. Sixth, fowl seminal plasma is enriched with glutamate [17, 18]. Seventh, sperm that were immobile in vitro had a VSL 
30 µm/sec in addition to swollen mitochondria [7]. And finally, mitochondrial disorganization manifested a graded effect.
Therefore, in the present work, we deemed immobile sperm as moribund and suspected that their mobile counterparts would show a latent defect in the case of males that ejaculate large numbers of immobile sperm. We tested this possibility by evaluating sperm egress from the oviduct's SST. Only motile sperm ascend the hen's vagina and enter these tubules, and they do over a time course of hours following an intravaginal insemination [19]. Furthermore, sperm egress is most readily explained by a decreased ability to move forward against a current within the tubular lumen as sperm cells age and velocity declines [6]. Consequently, we made the following argument: if sperm egress is more rapid in the case of low sperm-mobility males, then mitochondrial function may be compromised to a greater extent than what is evident in vitro. Sperm egress was estimated by plotting perforations of the oocyte's inner perivitelline layer as a function of time. These perforations arise from induction of the acrosome reaction following contact between sperm and N-linked glycans associated with the inner perivitelline layer [20]. As first illustrated by Wishart [21], perforation density approximates an exponential decay following a single intravaginal insemination.
Semen samples characterized by low sperm mobility manifest the following attributes when evaluated by computer-assisted sperm motion analysis [7]. The sample will contain sperm with a VSL >30 µm/sec. However, the proportion of these sperm is small because the majority of sperm are characterized by a low VSL whether motile concentration is low or high. In this regard, three distinctions are noteworthy. First, motile concentration denotes the number of motile sperm within a volume defined by sample chamber depth and the area of the analysis field. Second, motile concentration is independent of VSL. Third, our computer-assisted sperm-motion analysis technique utilizes an erythrocyte monolayer on the floor of the microcell [4]. Consequently, immotile sperm are obscured by the monolayer. Thus, motile concentration is similar to but distinct from the variable percent motile.
Hens were inseminated with three times as many total sperm when low sperm-mobility males were used as semen donors. This was done to maximize SST filling. This objective was realized as evidenced by the y-intercept of the predicted curve for the low sperm-mobility phenotype (Fig. 1). Nonetheless, these sperm emerged from the SST more rapidly than did sperm from high sperm-mobility roosters. We attributed this to a latent mitochondrial dysfunction. This experimental outcome warranted a mechanism that could account for graded loss of sperm cell function among sperm within a single ejaculate. We hypothesized that Ca2+ homeostasis was the key because fowl sperm are bathed in millimolar amounts of glutamate before ejaculation [18], fowl sperm appeared to have NMDA receptors [6], which are permeable to Ca2+ [14], and extracellular Ca2+ plays a critical role in neuronal glutamate-mediated toxicity, in which the mitochondrial PTP forms and mitochondrial function is lost [15, 16]. Therefore, we proposed the following argument: if sperm Ca2+ content differs between low and high sperm-mobility phenotypes, then Ca2+ homeostasis may contribute to phenotypic expression. We tested this hypothesis using confocal microscopy and rhod-2 AM, a fluorescent Ca2+ indicator (Fig. 2). A phenotypic difference was observed (Fig. 3), and localization of fluorescence was consonant with Ca2+ storage within the perinuclear space [22], a functional equivalent of the smooth endoplasmic reticulum's lumen [23]. Rendering sperm immotile with BAPTA (Fig. 4) reduced fluorescence by 50%, thereby demonstrating that this Ca2+ pool was dynamic.
These experimental outcomes warranted the investigation of the link between extracellular glutamate and intracellular Ca2+. Fowl sperm motility is maintained at body temperature in the absence of exogenous substrates only when extracellular Ca2+ cycles through mitochondria [6, 13]. Whereas glutamate is not required for maintenance of fowl sperm motility at body temperature, VSL was nonetheless increased by millimolar amounts of glutamate and NMDA in previous work [6]. These observations were consistent with the glutamate content of fowl seminal plasma [17, 18], the presence of a small molecular weight motility agonist in fowl seminal plasma that is distinct from Ca2+ [24], and the Ca2+ permeability of the NMDA channel [14]. In the present work, we used a potent, fast-acting agonist of the NMDA glutamate receptor to confirm the presence of NMDA receptors on fowl sperm. A micromolar dosage of D-homocysteinesulfinic acid increased both motile concentration and VSL (Table 1), presumably by enhancing Ca2+ uptake.
