| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Institute of Reproductive Medicine of the University D-48129 Münster, Germany
ABSTRACT
Male homozygous transgenic c-ros knockout mice are sterile by natural mating, lack a part of their epididymis, and the epididymal sperm exhibit tail angulation in vivo and in vitro. To ascertain if this abnormal tail form caused the infertility, the number and nature of sperm in the tract of females mated to knockout and wild-type mice were determined. Percentage motility and numbers of sperm in the uterus 1 h after mating were similar between genotypes. The majority of the uterine sperm from the wild-type males had straight flagella, whereas 46–86% of knockout sperm were bent at the cytoplasmic droplet even when motile. Motile knockout sperm showed a 54 and 37% reduction in the straightline and curvilinear velocities compared with straight wild-type sperm. Sequential flushings of the oviduct 4 h after mating with the wild-type males contained sperm: 591 ± 119 free, 371 ± 70 loosely, and 122 ± 47 tightly bound to the epithelium, but no knockout sperm were recovered from the oviduct or observed within the uterotubal junction in tissue sections. The infertility of c-ros knockout male mice can be explained by the sperm's inability to enter the oviduct, as a result of their bent tails forming the entangled sperm mass and their compromised flagellar vigor within the uterus.
epididymis, fertilization, oviduct, sperm maturation, sperm motility and transport, uterus
INTRODUCTION
The c-ros proto-oncogene encodes an orphan receptor with an intracellular tyrosine kinase domain that is expressed in embryonic epithelial structures of the kidney, lung, intestine, and the Wolffian duct [1, 2]. The expression of c-ros in these tissues thereafter declines but is upregulated in the proximal epididymis during prepubertal development, coincident with the epithelial differentiation of this region into the initial segment of the epididymis, such that this is the only organ synthesizing the protein in the adult [3]. Homozygous c-ros knockout mice develop normally into healthy adults, but the males are sterile and lack the initial segment despite normal testicular sperm output [3]. This transgenic mouse is thus a unique model for studying epididymal regulation of sperm function, because the epididymal abnormality seems solely responsible for the sterility [4]. The infertility of males by mating contrasts with the effectiveness of cauda epididymidal sperm in fertilizing eggs in vitro [3], and the most obvious explanation for these results would be that the motility of sperm from the knockout animals is impaired so that they fail to reach the oocytes in vivo. However, sperm released from the cauda epididymidis and diluted with culture medium were no less motile in percentages than those from the wild-type (WT) males in vitro, whereas swimming velocities and lateral head displacements were only about 20% lower than those of WT sperm [5]. Because sperm from the epididymis have not interacted with the female tract, it is necessary to examine sperm in various parts of the female tract to ascertain if motility or other factors contribute to the infertility.
A high proportion of sperm released from the cauda epididymidis of null mutant mice display severe tail angulation at the midpiece/principal piece junction associated with swollen cytoplasmic droplets [5]. This defect is thought to reflect an abnormality in the development of volume regulation ability during their epididymal sojourn [5]. Examination of female mice mated to knockout males in the earlier report [3] found no sperm in 40% of the oviducts and highly variable sperm numbers in the others. Because the majority but not all mature epididymal sperm from the knockout mice exhibit tail angulation, these findings suggest the possibility of some motile sperm with straight tails migrating into the oviduct, so the complete infertility of the knockout males remains unexplained. One crucial aspect of natural fertilization is the binding of sperm to the oviductal epithelium for survival and prevention of precocious capacitation [6–11]. This involves specific carbohydrate residues on the surface of sperm [12] that may be acquired during post-testicular modification in the epididymis [13] by the activities of epididymal glycosyltransferases and glycosidases responsible for modification of sperm proteins [14]. Because the initial epididymal segment is active in the synthesis of specific epididymal secretions, and this segment fails to differentiate in the sterile c-ros knockout males, it is possible that sperm from these males are not normally endowed and consequently incapable of normal interaction with the oviductal epithelium and fail to reach the oocytes in a potentially fertilizing state.
The present work was designed to clarify the cause of male infertility by evaluating the number, location, and nature of sperm within the female tract after mating to c-ros knockout mice and wild-type males as controls. Defects in the interaction of sperm with the oviduct were studied by the multiple flushing technique [6, 7] aimed at separating free sperm in the oviductal lumen from sperm attached to the epithelium.
