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Gamete Biology |
Center for Research on Reproduction & Women's Health7
Division of Reproductive Endocrinology & Infertility,8 Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6142
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gamete biology, in vitro fertilization, sperm, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Controversy regarding the necessity for glucose to act through these pathways (or through other, as-yet-unidentified mechanisms) exists at several stages during murine sperm capacitation and fertilization. Early studies suggested that glucose is essential for murine sperm capacitation, including the acquisition of hyperactivated motility and ability to undergo acrosomal exocytosis [5–7]. In support of these findings, ATP from glycolysis has been shown to provide the energy for the majority of protein tyrosine phosphorylation events associated with capacitation [2]. However, glucose might also influence downstream signaling through the PPP, because exogenous NADPH supports tyrosine phosphorylation events [8]. Paradoxically, Urner et al. [8] have reported that over prolonged periods, sperm proteins will become phosphorylated on tyrosine residues in the absence of glucose, although this response is reduced. Those authors also found that despite the appearance of delayed tyrosine phosphorylation events, glucose is required for a number of stages of fertilization, including the binding and fusion of sperm to the egg plasma membrane and successful penetration and decondensation of the sperm head [8–11]. Contrary to those findings, Redkar and Olds-Clarke [12] have reported that although sperm-egg binding and fusion are slowed in its absence, glucose is not essential either for these events or for earlier stages of capacitation.
The controversy over the importance of glucose in fertilization extends to the human as well. Several studies strongly suggest the importance of glucose in achieving fertilization [13, 14], but these findings are in disagreement with those of an earlier report [15]. Williams and Ford [16] found that a glycolyzable sugar is necessary for hyperactivation of human sperm and maintenance of optimal ATP levels, but they also found only a slight enhancement by glucose of the rate of acrosomal exocytosis and penetration of zona pellucida (ZP)-free hamster oocytes. To those authors, such data suggested that hyperactivation is the only capacitation-related phenomenon that depends on glycolyzable substrates. One observation that might account for conflicting results regardless of model system is that glucose has been reported to exert a "priming" effect on murine sperm [6]. Priming, or exposure to glucose and subsequent removal of this substrate, was suggested to be sufficient for fertilization in the mouse [6]. Therefore, the handling of sperm during the preliminary stages of an experiment, including subtle distinctions such as the time of exposure to epididymal/seminal fluid, washing protocols, or length of incubations, might influence the experimental results. In addition, extremely low concentrations of glucose (10–100 µM) are sufficient to support the full pattern of protein tyrosine phosphorylation events that are associated with capacitation [2]. This finding raises the possibility that media containing sperm washed free of glucose might actually still contain enough of this substrate to support aspects of capacitation and fertilization.
One historical problem with studies in both the mouse and human has been the lack of technically convenient biochemical markers for different stages of capacitation. Therefore, studies regarding capacitation often used fertilization as an endpoint, failing to distinguish between the maturational process conferring fertilization competence and the multistage process of fertilization itself [5]. The lack of this distinction has continued in recent studies as well, particularly in work with the human, in which it is common clinical practice to capacitate sperm in the same drop that is used for in vitro fertilization (IVF) [13, 14].
The ability to distinguish specific stages of fertilization that require glucose is possible because cumulus cell-free eggs are unable to utilize glucose as an energy source [17, 18]. In support of this statement, IVF may still be performed when either glucose transport mediated by the GLUT family of facilitative glucose transporters [9] or active glucose transport mediated by Na+/glucose transporters [9, 19] is blocked in eggs. Therefore, one can assess the effects of different concentrations of glucose on sperm apart from the potential effects on eggs. In the present study, we relate the results from a series of experiments designed to separate the effects of glucose on sperm during capacitation from the effects of glucose on sperm during different events associated with IVF. By determining the steps at which glucose might be required for sperm to fertilize an egg, we hope to gain insight not only regarding the specialized metabolic pathways required for the fusion of these cells but also concerning potential metabolic requirements for cell-cell recognition and fusion events in other cell systems.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because different endpoints have been utilized in previous studies to assess capacitation and fertilization, it is important to introduce strict definitions when trying to elucidate potential roles for substrates such as glucose in these different processes. For the present investigation, capacitation will be strictly defined as the process of functional maturation that results in the acquisition of fertilization competence (including changes in the head leading to the ability to undergo acrosomal exocytosis and changes in the flagellum leading to a hyperactivated pattern of motility). Fertilization will be defined as interactions between male and female gametes, including sperm-ZP binding, and binding and fusion of the plasma membranes up to the development of a 2-cell embryo.
