| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Institute of Reproductive Medicine of the University, D-48129 Münster, Germany
b Max-Delbrück Center of Molecular Medicine, D-13122 Berlin, Germany
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The protooncogene c-ros is an orphan receptor with an intracellular tyrosine kinase domain and a long extracellular domain [6, 7]. The original c-ros knockout mice were generated with the intention of examining the role of c-ros in morphogenesis, since it is expressed in specific temporal patterns in the embryonic epithelia of the kidney, lung, and intestine [7, 8]. It is also expressed in the wolffian duct, the embryonic genital tract. Whereas the expression of c-ros in other organs rapidly disappears in neonatals, that in the epithelium of the proximal epididymis (caput) is up-regulated during prepubertal development, before the arrival of spermatozoa from the testis, and persists in adults [4]. This coincides with the differentiation of the proximal caput epididymidis into the so-called initial segment. In the homozygous knockout mice, c-ros is not detected at the mRNA or protein level and there is no differentiation of the initial segment, which is the only abnormal phenotype found in these infertile animals. Heterozygous transgenic mice are indistinguishable from the wild type.
To elucidate the relationship between the specific epididymal abnormality and the infertility of these transgenic animals, it is necessary to characterize the sperm defects in these mice. Previous findings of reduced numbers of sperm in the oviduct of mated wild-type females despite normal mating and uterine deposition of sperm, and of successful reproduction using these sperm in conventional in vitro fertilization and embryo transfer techniques [4], suggest that sperm from the homozygous mice may have defective motility compromising their transport through the female tract. Therefore the maturation of sperm kinematics in the epididymis was examined in detail in the present study. Preliminary observations of abnormal forms of the sperm flagellum [5] were also investigated systematically. Angulation or coiling of the sperm tail is well recognized as a phenomenon of sperm cell swelling (e.g., [9–11]). There are several reports of infertility associated with the presence of coiled sperm in the ejaculates of stallions, boars, and bulls in which coiling originates in the epididymis [12], but the mechanism of volume regulation in spermatozoa has received hardly any attention. Since mammalian spermatozoa are stored in a hypertonic environment in the epididymis [13] and would experience relative hypoosmotic conditions upon ejaculation, consideration of regulatory volume decrease (RVD) is particularly pertinent. The present work is the first to characterize the maturational development of RVD in mammalian sperm and the involvement of ion channels, and to reveal defects in them that are possibly linked to infertility.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animals and Sperm Preparation
Homozygous and heterozygous transgenic mice with targeted deletions of c-ros were produced in the Max-Delbrück Center. Since the deletion of either the intracellular kinase domain or the extracellular domain resulted in complete infertility in homozygous mice [4], the two genotypes were used indiscriminately in the present study. Wild-type mice of the same strain (C57/BL6) were obtained for control experiments from Charles River (Sulzfeld, Germany). Animals were anesthetized with ethyl carbamate, and several loops of tubule were excised from each defined region of the epididymis. Sperm were gently expressed into a drop of experimental medium, transferred into an Eppendorf (Hamburg, Germany) tube kept in an incubator at 37°C with 5% CO2, and examined at various times.
Measurement of Motility and Flagellar Angulation of Diluted Sperm
Sperm were obtained from 5 regions of the epididymis: the initial segment (or the topologically equivalent zone in the homozygous mice), distal caput, proximal corpus, and proximal and distal cauda. After dilution and dispersion for 1 min at 37°C, the sperm were loaded onto a 40-µm-deep siliconized chamber and examined on a microscope stage warmed to 37°C. One hundred cells were classified as motile (flagellating) or immotile, and the tail form was categorized as straight, angled, or hairpin (see Results for description). A video recording was made from several microscopic fields over 3 sec and analyzed later by tracking 100–200 motile cells for 30 video frames at a rate of 25 Hz, using the computerized sperm analysis system (HTM-C version 10.6; Hamilton Thorne Research, Beverly, MA) as described previously [14]. Kinematic parameters measured included curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN = 100% x VSL/VCL), and amplitude of the lateral displacement of sperm head from the averaged swim path (ALH). Median values of the kinematic parameters were obtained for each sample.