MATERIALS AND METHODS
Animals
A colony of c-ros mice, generated as described in Sonnenberg-Riethmacher et al. [3] as offspring of cross-breeding between C57BL6 and Ola129 strains, was established from two breeding heterozygous pairs generously donated by Dr. E. Riethmacher and Prof. C. Birchmeier of the Max-Delbrück Centre of Molecular Medicine, Berlin, Germany. Genomic DNA was extracted from the tip of the tails of 3–4-wk-old mice. Genotyping was performed by polymerase chain reaction using primer pairs that were specific for sequences in the kinase domain of the c-ros gene (primers 1 and 2), for the detection of the wild-type allele, and sequences in the inserted neo cassette (primers 3 and 4), for the detection of the knockout allele: primer 1, 5'-GGCTGCGTCTACTTGGAGC-3'; primer 2, 5'-CCATACATCAGAC-TGAGAAGTA-3'; primer 3, 5'-CCGAGAGTTAAAATCTCCCACA-3'; primer 4, 5'-TCGCCAATGACAAGACGCTGG-3').
All animals were kept in 12L:12D cycles. In this study 10- to 15-wk-old adult male mice including 20 homozygous knockouts and 24 wild type (15 littermates of the transgenic mice and 9 C57BL6 supplied by Charles River, Sulzfeld, Germany) were used for one or two of the study designs described below. All males were caged individually and given adult females for 5–10 days for mating practice that were removed 2–10 days before the study. None of the female mice caged with the knockout males became pregnant.
Estrus and ovulation were induced in 30- to 45-day-old females (C57BL6, Charles River) by the injection of 10 IU FSH (Humegon; Organon GmbH, Oberschleissheim, Germany) at 1200 h followed 47 h later by 10 IU hCG (Choragon; Ferring Arzneimittel GmbH, Kiel, Germany) giving an estimated ovulation time between 2100 and 2400 h on the same day according to Hogan et al. [15]. Each female mouse was placed into the cage containing a wild-type or knockout male before the lights were turned off (1900 h), which is considered the earliest time for copulation to occur. The females were examined under red light for the presence of vaginal plugs with a sealed glass pipette tip as a vaginal probe, as an indication of successful mating.
Recovery of Sperm from Uterus and Oviduct
Study I. Assessment of sperm motility and tail morphology and osmotic pressure of uterine contents 1 h postcoitum (10 wild-type and 6 knockout males) The first examination for the presence of vaginal plug was at 2000 h (1 h after darkness commenced) and at 60- to 80-min intervals thereafter. Positive females were killed by cervical dislocation and the female tracts were exposed. A positive displacement pipette was inserted into the uterine horn through a side incision close to the bifurcation and the contents were withdrawn. A 3-µl aliquot of the neat uterine contents was taken for osmotic pressure measurement (see below). The remaining uterine contents were examined on a siliconized slide for the tail form of motile and immotile sperm (categorized as straight, bent at the mid-/principal piece, or in a hairpin form) immediately after dilution with medium H340, which is a bicarbonate-buffered physiological solution supplemented with glucose, lactate, pyruvate (medium H in Yeung et al. [16]), and 12 mg BSA/ml. The osmolality of this medium was increased by NaCl from 310 to 340 mmol/kg to mimic uterine content fluid osmolality measured in preliminary studies.
Study II. Measurement of kinematics of the dispersed sperm and counting of the dispersable and total sperm recovered from the uterus 1 h postcoitum (9 wild-type and 10 knockout males) Mated female mice were treated and killed as in study I. After ligation of the uterine cornua to retain the spermatozoa, the bilateral reproductive tract was isolated and dissected clean. Each horn in turn was tied off at the bifurcation, held above a tube, and cut at the cervical end and its contents drained and washed through by injecting 150 µl medium H330 (medium H containing 12 mg BSA/ml with osmolality adjusted to 330 mmol/kg similar to the overall average of uterine fluids; see Results) into the lumen at the oviductal end. Any sperm clumps trapped in the cornua were added to the uterine flushings. The tube was inverted three times to mix the contents, and clumps were allowed to settle by standing for up to 8 min. A 10-µl aliquot from the supernatant was examined on a siliconized slide for sperm tail morphology and motility and another aliquot added to 50 µl fixative (4% w/v formalin or 2.5% v/v glutaraldehyde) for the sperm count. Another aliquot was further diluted, loaded in a 40-µm deep chamber on a 37°C microscope stage (Olympus BH-2; Olympus Optical Co. GmbH, Hamburg, Germany), and recorded on videotape for analysis of motile sperm using a computerized system (HTM-C; Hamilton-Thorne, Beverley, MA) as described in Soler et al. [17]. For each sample, about 150 sperm tracks from several microscopic fields were measured for the straight-line (VSL) and curvilinear velocities (VCL), linearity (100 x VSL/VCL), amplitude of the lateral displacement of the sperm head (ALH), and the beat cross frequency (BCF). The volume of the uterine flushings was obtained by weighing, and the whole tube contents were digested by addition of 150 µl trypsin (Sigma type II, 40 mg/ml saline; Sigma, Deisenhofen, Germany) overnight at room temperature. The fixed dispersed sperm and sperm heads in the digested sample were counted in an improved Neubauer hemocytometer (Fischer Scientific, Schwerte, Germany), and the total number of sperm recovered from the uterus was calculated. Mean coefficient of variation in sperm counts, calculated from duplicates, was 4.5%.
