Hyperactivated motility induced in mouse sperm by calcium ionophore A23187 is reversible

The reversibility of hyperactivated motility was tested in caudal epididymal mouse sperm by treating them with 1 microM calcium ionophore A23187 in dimethyl sulfoxide (DMSO), followed 2 min later by the addition of medium containing high levels of bovine serum albumin (BSA) (final concentrations: 0.5 microM A23187, 22 mg/ml BSA). Controls received DMSO alone, followed by BSA. Immediately following treatment with A23187, motility was weak and vibratory. Two minutes after the addition of high levels of BSA, motility was hyperactivated, as determined by videotape analysis of linearity of trajectory and acuteness of flagellar bending. Ten minutes after the addition, the movement pattern returned to that of fresh, uncapacitated epididymal sperm. Control sperm retained the linear swimming pattern of fresh caudal epididymal sperm during the 10 min of observation. Ninety minutes later, however, both control and treated sperm became hyperactivated. The percentage of motile sperm was not affected by treatment or time. Thus, ionophore-induced hyperactivation is reversible and does not interfere with the normal development of hyperactivation during incubation under capacitating conditions in vitro.     

Unexpected flagellar movement patterns and epithelial binding behavior of mouse sperm in the oviduct

In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca(2+) from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site.     

Role of the oviduct in sperm capacitation

Following insemination of spermatozoa pre-ovulation, the mammalian oviduct ensures, by the formation of a functional sperm reservoir (SR), that suitable (low) numbers of viable and potentially fertile spermatozoa are available for fertilization at the ampullary isthmic junction (AIJ). As ovulation approaches, a proportion of the SR-stored spermatozoa is continuously distributed towards the AIJ and individually activated leading to step-wise capacitation and the attainment of hyperactivated motility. This paper reviews in vivo changes in the intra-luminal milieu of the oviduct of pigs and cows, in particular the SR and the AIJ which relate to the modulation of sperm capacitation around spontaneous ovulation. In vivo, most viable spermatozoa in the pre-ovulatory SR are uncapacitated. Capacitation rates significantly increase after ovulation, apparently not massively but concurrent with the individual, continuous sperm dislocation from the SR. Bicarbonate, whose levels differ between the SR and the AIJ, appears as the common primary effector of the membrane destabilizing changes that encompasses the first stages of capacitation. Sperm activation can be delayed or even reversed by co-incubation with membrane proteins of the tubal lining, isthmic fluid or specific tubal glycosaminoglycans, such as hyaluronan. Although the pattern of response to in vitro induction of sperm activation - capacitation in particular - is similar for all spermatozoa, the capacity and speed of the response is very individual. Such diversity in responsiveness among spermatozoa insures full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation at the AIJ, thus maximizing the chances of normal fertilization.     

Tubulin synthesis during ciliogenesis in the mouse oviduct.

We reported earlier that tubulin levels increase in the developing mouse oviduct during that period after birth when ciliogenesis is at a maximum (Staprans, I., and Dirksen, E. R. (1974) J. Cell Biol., 62, 164). To determine the degree to which de novo synthesis and tubulin pools contribute to this increase, [³H]leucine-incorporation experiments were performed in vivo and in culture. Soluble, particulate and axonemal fractions, obtained from homogenized oviducts of 3-, 5-, 8- and 12-day-old suckling mice, were electrophoresed on sodium dodecyl sulfate gels and the specific activity of the tubulin band determined. The present work shows that more than 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts is synthesized de novo. From both the in vivo and in culture experiments we conclude that although tubulin pools are present in mouse oviduct, they are continuously being replenished by newly synthesized protein as there is a rapid outflow from the soluble and particulate to the axonemal fraction into structures such as basal bodies and cilia. This burst of de novo tubulin synthesis corresponds to evidence from electron microscopic autoradiography, where label is present to a greater extent over centriole precursors and basal bodies than over other cell organelles. [³H]leucine incorporation into tubulin was inhibited by cycloheximide, demonstrating that we are dealing with synthesis, while colchicine below 10^?3, M concentration had no effect on tubulin assembly into axonemes.     