All experiments were conducted under the approval of the Institutional Animal Care and Use Committee of the University of Pennsylvania in accordance with the Guide for Care and Use of Laboratory Animals.
Reagents and Medium
All reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. A modified Whitten medium (ModW; 22 mM Hepes, 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid hemicalcium salt, pH 7.35) was used for sperm collection. This medium was designed in previous studies regarding the effect of glucose on sperm capacitation [2] to support sperm function yet not interfere with spectrophotometric assays. Modified Whitten medium containing 15 mM Hepes, 15 mg/ml of bovine serum albumin (AlbuMAX I; Gibco BRL, Grand Island, NY), 22 mM NaHCO3, 50 µg/ml of gentamicin (Gibco BRL), and 0.001% phenol red at pH 7.4 (ModW-IVF) was utilized for IVF assays.
Sperm Collection
Cauda epididymal sperm were collected from retired breeder CD-1 male mice (Charles River, Wilmington, MA), B6SJLF1/J male mice (Jackson Laboratories, Bar Harbor, ME), or transgenic mice expressing a proacrosin/enhanced green fluorescent protein (EGFP) construct (courtesy of Dr. George Gerton, University of Pennsylvania, Philadelphia, PA) [20, 21] by swim-out into ModW as described previously [2]. Both CD-1 and B6SJLF1/J strains of mice were tested in preliminary experiments to confirm that effects were not strain specific. The CD-1 mice were used for the majority of experiments to maintain consistency with previously published work concerning the effect of glucose on sperm capacitation [2].
Egg Collection and IVF Assays
For all IVF experiments, metaphase II-arrested eggs were obtained from superovulated CF-1 mice (Harlan Sprague Dawley, Wilmington, MA) according to standard methods [22]. Cumulus cells were removed from ZP-intact eggs by treatment with 0.1% hyaluronidase, and the eggs were washed three times in ModW-IVF containing no glucose. For ZP-free IVF, zonae were removed by brief incubation in acidic Tyrode medium (pH 1.6) and then allowed to recover in ModW-IVF for 60 min before insemination.
Fertilization was performed in 250-µl droplets under oil at 37°C in an environment of 5% O2, 5% CO2, and 90% N2. For ZP-intact IVF, the eggs were washed free of unbound sperm 3 h after insemination and then cultured in CZB medium [23]. The success of fertilization was determined 27 h after insemination by counting the number of 2-cell embryos. Unfertilized eggs (as determined based on morphological criteria) were assessed for sperm-egg binding and fusion by 4',6'-diamidino-2-phenylindole (DAPI)-staining as described previously [24]. For ZP-free IVF, the eggs were incubated with sperm for 90 min and then washed free of unbound sperm. The eggs were fixed, stained with DAPI, and evaluated for sperm-egg binding and fusion by epifluorescence microscopy [24]. The ZP-free eggs were considered to be fertilized if at least one decondensing sperm head was observed in the egg's cytoplasm and the egg chromatin exhibited an anaphase configuration.
Sperm Capacitation and Washing
To perform the experiments described below, it was necessary to wash sperm free from medium containing glucose. Because centrifugation and resuspension can cause sublethal membrane damage to sperm [25], a number of different wash protocols were tested in an attempt to minimize such damage. For each type of experiment, a wash protocol is described that involved the fewest manipulations while still resulting in appropriately low glucose concentrations such that when the sperm were added to the fertilization drops, the resulting final concentration of glucose was 5 µM or less.