Study III. Evaluation of oviductal sperm and uterine contents 4 h postcoitum (10 wild-type and 9 knockout males) Female mice were examined for the presence of a vaginal plug at 2300 h and, when positive, were killed at a mean time of 2350 h ± 14 min in the wild-type group and 2356 h ± 15 min in the knockout group. The two sides of the female tract were separately removed after ligating the base of each uterine horn at the bifurcation. One side was transferred to medium H310 (osmolality 310 mmol/kg [16]) containing 12 mg/ml BSA and kept at 37°C in 5% CO2, while the other side was processed as follows. After cleaning, the oviduct was cut where it emerged from the uterus and transferred with the attached ovary to a clean dish of medium. During dissection, extreme caution was taken to avoid damage to the uterus and any contamination of the tissues with sperm from the cut end of the uterus. The uterine horn was held at the uterotubal end, blotted, and cut at the cervical end to drain the contents into a tube. A 3-µl aliquot of the undiluted uterine contents was used to measure osmotic pressure (see below), and the remainder was fixed in 2.5% glutaraldehyde overnight for examination of sperm tail morphology after a 0.5-sec sonication to disperse the sperm. The oviduct was transferred to a siliconized glass slide and a side incision was made at the fimbrial end of the distended ampulla to pull out the cumulus mass for counting the number of oocytes and detecting the presence of sperm. Sperm in the oviduct were obtained by the three-step flushing technique developed by Smith and Yanagimachi [6, 7] for hamsters with volumes modified for mice. Twenty microliters of medium was flushed into a drawnout PVC catheter (Dural Plastics, Silverwater, N.S.W. Australia) tied into the distended ampulla with a 10/0 suture. The fluid flushed out from the uterine end of the isthmus onto a siliconized slide was covered with a 22- x 22-mm coverslip and the total area scanned to assess the total number of spermatozoa and their motility. After 4 min a second flush was made in the same way to recover loosely bound sperm that had detached from the oviductal epithelium in that period. The oviduct was then transferred to a tube and 40 µl medium H containing Triton X-100 (0.2%, v/v) was flushed through to fill the lumen, causing detachment of the remaining tightly bound sperm. After a minute interval, another 40 µl was flushed through to recover the sperm. The tube was centrifuged at 1000 x g for 5 min, and the pellet was resuspended in about 10 µl of supernatant after removal of the bulk. The number of sperm in the whole pellet was counted by microscopic examination.
Examination of the Uterotubal Junction
The uterine horns with the attached oviduct and ovary from one side of the females mated to five wild-type and three knockout mice were fixed in Bouin's fluid for 1 h (n = 6) or 4 h (n = 2) postcoitum. The end of the uterus together with the oviduct was cut out and processed for embedding in paraffin from which serial sections were cut at 7 µm until the colliculus tubarius and intramural uterotubal junction were found. Sections were stained with PAS and examined in a light microscope for the presence and location of sperm in the oviductal lumen.
Measurement of Osmotic Pressure of Uterine Contents
A 3-µl aliquot of the neat uterine contents collected at 1 and 4 h postcoitum as described in studies I and III above was loaded into a small measuring chamber of a vapor pressure osmometer (Wescor Vapro 5520; Schlag GmbH, Bergisch Gladbach, Germany [5]), and replicate measurements of osmolality were made after 10 and 15 min equilibration. If necessary, readings were continued at 5-min intervals to achieve agreement of <2 mmol/kg between readings, to ensure saturation of the measurement chamber. A standard of 290 mmol/kg was measured on the same day under the same conditions to provide a correction factor.