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization

Dear Editor, Sperm cooperation has been observed in multiple species(Pizzari and Foster,2008),yet its existence and benefit for reproductive success in mammals remains underexplored.Here,combining tissue-clearing with deep three-dimen-sional (3D) imaging,we demonstrate that postcopulatory mouse sperm congregate into unidirectional sperm cooper-ative clusters at the utero-tubal junction (UTJ),a key physical barrier for passage into the oviduct.Reducing sperm number in male mice by unilateral vasoligation (Uni-Vas) or busulfan-treatment impairs sperm cluster formation and oviduct entry.     

Regulation of sperm storage and movement in the mammalian oviduct

The oviduct plays a vital role in ensuring successful fertilization and normal early embryonic development. The male inseminates many thousands or even millions of sperm, but this alone does not ensure that fertilization will be successful. The female tract, particularly the oviduct, provides filters that select for normal vigorously motile sperm. In conjunction with molecules in the seminal plasma and on sperm, the female tract regulates how and when sperm pass though the tract to reach the site of fertilization. Various regulatory processes control sperm passage into and through the oviduct. In some species, the uterotubal junction opens and closes to regulate when sperm may enter; furthermore, passage through the junction requires certain proteins on the sperm surface. Most of the sperm that manage to enter the oviduct soon become trapped and held in a reservoir. In marsupials and insectivores, this involves trapping sperm in mucosal crypts; while in most other mammalian species, this involves binding sperm to the oviductal epithelium. As the time of ovulation approaches, the sperm in the reservoir undergo capacitation, including motility hyperactivation. Capacitating sperm shed proteins that bind them to the mucosal epithelium, while hyperactivation assists the sperm in pulling off of the epithelium and escaping out of mucosal pockets. The process of sperm release is gradual, reducing chances of polyspermic fertilization. Released sperm may be guided towards the oocyte by secretions of the oviduct, cumulus cells, or oocyte. Hyperactivation likely assists sperm in penetrating the cumulus matrix and is absolutely required for penetrating the oocyte zona pellucida and achieving fertilization.     

Effect of ovulation on sperm transport in the hamster oviduct

When hamsters mate shortly after the onset of oestrus (4.5-6 h before the onset of ovulation), spermatozoa are stored in the caudal isthmus of the oviduct until near the time of ovulation. At this time, a few spermatozoa ascend to the ampulla to fertilize the eggs. Superovulation resulted in a significant increase in the number of spermatozoa in the caudal isthmus at 6 h post coitus (p.c.) and in the ampulla and bursal cavity at 12 h p.c. Precocious ovulation resulted in a highly significant reduction in the total number of spermatozoa in the oviduct at 3 and 6 h p.c. This effect was completely overcome by intrauterine artificial insemination, suggesting lack of cervical patency as the block to sperm transport in precociously ovulated animals. Ligation of the ampulla-infundibulum junction in naturally ovulating hamsters resulted in significantly fewer spermatozoa in the caudal isthmus and ampulla at 12 h p.c. Preclusion of ovulation also resulted in fewer spermatozoa in the caudal isthmus and ampulla at 12 h p.c., suggesting that the products of ovulation stimulate sperm transport in the oviduct.     

Endocytosis in the epithelium of the mouse oviduct

We studied the pathway of serum protein transport into the lumen of the mouse oviduct by localizing several tracer proteins in the oviduct after intravenous injection on days 1, 5, and 11 of pregnancy. Fluorescent proteins were observed in the lamina propria and in vesicles in the lumenal epithelial cells mainly in the preampulla segment on days 5 and 11 of pregnancy. In the isthmus, there was much less fluorescence in the lamina propria and no fluorescent vesicles in lumenal epithelial cells. This is similar to previous observations on day 1 and indicates that the uptake of serum proteins into lumenal epithelial cells in the preampulla is not limited to the time when embryos are present in the oviductal lumen. Horseradish peroxidase (HRP) was present in the lamina propria of the preampulla on days 1 and 5, but direct tracer movement into the oviductal lumen was blocked by the epithelial junctional complexes. Within the epithelial cells, HRP was localized in endocytic vesicles along the basolateral membrane, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. Ferritin was also used as a tracer and was observed in the same locations as HRP. Acid phosphatase in the epithelial cells of the preampulla on day 1 was localized in mvb and bdb, indicating that these structures are lysosomes. It appeared that HRP and ferritin followed two pathways after basolateral endocytosis by the epithelial cells in the preampulla: 1) they were transported to apical vesicles that may release their contents into the oviductal lumen, or 2) they were transported to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP

Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization in vivo. It is characterized by high-amplitude flagellar waves and, usually, highly asymmetrical flagellar beating. It had been suggested, but not tested, that Ca2+ and cAMP switch on hyperactivation by directly affecting the flagellar axoneme. In this study, the direct affects of these agents on the axoneme were tested by using detergent-demembranated bull sperm. As confirmed by TEM, treatment of sperm with 0.2% Triton X-100 disrupted the plasma, acrosomal, and inner mitochondrial membranes, leaving axonemes intact. In the presence of 2 mM ATP, the percentage of reactivated sperm that were hyperactivated increased to 80% when free Ca2+ was increased from 50 to 400 nM. The effect of the Ca2+ in this range was to increase beat asymmetry by increasing the curvature of the principal bend. No additional increases were observed above 400 nM free Ca2+, but motility was suppressed at 1 mM. The ability of Ca2+ to produce hyperactivation depended on ATP availability, such that more ATP was required to produce the high amplitude flagellar bends characteristic of hyperactivated motility than to produce activated motility. Cyclic AMP was not required for reactivation, nor for hyperactivation. Production of hyperactivated motility also required an alkaline environment (pH 7.9-8.5). These results suggest that, provided sufficient ATP is present and pH is sufficiently alkaline, Ca2+ switches on hyperactivation by enabling curvature of the principal bends to increase.     

Methyl parathion-induced sperm shape abnormalities in mouse.

Metacid 50, the commercial grade of methyl parathion (O,O-dimethyl-O-4-nitrophenyl phosphorothionate), a commonly used organophosphorus insecticide, was tested for its genotoxicity in Swiss albino mice using the sperm abnormality assay. Sperms of albino mice were examined at two time intervals, 1 week and 5 weeks after a single acute oral treatment with the pesticide at four dose levels, viz., 75.0, 37.5, 18.75 and 9.375 mg/kg body weight corresponding to 1/2 LD50, 1/4 LD50, 1/8 LD50 and 1/16 LD50 values respectively. A dose-related statistically significant increase in the percentage of abnormal sperm observed indicates the genotoxic potency of methyl parathion.     

Physical characteristics of mouse sperm nuclei.

The nuclei of epididymal sperm, isolated from C57BL/6J and CBA/J inbred mice by their resistance to trypsin digestion, retain the shape differences of the intact sperm head. Various physical characteristics of these nuclei were measured and compared. The measurement of the projected dimensions of nuclei showed that the CBA nuclei are 13.5% longer than C57BL/6 nuclei (8.64 +/- 0.02 mum compared with 7.61 +/- 0.02 mum), 0.8% narrower (3.51 +/- 0.01 vs. 3.54 +/-0.01 mum) with 6.8% more area (22.34 +/- 0.10 vs. 20.91 +/- 0.09 mum2). However, the volumes of the nuclei as based on reconstructing calibrated electronmicrographs of serial sections of the nuclei indicated that CBA are about 7% smaller than C57BL/6 nuclei (3.72 +/- 0.08 vs. 4.01 +/- 0.03 mum3). The buoyant density of the CBA nuclei is 1.435 +/- 0.002 g/cm3 compared with 1.433 +/- 0.002 g/cm3 for the C57BL/6 nuclei as determined on linear CsCl and Renografin-76 density gradients and confirmed by a technique utilizing physiological tonicities. Therefore, the average mass of the CBA nuclei is less than that of the C57BL/6 nuclei (5.34 +/- 0.12 vs. 5.75 +/- 0.05 pg). The sedimentation velocities at unit gravity of nuclei from 11 inbred strains differ over a range of more than 6% with CBA nuclei sedimenting about 2.0% more slowly than C57BL/6 nuclei. We show that for these nuclei the sedimentation velocity can be related to their buoyant density, volume and a sedimentation shape factor. Within the errors of our measurements of these various characteristics, it was found that C57BL/6 and CBA nuclei have similar sedimentation shape factors. Therefore, the difference in sedimentation velocity between these nuclei appears to be primarily a result of differences in volume. The possible applications of these techniques to the physical separation of sperm are evaluated in the discussion.     