Examination of Sperm Flagellar Angulation In Situ
To determine whether sperm were angulated in the epididymal lumen or whether the phenomenon appeared only when sperm were released from the lumen and diluted, the entire epididymis was fixed by immersion in 5% glutaraldehyde in PBS (Gibco, Eggerstein, Germany). The fixed tubule segments from 7 regions were coarsely minced, and the sperm were dispersed by mild sonication (0.5 sec at 50 W; Zinsser Analytic, Frankfurt, Germany). The form of sperm tails was examined in wet preparations or through preparation of smears and staining by the modified Papanicolaou method [15].
Effect of Ion Channel Blockers on Flagellar Angulation
Sperm from the distal caput and the mid cauda epididymidis were released into the basal medium, and each of the blockers (quinine, 1 mM; NPPB, 50 µM; BaCl2, 5 mM; ouabain, 0.1 mM; glibenclamide, 100 or 250 µM) was added separately to the sperm suspension after dispersion, as described above. The sperm were incubated for 20 min, and an aliquot was analyzed for motility and flagellar angulation. To test whether the kinking of the flagellum was a result of cell swelling, Triton X-100 (0.1% v:v) was added to another aliquot to remove the restraint due to the cell membrane so the axoneme could unbend, and the form of the sperm tail was reexamined.
Measurement of the Osmolality of Luminal Epididymal Fluid
Osmolality was examined using a vapor pressure osmometer (Vapro, model 5520; Wescor, Schlag GmbH, Bergisch Gladbach, Germany), which can measure 2- to 10-µl samples between 100 and 3000 mmol/kg with an error of about ± 3 mmol/kg; measurements of dew-point depression are not affected by sample viscosity or the presence of suspended particulates [16]. The vas deferens was cannulated and perfused in retrograde with PBS. The undiluted viscous luminal contents exuded from a tubule cut in the proximal cauda epididymidis were drawn up a positive displacement pipette. Luminal contents (3 µl) were transferred onto a 2 x 1.5-mm strip of Kimwipe (Kimberly-Clark Corp., Irving, TX) tissue paper in the loading chamber of the vapor pressure, and readings were taken after a delay of 10 and 15 min (to ensure saturation of the chamber as reflected by a difference of < 2 mmol/kg between the two readings). Calibration of the osmometer was performed using standard osmolality solutions of 290, 400, and 1000 mmol/kg via similar procedures before and after measurement of the samples.
Statistics
Comparisons between sperm kinematic parameters of wild-type and knockout animals were made with Student's t-test. Statistics on percentages were obtained after arcsine transformation. Mean values and upper standard errors (mean + SEM) given in the paper were retransformed into percentages for presentation. Differences between wild-type and knockout animals, and between treatments, were tested for statistical significance (P < 0.05) using one-way ANOVA followed by the Student-Newman-Keuls test.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Only a few spermatozoa were recovered from the initial segment. These cells from mice of all genotypes were straight; they exhibited no regular flagellation, with about 10% showing barely discernible twitching of the tail. Motility potential developed along the length of the epididymis with the acquisition of flagellation and then forward progression. The only difference in the percentage motile sperm between the homozygous transgenic and the heterozygous/wild-type groups was found in the corpus region after 3-h incubation, but this slight deficiency in the sperm of the knockout mice was not persistent through maturation, since there was no difference in cauda sperm. Detailed analysis of kinematics revealed only slight decreases in velocities of the mature spermatozoa, with concomitant increases in linearity and decreases in lateral head displacement (Fig. 1). Kinematic deficiencies of the same extents were shown in the knockouts when glycolysis, which is essential for fertilization processes, was made compulsory using medium containing only glucose as substrate (data not shown).