Capacitation and IVF at the same glucose concentrations Sperm were collected by a swim-out method from the cauda epididymides of male mice (age, 3–8 mo) into ModW-IVF medium lacking BSA and glucose. For the first set of experiments, the sperm (150 µl) were dialyzed for 15 min against 300 ml of ModW-IVF lacking BSA and glucose using a Slide-A-Lyzer Mini Dialysis Unit (10 000 molecular weight cutoff; Pierce, Rockford, IL) to reduce any glucose from epididymal fluid. Sperm were counted and then capacitated in 250 µl of ModW-IVF containing varying concentrations of glucose for 2 h at a concentration of 4 x 105 sperm/ml. Fertilization was performed at a concentration of 2 x 105 sperm/ml with the same final concentration of glucose that had been used for capacitation.
Capacitation at 5.5 mM glucose and ZP-intact IVF at varying glucose concentrations During initial trials, the sperm were allowed to swim into ModW-IVF plus 5.5 mM glucose and then capacitated for 2 h after removal of the epididymal tissue. This protocol was designed to match typical treatment of sperm before IVF. Glucose was removed from the sperm by the following wash procedure: 150 µl of the sperm suspension were placed into a 5-ml styrene, round-bottomed tube, and 850 µl of ModW-IVF were added. These sperm were centrifuged for 2 min at 300 x g utilizing a swinging-bucket rotor at 37°C. The supernatant was removed, and the wash was repeated, with sperm being resuspended in a final volume of 500 µl. These sperm were counted and 5 x 104 cells were aliquoted into fertilization droplets containing varying concentrations of glucose. In the absence of added glucose, this wash procedure resulted in dilution of the glucose to 5 µM or less in the final fertilization drop.
Alternatively, 6 x 106 sperm were capacitated in a 900-µl droplet of ModW-IVF plus 5.5 mM glucose so that the sperm concentration during capacitation would match the concentration of 2 x 106 sperm per 300 µl used for previous spectrophotometric assays and immunoblots for protein tyrosine phosphorylation patterns [2]. After transfer to a 5-ml styrene, round-bottomed tube, ModW-IVF was added to the 900 µl to a volume of 1100 µl. The sperm were centrifuged as described above for 2 min at 300 x g, and 1000 µl of supernatant were removed, the sperm resuspended to 1100 µl, and the wash repeated. After this spin, the sperm were resuspended in a final volume of 500 µl. Again, this procedure resulted in dilution of glucose to 5 µM or less in the final fertilization drop.
Capacitation at 5.5 mM glucose and ZP-free IVF at varying glucose concentrations For these trials, 6 x 106 sperm were capacitated in a 900-µl droplet of ModW-IVF plus 5.5 mM glucose. The sperm were transferred to a 5-ml styrene, round-bottomed tube and washed twice as described above by centrifugation and resuspension, yielding a glucose concentration of 5 µM or less in the final fertilization drop.
Controls for Capacitation
Ability to support capacitation was verified for every batch of medium used throughout the present study by incubating 2 x 106 sperm in both 300 µl of ModW (noncapacitating) and ModW supplemented with 3 mM 2-hydroxy-propyl cyclodextrin and 10 mM NaHCO3 (capacitating). The pattern of protein tyrosine phosphorylation events, which is correlated with capacitation status [26, 27], was assessed as described previously [2]. Briefly, the sperm were homogenized and the proteins were solubilized by boiling in sample buffer [28] and then separated by SDS-PAGE under reducing conditions. Immunoblotting and detection by chemiluminescence were performed as described previously [29]. These data are not shown. In each experiment, sperm motility was assessed first at the time of swim-out and then when appropriate at the end of the capacitation incubation (for the appearance of hyperactivated flagellar motion). Finally, motility was assessed again after washing before insemination of the fertilization droplets. No IVF was performed if overall sperm motility after washing was less than 40%.
Assessment of Acrosomal Status
Sperm that contain soluble EGFP in the acrosome were obtained from transgenic mice expressing a proacrosin-EGFP fusion protein under control of the proacrosin promoter [20, 21]. The sperm were capacitated for 2 h in ModW-IVF plus 5.5 mM glucose (6 x 106 sperm/900 µl) and then washed free of glucose as outlined above for ZP-free IVF. The sperm were counted, and 2.5 x 104 cells were aliquoted into 250-µl droplets containing the same concentrations of glucose as those used for the ZP-free IVF experiments. After 45 min, the sperm were collected, fixed in 2% paraformaldehyde, and mounted on a cover slip. The percentage of sperm that had undergone acrosomal exocytosis was assessed by epifluorescence microscopy. At least 80 sperm were counted in each of two independent experiments for all groups (except for the 0 mM glucose group in one experiment, in which 30 sperm were counted). As a positive control, 10 µM progesterone was added to sperm incubated in 5.5 mM glucose at the end of the 45-min capacitation period to induce a maximal acrosomal exocytosis response. After 15 min of progesterone treatment, these sperm were fixed and assessed as described above.