In vivo culture of embryos in the immature mouse oviduct

In-vitro culture of mammalian preimplantation embryos is associated with subsequent decreased viability. This phenomenon is more pronounced with the domestic species embryos as culture conditions are at present unable to sustain cleavage of early preimplantation embryos for more than one or two cell divisions. In this study, the immature mouse oviduct is shown to be capable of supporting cleavage and morphological development of rabbit and porcine embryos. The immature mouse oviduct was shown to be comparable to in vitro culture as 76% and 60% of the transferred zygotes developed to the morula stage after 2 and 3 d respectively. The porcine zygotes, however, failed to develop beyond the 4-cell stage in either the immature mouse oviduct or in vitro. Porcine morula showed better tolerance of the oviduct environment and when recovered after 2 d contained an average of 64 cells, which was significantly more than in in vitro cultured morulae (40 cells). Early porcine blastocysts transferred to the mouse oviduct had over a two-fold increase in cell division (104 cells) over comparable blastocysts grown in vitro (57 cells). The immature mouse oviduct is, therefore, a potential surrogate environment for short-term storage of embryos of other species.     

Expression of villin in the mouse oviduct and the seminiferous ducts

Summary Villin, a 95-kD cytoskeletal protein selectively expressed in the microvilli of some absorptive cells was localized immunohistochemically in the oviduct and the seminiferous excretory ducts of the mouse. Villin was found in the proximal part of the oviduct, comprising the preampulla, ampulla, and part of the isthmus. Distal to the isthmus the oviductal cells lining the junctura and the intrauterine colliculus tubaris were devoid of villin. No villin could be detected in the uterine cells.Ductuli efferentes, connecting the rete testis with the epididymis were the only portion of the male seminiferous ductal system expressing villin. The cells lining the epididymis and the vas deferens were devoid of villin. These data show that villin is selectively expressed in male and female reproductive systems and that it is limited to anatomically defined proximal portions of the reproductive ducts.     

Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats

Sperm migration in estrous cervical mucus can be used to measure the ability of spermatozoa to migrate through the genital tract. The relationship of this test with the sperm colonization of the isthmus, and its impact on fertility has not been evaluated in goats. Our objectives were to determine the differences among spermatozoa of different bucks in their ability to penetrate homologous cervical mucus in vitro and to determine the relationship between sperm displacement through cervical mucus and the ability of spermatozoa to colonize the oviduct and penetrate IVM oocytes, in vivo. Sperm migration in cervical mucus was assessed in flat capillary tubes with a phase contrast microscope. In the first experiment, fresh semen was used to establish differences between males in the ability of their spermatozoa to migrate in cervical mucus. In the second experiment, goats in estrus were inseminated with fresh spermatozoa from males with significant differences in mucus migration ability, and sperm numbers were evaluated at the UTJ. In the third experiment, the fertilization efficiency of IVM oocytes transferred to the oviduct of estrous females inseminated with semen from the same males as earlier, was used to assess the relationship between the mucus migration test and the in vivo fertilization performance of their spermatozoa. Spermatozoa from different males varies significantly in sperm migration efficiency in cervical mucus (15.5a +/- 1.2; 14.9a +/- 1.4; 17.5ab +/- 1.2; 17.0ab +/- 1.5; 19.7b +/- 1.2; 20.1b +/- 1.4 mm; media +/- S.E.M. for males A-F, respectively, P < 0.05). Spermatozoa from males with different mucus migration efficiency values produced different sperm populations at the oviduct reservoir of inseminated females (1,233 +/- 92.3 versus 28.8 +/- 17.0 spermatozoa of males with high and low relative migration efficiency, respectively, P < 0.02). Spermatozoa from males with different mucus migration efficiency values have different fertilization rates of IVM oocytes transferred to oviduct (47/96 (49.0%) versus 25/91 (27.5%) for males with high and low relative migration efficiency, respectively, P < 0.05). Cumulative results suggest that sperm migration in cervical mucus is related to the ability of spermatozoa to colonize the oviduct and to fertilize matured oocytes in vivo.     