|
Flagellar Angulation in Maturing Sperm
In the heterozygous and wild-type mice, about 10% of the epididymal sperm displayed flagellar angulation when freshly released and dispersed in the basal medium (310 mmol/kg). These bent forms showed various extents of kinking of the tail at the location of the cytoplasmic droplet, up to the extreme of hairpin forms (Fig. 2). Upon incubation, there was a slight increase in bent forms for cauda sperm and a greater increase for corpus sperm, whereas the majority of caput sperm showed angulation. Hence in the normal epididymis, the phenomenon of flagellar angulation in immature sperm incubated in vitro subsided on maturation (Fig. 3). Angulation was manifested by both motile and immotile cells and was more commonly found in the immotile than in the motile sperm with the exception of caput sperm. For instance, after 3-h incubation, 20 + 5% (mean + SEM) of the total but only 5 + 1% of the motile cauda sperm, and 26 + 8% of the total but 15 + 13% of the motile corpus sperm, were in bent forms. Since interpretation of angulation in immotile sperm could be complicated if cell shape were to change on dying, only data on the motile population are presented unless stated otherwise.
|
|
In contrast to observations in the wild type, the large majority of cauda sperm from the knockout mice were bent upon release, although the percentage decreased to about 40% on incubation (Fig. 3). Fresh corpus and caput sperm also showed higher percentages of bent forms than the wild type but upon incubation exhibited the same extents of further increase as did controls.
Effects of Osmolality on Flagellar Angulation
Since flagellar angulation is a phenomenon of hypotonic swelling of spermatozoa, responses of mature sperm to changes in tonicity of the medium were compared between the knockout and the wild-type groups. As expected, 70% of the wild-type cauda sperm were in the bent form when released into the 230 mmol/kg medium. Flagellar angulation decreased drastically with increasing osmolalities to barely 2% at 430 mmol/kg (Fig. 4, bottom). On the other hand, there was no such large decrease over the same range of osmolalities in the knockout group (only from 86% to 56%, Fig. 4), with the majority retaining the bent form even at 510 mmol/kg (53 + 11%; mean + SEM, n = 3). The percentage motility remained unchanged for all osmolalities tested for both wild type and knockouts (Fig. 4, top).
|
Flagellar Angulation In Situ and Osmolality of Luminal Fluid
In the wild-type mice, about 80% of sperm in the initial segment possessed straight tails. The occurrence of kinked sperm slightly increased as they passed through the caput before these forms gradually decreased such that only about 10% retained their flagellar angulation in the distal epididymis (Fig. 5). In the knockout mice, sperm in the proximal epididymis showed the same percentages of straight tails; yet the initial trend of decrease was not reversed as in the wild type but continued up to the proximal corpus region, such that only 60% of mature sperm had straight tails. Among the bent sperm, one third to one half showed only a very slight kink at the site of the cytoplasmic droplet; the other flagella were bent into either a conspicuous angle or a hairpin bend. This in situ angulation in the mature sperm, compared to that exhibited in vitro upon release and dilution with the basal medium, occurred less frequently (40 + 9% vs. 55 + 5% bent, respectively) and to a lesser extent (9 + 3% vs. 25 + 6% in hairpin form).
|
Cauda fluid from the wild-type epididymides had an osmolality of 415 ± 12 mmol/kg (mean ± SEM, n = 11), whereas that from the c-ros knockout mice was 427 ± 27 mmol/kg (n = 7), indicating no difference between the two genotypes.
Sperm Volume Regulation and Responses to Ion Channel Blockers
The concentrations of reagents used were those shown to have maximal effects in somatic cells; they did not affect the percentages of motile sperm. NPPB and BaCl2 (Cl-- and K+-channel blockers, respectively) significantly induced flagellar angulation in the cauda sperm from the wild type, and quinine showed a stronger effect, with only 38% of motile sperm maintaining straight tails compared to 91% in the control basal medium (Fig. 6A). In all these cases the majority of kinked flagella were hairpin forms. In the knockout group, 40% of motile sperm were straight and 60% were bent, as shown above, but none of the blockers caused any further angulation (Fig. 6B). This lack of response of the cauda sperm in the knockout male was similar to that shown by the immature caput sperm of the wild type (Fig. 7A). Caput sperm from the c-ros knockout also did not respond to NPPB or BaCl2, and when treated with quinine they showed a reverse effect with a decrease of hairpin tails resulting in an increase in straight motile sperm (Fig. 7B).