Statistics
Differences between the knockout and wild-type mice were analyzed by one-way analysis of variance and the Tukey test and considered statistically significant at P < 0.05.
RESULTS
Sperm Motility and Tail Morphology in Uterine Fluid 1 h Postcoitum
Sperm in neat uterine contents recovered from females mated to wild-type mice, observed immediately upon dilution (study I), showed about 40% motility and displayed mainly the normal straight forms of the flagellum whether motile or not (Figs. 1a and 2a). Sperm from knockout males were equally motile but clearly distinct because the large majority of these uterine sperm displayed angulation of the tail at the mid-/principal piece junction, bending into hairpin forms in the extreme, often forming entangled aggregates of sperm revealed in the fixed uterine contents (Figs. 1a and 2b). The hairpin configuration was more predominant (80%) in the motile than the immotile sperm (Fig. 1a).
|
When uterine contents were flushed out using medium H330 and allowed to disperse before examination (study II), a significantly greater percentage of sperm from the wild-type males were straight compared to the knockouts (P < 0.05, Fig. 1b), and twice as many motile sperm from the knockout males were in hairpin form, although they showed no difference in the percent motility (Fig. 1b). The extent of these differences was less than those found in study I where examination was made immediately upon dilution of uterine contents.
Kinematics of Uterine Sperm 1 h Postcoitum
Computer-assisted analysis of the dispersed motile uterine sperm after dilution revealed in the knockout males a significant reduction of VSL to 46%, and VCL to 63%, of the values in the wild-type group (Fig. 3). The ALH was also decreased but there was no significant difference in their beat cross frequency and linearity of the swim path.
|
Tail Morphology of Uterine Sperm 4 h Postcoitum
The predominance of flagellar angulation in uterine sperm derived from the knockout males observed 1 h postcoitum persisted at this later time point as revealed in the fixed uterine contents (study III), such that only 16% of spermatozoa displayed straight tails in contrast to 70% in the wild type (Table 1). Sperm recovered from the uterus after mating with knockout mice were still motile at this time (27.6 ± 10.3%; n = 5).
|
Osmotic Pressure of Uterine Contents
Tail bending of the uterine sperm could be a consequence of abnormal osmolarity of the uterine fluid. However, there was no significant difference between the osmotic pressure of the uterine contents collected from females mated to knockout or wild-type males, either 1 h (349 ± 27 and 327 ± 8 mmol/kg, respectively) or 4 h (332 ± 4 and 331 ± 6 mmol/kg) postcoitum.
Numbers of Spermatozoa Recovered from the Uterus and Oviduct
Almost 10 million sperm were recovered from the uterus of each female mouse 1 h after mating to wild-type or knockout males, and there was no difference in the number of sperm dispersed from the uterine masses when diluted in medium in vitro (Table 1).
At 4 h postcoitum, oocytes were found in 85% and 94% of oviducts from females mated to the wild-type and the knockout group, respectively. The number of oocytes recovered in the group mated to wild-type males was significantly higher (7.2 ± 1.2 per oviduct) than those mated to the knockout group (4.9 ± 0.6 per oviduct). The presence of sperm around the eggs in the distended ampulla was rare and only occurred in females mated to wild-type males (4 of 10 females).
The total number of spermatozoa from wild-type males recovered by flushing of the oviducts of the mated females 4 h postcoitum was around 1000 per oviduct (Table 1). There were more sperm recovered in the first flush (~600) than the second (~400), but there was no significant difference in percentage of motile cells in these two fractions (36 ± 6 and 37 ± 4%, respectively). Over 90% of both motile and immotile sperm had straight flagella. Additional sperm (as much as 30% of the second flush) were recovered after flushing with Triton X-100. In sharp contrast to the mating with wild types, no spermatozoa were recovered from all three flushings of the oviducts of any of the females mated to any of the knockout males. In the single instance of four spermatozoa in the second flush from one oviduct, but none in the first or third flush from the same organ, contamination with uterine sperm during dissection cannot be ruled out.