Neuronal glutamate excitotoxicity entails excessive Ca2+ loading, subsequent formation of the mitochondrial PTP, and mitochondrial demise [25]. Likewise, it is noteworthy that Ca2+ homeostasis is affected by the endoplasmic reticulum and associated proteins such as the SERCA pump [26–30]. This ATPase is inhibited by thapsigargin and depletion of Ca2+ stores results from enzyme inhibition [31]. Therefore, we used thapsigargin and cyclosporin A to demonstrate a cause-and-effect relationship between Ca2+ efflux from the perinuclear space and formation of the mitochondrial PTP. Motile concentration and VSL were predictably compromised when sperm were treated with 20 µM thapsigargin (Table 2). This effect was counteracted by cyclosporin A, which inhibits the formation of the PTP in response to excessive Ca2+ accumulation in the mitochondrial matrix [32]. Thus, we induced formation of the mitochondrial permeability transition pore by manipulating the Ca2+ pool found within the perinuclear space. In this regard, the effect of rendering sperm immotile by treatment with BAPTA is distinct from that of thapsigargin; for sperm rendered immotile by treatment with BAPTA can be reactivated by addition of excess Ca2+ [6], whereas the effect of thapsigargin is terminal apart from the presence of cyclosporin A.
In review, the hypothesis proposed by Froman and Kirby [7] linked seminal plasma glutamate with sperm cell dysfunction. As shown in Table 3, glutamate concentration was 1.5-fold greater in the seminal plasma of low sperm-mobility males when compared with that of high sperm-mobility males (P < 0.05). Consequently, secretion of glutamate into the effluent of the male reproductive tract appears to be enhanced in the low sperm-mobility phenotype. In contrast, elevated levels of Ca2+ and Mg2+ observed in the seminal plasma of these males (Table 3) may most readily be explained by release from moribund sperm cells.
In summary, we propose that 1) glutamate in deferent duct fluid enables Ca2+ uptake through sperm NMDA channels, 2) excessive Ca2+ uptake triggers formation of the PTP in a subpopulation of sperm, and 3) as a result, sperm mobility is decreased in proportion when sperm become motile at ejaculation. Even though seminal plasma glutamate concentration differed between sperm mobility phenotypes (Table 3), we propose that glutamate is a contributing factor rather than a causative agent. Sperm mobility is a quantitative trait [1] subject to genetic selection [7, 33]. As such, multiple genes are likely to affect phenotypic expression, which necessarily complicates the attempt to explain phenotypic variation. Nonetheless, a single critical control point may be formation of the mitochondrial PTP. Even though some aspects of the PTP remain controversial [25], the generally accepted trigger is excessive mitochondrial uptake of Ca2+ [32]. Therefore, we propose that formation of this pore is pivotal to expression of sperm mobility phenotype.
FOOTNOTES
2 Correspondence: David P. Froman, 112 Withycombe Hall, Department of Animal Sciences, Oregon State University, Corvallis, OR 97331. FAX: 541 737 4174; david.froman{at}oregonstate.edu 
1 Supported by National Research Initiative Competitive Grant 2003-35203-13343 from the USDA Cooperative State Research, Education, and Extension Service.
Received: 17 August 2005.
First decision: 13 September 2005.
Accepted: 11 November 2005.
REFERENCES
J, Kaplán P, Bubu
íková E, Strapková A, Murín R, Molecular pathways of endoplasmic reticulum dysfunctions: possible cause of cell death in the nervous system. Physiol Res 2003 52:269-274[Medline]This article has been cited by other articles:
|
|
 |
|
  J. Pelaez and J. A. Long Characterizing the Glycocalyx of Poultry Spermatozoa: II. In Vitro Storage of Turkey Semen and Mobility Phenotype Affects the Carbohydrate Component of Sperm Membrane Glycoconjugates J Androl, July 1, 2008; 29(4): 431 - 439. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