|
|
Evidence for Cell Swelling as a Cause of Angulation
To verify that flagellar angulation was a manifestation of cell swelling, percentages of all sperm in straight or kinked forms were compared in the absence and presence of Triton X-100 used to perforate the cell membrane. Flagellar angulation in the cauda sperm of both the wild type (induced by quinine) and knockout (in basal medium) largely disappeared in Triton X-100 (from 62 + 6% and 60 + 8% to 11 + 2% and 20 + 5%, respectively). The hairpins in the knockout caput sperm opened up (51 + 7% vs. 4 + 1% hairpin in the absence and presence of Triton, respectively) into the angled form but did not straighten further, resulting in only 50% with straight tails when demembranated compared to 6% while intact. Quinine-treated caput sperm from both wild type and knockouts straightened out in Triton X-100 to 85 + 5% and 82 + 3%, respectively.
Further Characterization of RVD in Wild-Type Mature Sperm
Ouabain did not alter the motility or the form of the motile cauda sperm (90 + 3% straight forms vs. 92 + 1% in controls; mean + SEM, n = 6); neither did glibenclamide at 100 µM (Fig. 8) or at a higher concentration at which motility was suppressed (250 µM; data not shown).
|
When Ca2+ was removed from the medium, beat amplitude decreased markedly (not quantified), although the percentage of motile sperm remained unchanged. Flagellar angulation gradually increased with incubation time, such that by 2 h only 3% of the feebly flagellating motile sperm still retained a straight form (Fig. 8). Whereas the increased angulation was first detected largely in angled forms at 20 min, 85% and 92% of kinked flagella were in hairpin forms by 1 and 2 h of incubation, respectively. In Ca2+-free medium, the effect of quinine was reduced (Fig. 8, histograms) such that the decrease in straight forms was only 16 percentage points compared with 53 in the control medium. Significantly fewer hairpin forms were observed when quinine treatment was accompanied by the absence of Ca2+ (6 + 2% vs. 20 + 6% in the presence of Ca2+).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One possible explanation for the absence or reduced number of sperm in the oviduct of wild-type females after mating with the infertile homozygous c-ros knockout mice is a reduction in sperm motility that compromises passage in the female tract. However, the present findings of only a 20% decrease in velocities and lateral head displacement, and no decrease in percentage motility of mature sperm, are unlikely to account for the complete infertility. The slight kinematic differences were probably a consequence of the kinked sperm tails, which could compromise the efficiency of propulsion. It is interesting to note the implication that the initial segment, which is not developed in the knockout mice, was not essential for the development of flagellating ability of immature sperm. The nearly normal velocities and forward progression of the more mature sperm are consistent with the finding that these kinematic changes occur mostly downstream in the corpus epididymidis [14], which appeared normal in the knockouts.
In contrast to the slight abnormality in motility, a marked defect of the motile mature (cauda) sperm from the homozygous transgenic mice was revealed as flagellar angulation. Although sperm are transported passively as far as the uterotubal junction, there is no en masse passage through the junction into the oviduct. It has been observed in rats [17] that motile sperm pass through individually at intervals of a few minutes, implying that the junction is a selection barrier. The kinked sperm would have difficulty passing through the uterotubal junction or the cumulus mass to reach the oocytes. Spermatozoa are exempted from these barriers in the conventional in vitro fertilization procedure, which revealed no difference in the fertilization rate between the wild-type and the knockout sperm [4].
The observed tail angulation was an indication of cell swelling and not an intrinsic axomenal abnormality, since it was largely reduced by demembranation using detergent and since wild-type sperm showed the same phenomenon to an increasing extent when exposed to media of decreasing tonicity. Osmolarity of epididymal luminal fluids from laboratory animals ranges from 330 to 420 mmol/kg and is highest in the corpus [13]. The present study documents for the first time that the osmolality of the cauda epididymal fluid is 415 mmol/kg in the wild-type mouse, and the same as in the knockouts. This means that the dilution of epididymal sperm upon release with the commonly used experimental media (290–310 mmol/kg) presents a similar hypotonic stress to sperm from both the wild type and knockouts. That flagellar angulation was not seen in the majority of mature sperm from the wild-type mice suggests that there is normally a rapid occurrence of volume regulation.