Examination of the Uterotubal Junction
Bouin-fixed, paraffin-embedded tissue of the uterus-oviduct junction (Fig. 4, a and b) revealed that uterine sperm from wild-type males had migrated through the folds of the colliculus tubarius and were found within the intramural oviduct by 1 h postcoitum (Fig. 4c), and sperm could be seen within the folds of the junction (Fig. 4e). In complete contrast, and despite the close contact of the sperm-laden uterine contents with the junction (Fig. 4b) and migration of the occasional sperm into the initial luminal folds of the colliculus tubarius (Fig. 4d), there was never entry of sperm from the knockout males into the oviduct beyond the junction (Fig. 4f), whether tissues were observed at 1 h (Fig. 4) or 4 h postcoitum (not shown).
|
DISCUSSION
The results from this study strongly suggest that the infertility that characterizes the male c-ros tyrosine kinase knockout mice is due to the inability of the ejaculated sperm to leave the uterus and enter the oviduct. The basis for this conclusion lies in examination of the uterine and oviductal contents and tissue at two time points following natural mating. The earlier time point, averaging 1 h postcoitum, was chosen for the recovery of fresh sperm deposited in the uterus. The later time point, around midnight (13 h post-hCG stimulation), was chosen to allow enough time for the migration of uterine sperm into the oviduct, but not too long after ovulation, because in mice sperm transport in the oviduct is optimal around ovulation [18, 19], and sperm number declines 2–3 h afterward [18]. The hormonal stimulation schedule in the present study was aimed at inducing ovulation around 2200 to 2300 h and 90% of animals had ovulated when examined around midnight. From our records of similarly handled and successfully mated mice, 73% had mated by 2000 h (1 h after lights out) and the last 10% mated between 2130–2300 h. Therefore all mice would have had an optimal time of 2–4 h for sperm transport into the oviduct. The multiple flushing technique adopted for sperm recovery from the oviduct was aimed at obtaining in the first flush those sperm free in the lumen, in the second flush those attached to the oviductal epithelium, and in the last flushing with Triton X-100 those tightly bound or trapped in the crypts [6], for comparison between genotypes.
At both time points after mating, spermatozoa from the wild-type male mice were found in the uterine contents mostly with straight flagella and within the folds of the colliculus tubarius as well as within the oviductal isthmus. As shown for the hamster [6, 7], most sperm were recovered from the murine oviduct in the first flush and more in the second than third. Unlike the hamster, cell viability, as judged from their motility, was equal in the two fractions. The present finding of sperm migration through the intramural uterotubal junction and their presence in the lumen of the oviductal isthmus of females mated to wild-type mice as early as 1 h postcoitum is in agreement with the observations of Suarez [19] on murine sperm in the oviduct in situ.
By contrast, the infertile c-ros knockout males deposited sperm in the uterus with their flagella predominantly bent at the mid-/principal piece junction at both time points examined. The entangling of bent sperm in aggregates observed in the fixed uterine contents is likely to handicap motile sperm swimming free from the aggregates. The similar numbers of dispersed sperm in the knockout and wild-type groups counted after flushing out and diluting the uterine contents could be due to agitation in the experimental procedure that dispersed the sperm. After mating to knockout males, sperm were never found in the oviducts even after ovulation, whether oviductal contents or sections of the uterotubal junction were examined. This suggests that there was an inability of the knockout sperm to negotiate the uterotubal junction, despite the deposition of a normal number of sperm that were motile in the uterus. The initial observation with knockout males 4 h postcoitum that 1000–10 000 sperm were flushed out from 16 of 26 oviducts, with none from the others [3], was probably due to contamination with uterine contents during tissue preparation, because the reported number of oviductal sperm from the wild-type group varied enormously from 1000 to 60 000 per oviduct, far higher than the two thousands per oviduct estimated from serial tubule sections [18] and the thousand estimated in the present study by multiple flushing.
The dispersion and dilution of uterine contents that are necessary for the analysis of sperm kinematics may not reflect the situation in situ, but a 50% reduction of swimming velocities of the motile knockout sperm and a decrease in the amplitude of the lateral head movement were found. These decreases were more than the 20% found for mature sperm released directly from the epididymis [5], suggesting that the uterine environment is more challenging than the in vitro culture medium. Indeed some of these sperm can achieve fertilization in vitro [3], indicating that the in vivo defects are alleviated to certain extents in vitro.