Statistical Analyses
Uncorrected 95% confidence intervals were computed to allow comparisons between groups. Kruskal-Wallis rank sum tests were used to evaluate the nonparametric data regarding binding and fusion events during ZP-free IVF. Post-hoc tests between individual groups were performed as indicated in the figure legends. All statistical analyses were performed with JMP (version 5) statistical software (SAS Institute, Cary, NC). Graphs were drawn with Delta Graph 4.0 (DeltaPoint, Inc., Monterey, CA) and with JMP.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To evaluate better the effect of glucose concentration on fertilization separate from its effects on capacitation, sperm were first capacitated with 5.5 mM glucose, a concentration that is found in many media utilized for murine sperm capacitation. After incubation under capacitating conditions, the sperm were washed by centrifugation and resuspension such that the amount of glucose added to the fertilization drops by media accompanying the sperm resulted in a final concentration of 5 µM or less. The fertilization drops themselves had differing concentrations of glucose, again ranging from 0 to 5.5 mM. Despite previous exposure to glucose, the spermatozoa required final glucose concentrations of 1 mM or greater in the fertilization drops to result in fertilization rates of 50% or more (Fig. 2).
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Although these experiments were performed according to established methods for the handling of gametes during IVF, it was possible that performing capacitation at a different concentration of sperm relative to our previous work on capacitation [2] might affect the results. To control for possible confounding effects because of differences in the total amount of glucose available per sperm, the IVF assays were repeated after capacitating the sperm at the same concentration as used in previous experiments (i.e., 2 x 106 cells/300 µl). No differences in fertilization were seen using this protocol (data not shown).
Having determined that glucose was required for fertilization apart from its presence during capacitation, we next sought to identify those processes or stages during fertilization that required glucose. We performed IVF using ZP-free eggs with sperm that previously had been capacitated with 5.5 mM glucose and then washed "free" of glucose as described above. The concentration of sperm used to inseminate the eggs was adjusted downward from the previous experiments with ZP-intact eggs to reduce the incidence of polyspermic fertilization (from 2 x 105 sperm/ml for ZP-intact eggs to 1 x 105 sperm/ml for ZP-free eggs). The ZP-free eggs were considered to be fertilized if at least one decondensing sperm head was observed in the egg cytoplasm and the egg chromatin exhibited an anaphase configuration. Concentrations of glucose greater than 1 mM were required to obtain fertilization rates greater than 50% (Fig. 3). The fertilization rate at 5.5 mM glucose with the washed sperm was essentially the same as that with sperm capacitated at 5.5 mM glucose and then used for fertilization without washing. Data from this unwashed control group demonstrated that any sublethal damage caused by the washing protocol did not cause a significant decrease in overall fertilization rates at this concentration.
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These experiments using IVF performed on ZP-free eggs also provided information regarding the effect of glucose concentration on sperm-egg binding and fusion. A significant difference in sperm binding was seen when concentrations of glucose less than 1 mM were compared to concentrations of 1 mM or greater (Fig. 4). Interestingly, minimal differences were noted between any two concentrations below or above the apparently critical concentration of 1 mM (data not shown). In contrast to the binding data, the concentration of glucose had a more graduated effect on sperm-egg fusion for concentrations of 1 mM or greater (Fig. 5). However, all concentrations less than 1 mM had similarly low mean numbers of sperm fusing with individual eggs, again suggesting a threshold effect. Although the washing protocol did not significantly affect fertilization rates or sperm-egg binding, we did find an effect on the number of sperm that fused with ZP-free eggs (Fig. 5A). These data therefore suggest that the sublethal damage accompanying washing protocols can be reflected in subtle ways.