The present observations of kinking of the mutant sperm upon dilution might reflect an in vitro response as a consequence of prior exposure to some abnormal luminal components not experienced by normal fertile sperm. Indeed, abnormal flagellar angulation was already detectable in situ. In the wild-type epididymis, caput sperm fixed in situ showed the highest level of kinking, which was no longer observed in sperm fixed in the distal half of the epididymis—indicating a development of volume regulation during normal epididymal maturation. In the homozygous knockout mice, slight kinking attained in the caput was further increased and maintained in the distal epididymis, suggestive of a lack of such volume regulation. Furthermore, upon release and dilution of the cauda sperm, the defect was immediately exaggerated by rapid swelling. Increasing the osmolality of the medium only slightly relieved the angulation, which persisted up to 510 mmol/kg. In comparison, wild-type sperm in hypotonic media showed swelling-induced kinking, as expected, which was drastically decreased to low levels at 310 mmol/kg and to a few percent at hyperosmolarity. This confirms that the defect in the knockouts is in sperm volume regulation rather than the osmolality of epididymal luminal fluid.
Sperm Volume Regulation in Wild-Type Mice
In wild-type epididymal sperm, the manifestation of flagellar angulation upon in vitro incubation, to decreasing extents with increasing maturity (caput > corpus > cauda), also indicates a normal maturational progress in the development of the volume regulation mechanisms.
Different pathways used by different cell types for RVD include K/Cl cotransport (best studied in erythrocytes and regulated by tyrosine kinase and phosphatase [18, 19]), Ca-sensitive K+ channels [20], separate Cl and K channels [18, 21], voltage-gated Cl channels, and volume-sensitive organic osmolyte anion channels [22, 23]. The latter permit the efflux of amino acids, myoinositol, betaine, and glycerophosphocholine in renal epithelial cells, which are sensitive to anion blockers [24]. Interestingly, these substances or their derivatives are secreted by the epididymis in high amounts [13, 25]. The mechanism for the regulation of cell volume by sperm is virtually unknown. Early work on bovine ejaculated sperm demonstrated that extracellular Ca2+ is required for the stability of sperm volume as well as motility [26, 27]. The control of ejaculated bovine sperm volume involves quinine-sensitive K channels [28] and is modulated by oxidative metabolism and the Na/K pump [27]. Water permeability in ram sperm decreases upon inhibition of glucose transport, but the water channel aquaporin CHIP28 cannot be found on the sperm surface [29]. Both cation and anion channels with various putative roles, such as chemotaxis, acrosomal exocytosis, and flagellar motion, have been reported in spermatogenic or sperm cells [30–32]. However, it is not known whether any of these are involved in RVD, except for a Cl channel (pICln) [33] suggested to regulate cell volume-induced Cl- efflux [34]. It is currently debated, however, whether this is a swelling-sensitive chloride channel or a channel regulator or a cation channel [35–37]. Although the protein has been immunocytochemically localized in abundance in the rat testis, epididymal epithelium, and sperm [38], there is as yet no direct evidence that this protein regulates cell volume of sperm.