In addition to the angulation of the sperm tail in the uterus, forming entangled sperm aggregates, the deficiency in the vigor of flagellation (decreased VCL) and inefficiency of forward progression (decreased VSL) could account for the inability of the knockout sperm to leave the aggregated sperm masses or physically to squeeze between the close folds of the uterotubal junction. In the rat only progressively swimming sperm are observed leaving the junction on the oviductal side [20], and in hamsters, caput sperm that swim in circles in vitro and cannot fertilize after intrauterine insemination achieve 22% fertilization after induction of forward motility and enhanced flagellation vigor [21]. Sperm from the knockout mice with hairpin bends do progress forward when examined in vitro but swim backward in the sense that the leading point is the swollen cytoplasmic droplet at the flexure of the midpiece/principal piece junction instead of the hook-shaped head, compromising the forward thrust. Tail angulation less extensive than a hairpin bend would also create difficulty for sperm entering the closely applied crypts of the colliculus tubarius illustrated in Figure 4.
Angulation and hairpin bending of the sperm tail in rats [22] and mice [23] are thought to indicate swollen cells that change their shape to avoid increases in surface area, because coiling of the sperm tail is also demonstrated as osmotic swelling in other mammalian spermatozoa [24, 25]. Angulation in caput sperm stimulated to swim forward has been demonstrated in hamsters [26, 27], with the suggestion that this is the result of a lack of stiffness in the immature sperm flagellum that has fewer disulfide bonds than mature sperm [28]. For epididymal sperm from the c-ros knockout mice, it has been shown that the tail angulation could be straightened out upon demembranation by Triton X-100 [5] and is therefore not an inherent flagellar defect but a cell-swelling phenomenon. Neither could the oxidizing agent diamide or reducing agent dithiothreitol affect the tail form of the wild-type sperm [4] whose angulation could be induced by blocking ion channels known to be involved in volume regulation [5]. An association of infertility with coiled sperm in the ejaculate of domestic animals has long been reported [29], for instance in bull [30], boar [31], dog [32], and stallion [33]. The cause of such a defect is not known, although it originates from the epididymis [29]. The present work is the first direct evidence that such sperm with bent tails fail to migrate into the oviduct.
For the c-ros knockout male mice, the bent forms of sperm tails observed in the uterus cannot be due to an abnormally low osmotic pressure of uterine fluid because 1) this was measured and did not differ between females mated to the two genotypes, 2) the fluid into which the sperm were diluted for observation was made to approximately the same osmotic pressure as uterine contents, and 3) the bends were present in fixed undiluted uterine contents or when fixed in situ. As uterine contents are of lower osmolality (around 330 mmol/kg) than fluid from the tail of the epididymis (around 420 mmol/kg for both genotypes [5]) sperm cell swelling may well ensue after ejaculation as a result of hypo-osmotic stress. Nevertheless, wild-type sperm are able to undergo the normal regulatory volume decrease as in somatic cells [34, 35] via activities of potassium and chloride channels [5] and maintain straight flagella in the uterus. On the other hand, sperm from the knockout mice were unable to do so, therefore swelled in the uterus, causing the tails to bend and entangle with each other in the sperm mass. It is most likely that sperm tail angulation caused by a defect in volume regulation, when aggravated by the compromised sperm kinematics, effectively prevents the migration of sperm from the knockout males through the uterotubal junction of the mated females. The total failure of sperm transport through the female tract is sufficient to cause infertility of these transgenic mice, although there may be additional sperm defects yet to be identified.
|
ACKNOWLEDGMENTS
We thank Barbara Hellenkemper, Margrit Kloth, and Raphaele Kürten for technical assistance. We thank Dr. Eva Riethmacher and Prof. Carmen Birchmeier of the Max-Delbrück Centre of Molecular Medicine, Berlin, for their generous supply of animals that enabled the creation of our transgenic mouse colony.
FOOTNOTES
1 This work was supported by the Deutsche Forschungsgemeinschaft Confocal Grant number Ni-130/15 "The male gamete: production, maturation, function" and the Rockefeller and Ernst Schering Research Foundations' AMMPA Project. 
2 Correspondence: C.-H. Yeung, Institute of Reproductive Medicine of the University, Domagkstrasse 11, D-48129 Münster, Germany. FAX: 49 251 8356093; yeung{at}uni-muenster.de
Accepted: April 4, 2000.