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One interpretation of the data presented thus far might be that the different concentrations of glucose in the fertilization droplets supported different rates of sperm binding and fusion because those glucose concentrations differentially supported spontaneous acrosomal exocytosis. To test this possibility, acrosomal exocytosis was quantified using transgenic mice expressing soluble EGFP in the acrosome (generous gift of Dr. George Gerton). Sperm from these mice were capacitated in 5.5 mM glucose, washed, and incubated in droplets containing differing concentrations of glucose. Care was taken to replicate precisely all steps of the protocol for IVF with ZP-free eggs. Aliquots of the sperm were collected at four time points: 1) the beginning of the initial capacitation period, 2) the end of the 2-h capacitation period, 3) the end of the wash protocol to remove glucose, and 4) the midpoint of what would have been the incubation period given for fertilization. The status of the acrosomes of sperm collected from the different groups was assessed by epifluorescence microscopy. In two independent experiments, the incidence of spontaneous acrosomal exocytosis was the same throughout all groups after capacitation (
25% and 35% for the two experiments) regardless of the glucose concentration in the final droplet and whether the sperm were washed by centrifugation (data not shown). In fact, all spontaneous acrosomal exocytosis appeared to take place during the initial 2-h capacitation in 5.5 mM glucose. As a positive control for the method of assessing the acrosome, sperm were stimulated with progesterone after incubation in the final droplet, yielding greater than 75% acrosomal exocytosis in both experiments (data not shown). These data indicate that the observed differences in fertilization rates of ZP-free eggs in different concentrations of glucose could not be explained by differences in the rates of spontaneous acrosomal exocytosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Having shown that the concentrations of glucose required for fertilization were not equivalent to the concentrations required for this common marker of capacitation, we next tried to differentiate whether the higher concentrations of glucose were required for additional, as-yet-undefined steps in capacitation or whether they were required for fertilization itself. To achieve fertilization at 50% of the maximal rate or greater, glucose concentrations of 1 mM or higher were required in the fertilization drop, despite the fact that the sperm were already capacitated in the presence of 5.5 mM glucose. This finding contradicted the reported "priming effect" of glucose on fertilization, in which previous exposure to glucose was sufficient to allow fertilization in the absence of glucose [6]. Technical differences between the present study and that of Fraser and Quinn [6], such as the length of time required for the washes, the concentration of glucose left in the fertilization drop, the strain of mice, or some other factor, most likely account for the disparity in findings regarding such a priming effect on fertilization.
In addition to potential priming effects during capacitation, the necessity of glucose for other aspects of mouse IVF has been a source of some controversy. Williams and Ford [16] suggested that in the human, sperm hyperactivation is the only significant aspect of sperm function relevant to fertilization that relies on glucose. Hyperactivation has been suggested to be necessary in vivo for a variety of events, including release from interactions with oviductal epithelial cells, penetration of the expanded cumulus cell mass surrounding the egg, and mechanical advantage for penetration of the ZP. If hyperactivation were only required for the last of these purposes and the only aspect of capacitation/fertilization to rely on glucose, then capacitating the sperm in 5.5 mM glucose and performing ZP-free IVF should produce good fertilization rates regardless of the presence or absence of glucose. However, the results of the present study clearly showed that glucose was necessary for successful fertilization even when performing ZP-free IVF after capacitating sperm in the presence of 5.5 mM glucose (Fig. 3).
Using ZP-free eggs, Urner and Sakkas [9, 11] found that glucose or NADPH, an end product of the PPP, is required to support fertilization. On the other hand, Redkar and Olds-Clarke [12] found that sperm-egg plasma membrane binding still occurs in the absence of glucose, albeit at a slower rate. Our data suggested that binding of sperm to the plasma membrane of a ZP-free egg can take place in the absence of glucose but is improved by concentrations of 1 mM or greater (Fig. 4). Similarly, some sperm-egg fusion can take place in the absence of glucose, but it also is greatly improved by the presence of this substrate (Fig. 5). The observation that fusion varied more than binding with glucose concentrations greater than 1 mM suggested that the observed differences in fertilization (Fig. 3) were not just the result of different numbers of sperm interacting with the eggs. Similarly, none of these differences could be ascribed to different rates of spontaneous acrosomal exocytosis occurring in different concentrations of glucose. Together, these data support previous work demonstrating a requirement for glucose in protein tyrosine phosphorylation and hyperactivation and also provide new evidence that glucose greatly facilitates additional aspects of fertilization.