In the present study, the ion channels involved in normal sperm RVD were probed by means of various commonly used blockers on sperm from wild-type animals. RVD was found to be inoperative in immature sperm, since there was no response to any of the blockers tested. In wild-type mature sperm, RVD was rapidly inhibited by quinine, NPPB, and BaCl2. Quinine is well known as an inhibitor of many types of K channels involved in RVD [39, 40] and volume-sensitive anion channels [23, 25]. NPPB has been used as a blocker of the swelling-activated Cl channel, mostly showing maximal inhibition at 0.1 mM [22, 23]. It also blocks anion channels for organic osmolytes, such as the swelling-induced efflux of taurine, inositol, and gluconate [41, 42]. Ba2+ is a specific K-channel blocker frequently used to study K+ currents involved in RVD [40, 43]. The inhibition of sperm RVD to the same extent by NPPB and BaCl2, and to a greater extent by quinine, suggests that for spermatozoa, the mechanisms involve a K channel and a Cl or an organic anion channel. Further characterization showed no effect on RVD by the Na-channel blocker ouabain, or by glibenclamide up to 0.25 mM. The latter is a sulfonylurea effective in blocking the ATP-sensitive inwardly rectifying K channels, the Na-activated voltage-dependent K channels [40], and a Ca-independent CFTR-like Cl channel [44]. The present findings suggest no involvement of these channels in sperm RVD.
The involvement of Ca2+ in RVD is controversial and may vary for various channels and cell types. Intracellular Ca2+ release augmented by influx of extracellular Ca2+ has been implicated in some epithelial cells (e.g., [20, 45, 46]). On the other hand, it has been demonstrated that extracellular or intracellular Ca2+ is not involved in the activation of certain types of volume-sensitive Cl channels in submandibular gland cells [44]. RVD involving organic osmolyte efflux or K+ efflux in certain cell types is also Ca-independent [47, 48]. In leukocytes, prolonged depletion of extracellular Ca2+ leads to inhibition of RVD [49], but it is not clear whether this is secondary to the resultant decreased energy metabolism, which can cause gradual swelling of bull sperm [27]. Inhibition of volume-sensitive organic anion channels by mitochondrial inhibitors has been demonstrated [42]. The slow effect of Ca2+ depletion in inducing flagellar angulation in the present study, accompanied by a reduction in the vigor of flagellation, may reflect such an indirect mechanism.
Defect of RVD Mechanism in Spermatozoa from c-ros Knockout Mice as the Probable Cause of Infertility
The failure of sperm from the knockout mouse to respond to quinine, NPPB, and BaCl2 with further angulation suggests that the relevant ion channels were not functioning normally. Since the present findings showed the acquisition of the ability for RVD, and a development on maturation in the wild-type epididymis of the responsiveness to the relevant ion channel blockers by the immature sperm, it can be hypothesized that some epididymal factors normally present in the lumen may interact with maturing spermatozoa in the development of their RVD mechanisms. Flagellar angulation should greatly compromise the hydrodynamics of motile sperm in their migration through the restrictive uterotubular junction and could explain the absence of sperm from the oviduct in 40% of the wild-type females, and the reduced numbers in the others, after mating with the homozygous c-ros knockout males [4]. Penetration of the intact cumulus to approach the zona pellucida of the oocyte with the sperm head at the appropriate angle for binding would also be difficult. Therefore a defect in RVD is a plausible cause of the in vivo infertility, although other contributing factors cannot be ruled out. Since the knockout mice lack development of the initial segment, which is very active in resorption and secretion, including that of epididymal-specific proteins [50], the infertile sperm would probably have multiple defects. This study suggests that much effort is needed to define the epididymal factors leading to natural infertility, as well as the nature of the development of ion channels in regulating sperm cell volume, which seem to be important for normal sperm function in the female tract.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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: June 1, 1999.