Received: February 2, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  W. Si, H. Men, J. D Benson, and J. K Critser Osmotic characteristics and fertility of murine spermatozoa collected in different solutions Reproduction, February 1, 2009; 137(2): 215 - 223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Yeung, C. Callies, A. Rojek, S. Nielsen, and T. G. Cooper Aquaporin Isoforms Involved in Physiological Volume Regulation of Murine Spermatozoa Biol Reprod, February 1, 2009; 80(2): 350 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C Callies, T G Cooper, and C H Yeung Channels for water efflux and influx involved in volume regulation of murine spermatozoa Reproduction, October 1, 2008; 136(4): 401 - 410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T G Cooper, J P Barfield, and C H Yeung The tonicity of murine epididymal spermatozoa and their permeability towards common cryoprotectants and epididymal osmolytes Reproduction, May 1, 2008; 135(5): 625 - 633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A M Petrunkina, R A P Harrison, M Tsolova, E Jebe, and E Topfer-Petersen Signalling pathways involved in the control of sperm cell volume Reproduction, January 1, 2007; 133(1): 61 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Klein, T.G. Cooper, and C.H. Yeung The Role of Potassium Chloride Cotransporters in Murine and Human Sperm Volume Regulation Biol Reprod, December 1, 2006; 75(6): 853 - 858. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Fetic, C.-H. Yeung, B. Sonntag, E. Nieschlag, and T. G. Cooper Relationship of Cytoplasmic Droplets to Motility, Migration in Mucus, and Volume Regulation of Human Spermatozoa J Androl, March 1, 2006; 27(2): 294 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.P. Barfield, C.H. Yeung, and T.G. Cooper Characterization of potassium channels involved in volume regulation of human spermatozoa Mol. Hum. Reprod., December 1, 2005; 11(12): 891 - 897. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.H. Yeung, J.P. Barfield, and T.G. Cooper Chloride Channels in Physiological Volume Regulation of Human Spermatozoa Biol Reprod, November 1, 2005; 73(5): 1057 - 1063. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sofikitis, E. Pappas, A. Kawatani, D. Baltogiannis, D. Loutradis, N. Kanakas, D. Giannakis, F. Dimitriadis, K. Tsoukanelis, I. Georgiou, et al. Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment Hum. Reprod. Update, May 1, 2005; 11(3): 229 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.P. Barfield, C.H. Yeung, and T.G. Cooper The Effects of Putative K+ Channel Blockers on Volume Regulation of Murine Spermatozoa Biol Reprod, May 1, 2005; 72(5): 1275 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Carlson, X.-M. Xu, V. N. Gladyshev, and D. L. Hatfield Selective Rescue of Selenoprotein Expression in Mice Lacking a Highly Specialized Methyl Group in Selenocysteine tRNA J. Biol. Chem., February 18, 2005; 280(7): 5542 - 5548. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.G. Cooper Cytoplasmic droplets: the good, the bad or just confusing? Hum. Reprod., January 1, 2005; 20(1): 9 - 11. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Legare and R. Sullivan Expression and localization of c-ros oncogene along the human excurrent duct Mol. Hum. Reprod., September 1, 2004; 10(9): 697 - 703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nakanishi, A. Isotani, R. Yamaguchi, M. Ikawa, T. Baba, S. S. Suarez, and M. Okabe Selective Passage Through the Uterotubal Junction of Sperm from a Mixed Population Produced by Chimeras of Calmegin-Knockout and Wild-Type Male Mice Biol Reprod, September 1, 2004; 71(3): 959 - 965. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kotaja, D. De Cesare, B. Macho, L. Monaco, S. Brancorsini, E. Goossens, H. Tournaye, A. Gansmuller, and P. Sassone-Corsi Abnormal sperm in mice with targeted deletion of the act (activator of cAMP-responsive element modulator in testis) gene PNAS, July 20, 2004; 101(29): 10620 - 10625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Prins and W. Bremner The 25th Volume: President's Message: Andrology in the 20th Century: A Commentary on Our Progress During the Past 25 Years J Androl, July 1, 2004; 25(4): 435 - 440. [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Yeung, M. Anapolski, I. Setiawan, F. Lang, and T. G. Cooper Effects of Putative Epididymal Osmolytes on Sperm Volume Regulation of Fertile and Infertile c-ros Transgenic Mice J Androl, March 1, 2004; 25(2): 216 - 223. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Srivastav, B. Singh, A. Chandra, F. Jamal, M. Y Khan, and S. R Chowdhury Partial characterization, sperm association and significance of N- and O-linked glycoproteins in epididymal fluid of rhesus monkeys (Macaca mulatta) Reproduction, March 1, 2004; 127(3): 343 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Penttinen, D. A. Pujianto, P. Sipila, I. Huhtaniemi, and M. Poutanen Discovery in Silico and Characterization in Vitro of Novel Genes Exclusively Expressed in the Mouse Epididymis Mol. Endocrinol., November 1, 2003; 17(11): 2138 - 2151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. G. Cooper, A. Wagenfeld, G. A. Cornwall, N. Hsia, S. T. Chu, M.-C. Orgebin-Crist, J. Drevet, P. Vernet, C. Avram, E. Nieschlag, et al. Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice Biol Reprod, November 1, 2003; 69(5): 1750 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xu, C.-H. Yeung, I. Setiawan, C. Avram, J. Biber, A. Wagenfeld, F. Lang, and T. G. Cooper Sodium-Inorganic Phosphate Cotransporter NaPi-IIb in the Epididymis and Its Potential Role in Male Fertility Studied in a Transgenic Mouse Model Biol Reprod, October 1, 2003; 69(4): 1135 - 1141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. M. Andersen, C.-H. Yeung, H. Vorum, M. Wellner, T. K. Andreassen, B. Erdmann, E.-C. Mueller, J. Herz, A. Otto, T. G. Cooper, et al. Essential Role of the Apolipoprotein E Receptor-2 in Sperm Development J. Biol. Chem., June 20, 2003; 278(26): 23989 - 23995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.H. Yeung, M. Anapolski, M. Depenbusch, M. Zitzmann, and T.G. Cooper Human sperm volume regulation. Response to physiological changes in osmolality, channel blockers and potential sperm osmolytes Hum. Reprod., May 1, 2003; 18(5): 1029 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Shariatmadari, P. Sipila, M. Vierula, K. Tornquist, I. Huhtaniemi, and M. Poutanen Adenosine Triphosphate Induces Ca2+ Signal in Epithelial Cells of the Mouse Caput Epididymis Through Activation of P2X and P2Y Purinergic Receptors Biol Reprod, April 1, 2003; 68(4): 1185 - 1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wagenfeld, C.-H. Yeung, W. Lehnert, E. Nieschlag, and T. G. Cooper Lack of Glutamate Transporter EAAC1 in the Epididymis of Infertile c-ros Receptor Tyrosine-Kinase Deficient Mice J Androl, November 1, 2002; 23(6): 772 - 782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sipila, T. G. Cooper, C.-H. Yeung, M. Mustonen, J. Penttinen, J. Drevet, I. Huhtaniemi, and M. Poutanen Epididymal Dysfunction Initiated by the Expression of Simian Virus 40 T-Antigen Leads to Angulated Sperm Flagella and Infertility in Transgenic Mice Mol. Endocrinol., November 1, 2002; 16(11): 2603 - 2617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Butler, X. He, R. E. Gordon, H.-S. Wu, S. Gatt, and E. H. Schuchman Reproductive Pathology and Sperm Physiology in Acid Sphingomyelinase-Deficient Mice Am. J. Pathol., September 1, 2002; 161(3): 1061 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Yeung, M. Anapolski, and T. G Cooper Measurement of Volume Changes in Mouse Spermatozoa Using an Electronic Sizing Analyzer and a Flow Cytometer: Validation and Application to an Infertile Mouse Model J Androl, July 1, 2002; 23(4): 522 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. V. Holt and R. A. P. Harrison Bicarbonate Stimulation of Boar Sperm Motility via a Protein Kinase A--Dependent Pathway: Between-Cell and Between-Ejaculate Differences Are Not Due to Deficiencies in Protein Kinase A Activation J Androl, July 1, 2002; 23(4): 557 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Yeung, M. Anapolski, P. Sipila, A. Wagenfeld, M. Poutanen, I. Huhtaniemi, E. Nieschlag, and T. G. Cooper Sperm Volume Regulation: Maturational Changes in Fertile and Infertile Transgenic Mice and Association with Kinematics and Tail Angulation Biol Reprod, July 1, 2002; 67(1): 269 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Christova, P. S. James, T. G. Cooper, and R. Jones Lipid Diffusion in the Plasma Membrane of Mouse Spermatozoa: Changes During Epididymal Maturation, Effects of pH, Osmotic Pressure, and Knockout of the c-ros Gene J Androl, May 1, 2002; 23(3): 384 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Yeung and T.G. Cooper Effects of the ion-channel blocker quinine on human sperm volume, kinematics and mucus penetration, and the involvement of potassium channels Mol. Hum. Reprod., September 1, 2001; 7(9): 819 - 828. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