Chromosomal abnormalities and the morphology of mouse sperm heads.

In the mouse, numerous mutagens, teratogens and carcinogens have been shown to induce marked elevations in the fraction of sperm with head shape abnormalities. Since carcinogens and teratogens may act by causing genetic damage, a likely explanation of these results is that the sperm abnormalities are also caused by genetic damage. There are two more or less distinct classes of genetic damage, chromosomal aberrations and point mutations. In this paper, we provide evidence, that in general, chromosomal aberrations are not responsible for causing abnormally shaped sperm. Chromosomal aberrations could have caused abnormal sperm morphology in a number of ways. One possibility was that the mere presence of a translocated chromosome within the germ cell led to the malformation of the sperm head. A second possibility was that chromosomal imbalance, i.e., aneuploidy, duplications or deficiencies, within the spermatid or haploid cells caused abnormalities in shape. We tested these hypotheses by measuring the level of abnormally shaped sperm in mice homozygous and heterozygous for 24 various reciprocal and Robertsonian translocations. The diploid cells of these mice are known to be chromosomally balanced, containing translocated chromosomes. A predictable proportion of their gametes are, however, chromosomally unbalanced and carry translocated chromosomes. It was found that the levels of sperm abnormalities in these mice were convincingly unrelated to the levels predicted by any of the above hypotheses. Based on these results it seems that sperm abnormalities in mice are not due to the mere presence of translocated chromosomes in germ cells and also not due to chromosomal aneuploidy or duplication-deficiencies of chromosomal segments in the spermatid during development of the sperm.     

Quantitative fluorometry of abnormal mouse sperm nuclei.

An extensive quantitative analysis of deformed mouse spermatozoa was undertaken. Improvements over previous studies included the isolation and purification of sperm nuclei, a multifaceted analytical approach using several fluorochromes and the analysis of individual nuclei classified into shape categories. Malformed sperm nuclei in BALB/c mice could not be distinguished from normal ones in terms of total and basic proteins, sulfhydryl and disulfide group concentration, DNA concentration and chromatin organization. The shape of sperm nuclei is therefore probably determined by the manner in which the internal biochemical components are assembled.     

Early developing embryos affect the gene expression patterns in the mouse oviduct

Fertilization and development of mouse embryos occur in the ampullae of oviduct. We hypothesize that fetal-maternal communication exists in the preimplantation period, allowing optimal development of embryos. It is known that embryotrophic factors from oviduct affect the development of embryos. Although embryos affect their own transport in the oviduct, the mechanism of action is unknown. As a step toward understanding the action of embryos on oviductal physiology, we adopted suppression subtractive hybridization (SSH) to compare the gene expression in the mouse oviduct containing early embryos with that of oviduct containing oocytes. Ten to twelve 1-cell mouse embryos were transferred to one oviduct of a foster mother and similar number of oocytes were transferred to the contralateral oviduct. The animals were sacrificed after 48 h and their oviducts were excised for mRNA study. Using SSH, we screened out 250 putative positive clones from the subtracted embryo-containing oviduct library and 97 of them were screened positive by reverse dot-blot analysis. DNA sequence analysis identified genes that shared high homology with sequences in GenBank/EMBL database with unknown functions. Overall, 13 of the 90 high-quality sequences (14%) were homologous to 6 different genes previously described. Reverse Northern analysis confirmed that the expression of these genes were higher in the embryo-containing oviduct than in the oocyte-containing oviduct. About 12% of these clones (11/90) were novel. This article is the first to report identification of genes in the oviduct that are upregulated in the presence of embryos during the preimplantation period.