Received: February 23, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C.H. Yeung, F. Tuttelmann, M. Bergmann, V. Nordhoff, E. Vorona, and T. G. Cooper Coiled sperm from infertile patients: characteristics, associated factors and biological implication Hum. Reprod., June 1, 2009; 24(6): 1288 - 1295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lhuillier, B. Rode, D. Escalier, P. Lores, T. Dirami, T. Bienvenu, G. Gacon, E. Dulioust, and A. Toure Absence of annulus in human asthenozoospermia: Case Report Hum. Reprod., June 1, 2009; 24(6): 1296 - 1303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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, D Waberski, A R Gunzel-Apel, and E Topfer-Petersen Determinants of sperm quality and fertility in domestic species Reproduction, July 1, 2007; 134(1): 3 - 17. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Suzuki-Toyota, C. Ito, Y. Toyama, M. Maekawa, R. Yao, T. Noda, H. Iida, and K. Toshimori Factors Maintaining Normal Sperm Tail Structure During Epididymal Maturation Studied in Gopc / Mice Biol Reprod, July 1, 2007; 77(1): 71 - 82. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hoshii, T. Takeo, N. Nakagata, M. Takeya, K. Araki, and K.-i. Yamamura LGR4 Regulates the Postnatal Development and Integrity of Male Reproductive Tracts in Mice Biol Reprod, February 1, 2007; 76(2): 303 - 313. [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]
|
|
|
|
 |
|
 A. A. Y. Khalil, A. M. Petrunkina, E. Sahin, D. Waberski, and E. Topfer-Petersen Enhanced Binding of Sperm With Superior Volume Regulation to Oviductal Epithelium J Androl, November 1, 2006; 27(6): 754 - 765. [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]
|
|
|
|
|
|
Di Zhang and M. Gopalakrishnan Sperm Ion Channels: Molecular Targets for the Next Generation of Contraceptive Medicines? J Androl, November 1, 2005; 26(6): 643 - 653. [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]
|
|
|
|
|
|
A. M Petrunkina, B. Gropper, A.-R. Gunzel-Apel, and E. Topfer-Petersen Functional significance of the cell volume for detecting sperm membrane changes and predicting freezability in dog semen Reproduction, December 1, 2004; 128(6): 829 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.M. Petrunkina, R.A.P. Harrison, M. Ekhlasi-Hundrieser, and E. Topfer-Petersen Role of volume-stimulated osmolyte and anion channels in volume regulation by mammalian sperm Mol. Hum. Reprod., November 1, 2004; 10(11): 815 - 823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.H. Yeung, J.P. Barfield, M. Anapolski, and T.G. Cooper Volume regulation of mature and immature spermatozoa in a primate model, and possible ion channels involved Hum. Reprod., November 1, 2004; 19(11): 2587 - 2593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J-M Frenoux, P Vernet, D H Volle, A Britan, F Saez, A Kocer, J Henry-Berger, D J Mangelsdorf, J-M A Lobaccaro, and J R Drevet Nuclear oxysterol receptors, LXRs, are involved in the maintenance of mouse caput epididymidis structure and functions J. Mol. Endocrinol., October 1, 2004; 33(2): 361 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Davies, C. Baumann, C. Kirchhoff, R. Ivell, R. Nubbemeyer, U.-F. Habenicht, F. Theuring, and U. Gottwald Targeted Deletion of the Epididymal Receptor HE6 Results in Fluid Dysregulation and Male Infertility Mol. Cell. Biol., October 1, 2004; 24(19): 8642 - 8648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Shirley, S. Hayashi, S. Mounsey, R. Yanagimachi, and M. L. Meistrich Abnormalities and Reduced Reproductive Potential of Sperm from Tnp1- and Tnp2-Null Double Mutant Mice Biol Reprod, October 1, 2004; 71(4): 1220 - 1229. [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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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 M Petrunkina, M Hebel, D Waberski, K F Weitze, and E Topfer-Petersen Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa Reproduction, January 1, 2004; 127(1): 105 - 115. [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]
|
|
|
|
|
|
P. Sutovsky, R. M. Turner, S. Hameed, and M. Sutovsky Differential Ubiquitination of Stallion Sperm Proteins: Possible Implications for Infertility and Reproductive Seasonality Biol Reprod, February 1, 2003; 68(2): 688 - 698. [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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 P Sutovsky, R Moreno, J Ramalho-Santos, T Dominko, W. Thompson, and G Schatten A putative, ubiquitin-dependent mechanism for the recognition and elimination of defective spermatozoa in the mammalian epididymis J. Cell Sci., January 5, 2001; 114(9): 1665 - 1675. [Abstract] [PDF]
|
|
|
|
|
|
C.-H. Yeung, A. Wagenfeld, E. Nieschlag, and T. G. Cooper The Cause of Infertility of Male c-ros Tyrosine Kinase Receptor Knockout Mice Biol Reprod, August 1, 2000; 63(2): 612 - 618. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