Lastly, one interesting observation can be gleaned from reading the Materials and Methods sections of the reports on previous investigations of this issue. Namely, those studies that supported a strong role for glucose in fertilization were performed with concentrations of sperm similar to those used in the present investigation (0.05–0.1 x 106 sperm/ml for ZP-free IVF [8, 10, 11]), whereas those studies that did not support a critical role for glucose in fertilization tended to use much higher concentrations of sperm (107 sperm/ml [12] and 5–10 x 106 sperm/ml [16]). This observation suggests that under conditions supporting monospermic fertilization, the presence of glucose assumes a greater impact on the success of fertilization. In experimental situations supporting polyspermic fertilization, several conditions could exist that might lessen the concentration of glucose otherwise required. For example, it is unclear whether some metabolite of a pathway utilizing glucose is generated or carried by sperm heads that increases to a threshold amount on cumulative interactions between sperm heads and the egg. Alternatively, multiple interactions between sperm and the egg plasma membrane might make it easier for subsequent sperm to bind and/or fuse, or a small subpopulation of sperm might exist that do not require glucose for interacting with an egg. Finally, it is also possible that the simple increase in number of sperm-egg interactions might just increase the odds that an otherwise energetically unfavorable event would take place.
We have found that sperm require glucose for IVF at concentrations greater than those that support protein tyrosine phosphorylation events correlated with capacitation. The result that sperm-egg fusion in ZP-free IVF depended on glucose to a greater extent than did sperm-egg binding supports a role for glucose during later events of fertilization, such as membrane fusion and sperm head decondensation, as has been suggested previously [9–11]. Our finding, that in the presence of low concentrations of glucose some low degree of both binding and fusion remains, supports the notion that these events do not strictly require glucose but are significantly more likely to take place in the presence of this substrate. Under physiological conditions, our data support the hypothesis that glucose would be required for fertilization in the mouse.
How glucose supports these events during fertilization at the biochemical level is not clear, but several possibilities are suggested by what is known about the compartmentalization of metabolic proteins and pathways in murine spermatozoa. For example, glycolysis is restricted to the principal piece of the flagellum, and oxidative respiration is restricted to the midpiece. The localization of a germ cell-specific isoform of type 1 hexokinase in the midpiece and associated with the membranes of the sperm head suggests that the PPP is active in those regions [29]. Indeed, activity for glucose-6-phosphate dehydrogenase, the first enzyme specific to the PPP, has been quantified in murine sperm [2], and the PPP has been implicated in supporting protein tyrosine phosphorylation events in the sperm head as well as various events of fertilization [8–11]. One potential function of PPP-derived reducing power in sperm would be to protect the membranes from lipoperoxidative damage [31], and this specific function has been demonstrated in the head of bovine sperm [32].
Conversely, the activity of reactive oxygen species also has been suggested to promote protein tyrosine phosphorylation and sperm capacitation [33, 34]. Such conflicting uses for the same metabolic end product, NADPH, suggest that the role of the PPP might change during a sperm's transition from the epididymis through various regions of the female reproductive tract, or that the NADPH produced might be used for different purposes in different subcellular compartments of the sperm. However, to our knowledge, changes in PPP activity because of capacitation, or during specific events in fertilization, have not been quantified in the various regions of the sperm. A true understanding of the roles of glucose in complex cellular phenomena such as capacitation and fertilization will depend on techniques that can quantify pathway activities in separate subcellular regions and compartments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Carmen J. Williams, Center for Research on Reproduction & Women's Health, 1313 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6142. FAX: 215 573 7627; cjwill{at}mail.med.upenn.edu
3 Current address: The James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY
4 Current address: GATA Haydarpasa Egitim Hastanesi, Kadin Hastaliklari Ve Dogum Klinigi, 81327 Kadikoy-Istanbul, Turkey
5 Current address: Program in Developmental Biology, Baylor College of Medicine, Houston, TX
6 Current address: Women's Health Research Institute, Wyeth Research, Collegeville, PA
Received: 21 November 2003.
First decision: 8 December 2003.
Accepted: 24 February 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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