The transport of morphologically abnormal sperm in the female reproductive tract of mice

Mice of the PL/J strain exhibit a high percentage of morphologically abnormal sperm and provide a model for studying the function of abnormal sperm. The ability of such sperm to reach the site of fertilization within the female reproductive tract has been investigated. We have found a decrease in the percentage of structurally abnormal sperm within the population that reaches the oviduct. This observation suggests either that there is an active selection against abnormal sperm or that they are physiologically disadvantaged in reaching the site of fertilization.     

The onset and maintenance of hyperactivated motility of spermatozoa from the mouse

Estimates were made of the proportion of freely motile mouse spermatozoa displaying hyperactivated motility by an objective photographic method employing stroboscopic illumination under dark-field conditions and examining displacements of the sperm head and bend angles of the sperm tail. In media known to support in vitro fertilisation hyperactivation gradually appeared reaching about 40% by 6 hr incubation, and it was not promoted by 2 mM caffeine or 0.1 mM Bt₂ cAMP or washing the cells free of epididymal fluid. Raising the osmolarity of the medium to 400 mOSM with electrolytes, but not nonelectrolytes, did promote hyperactivation (60% by 2 hr) suggesting that the ionic strength of the medium was important. Hyperactivation in high ionic strength media could be prevented by removing or chelating Ca²⁺, or replacing Ca²⁺ with Ba²⁺ or Mg²⁺, when nonhyperactivated motility was maintained, but Sr²⁺, like Ca²⁺, permitted hyperactivated motility. Hyperactivation in low ionic strength medium could be promoted by the ionophore A23187, suggesting that Ca²⁺ movement into the cells is important. Of a range of glycolytic substrates tested supporting nonhyperactivated motility in the presence of lactate, only glucose supported hyperactivation. Addition to glucose— or Ca²⁺ — free, high ionic strength media after 2 hr increased hyperactivation immediately (glucose) or after a lag of 2 hr (Ca²⁺) suggesting that glucose acts on a Ca²⁺ — primed system. Removal from high ionic strength medium, chelation of Ca²⁺ or inhibition of glucose metabolism did not prevent hyperactivation continuing once it had been initiated, indicating different requirements for initiation and maintenance of this form of motility.     

Initiation,prolongation, and reactivation of the motility of salmonid spermatozoa

The motility of salmonid spermatozoa initiated by dilution of the milt with ovarian fluid or isotonic saline is brief duration; it was believed that it can be activated only once in the life of the spermatozoon. Dilution of the milt with an equal volume of isotonic saline (0.12 M-NaCl) containing 5 mM-3-isobutyl-1-methylxanthine (MIX) prolonged and intensified sperm motiliy. When motility had stopped after initial mobilization with saline or ovarian fluid, it could be reactivated by addition of MIX; reactivated spermatozoa fertilized eggs. Dilution with saline containing K⁺ (24 mEq/liter) did not initiate sperm motility even in the presence of MIX. The spermatozoa were mobilized by subsequent with 0.12 M-NaCl. The concentration of adenosine triphosphate (ATP) in sperm suspensions dropped on dilution with saline and rose as motility ceased, but declined without subsequent recovery following dilution with MIX-saline. The concentration of cyclic adenosine monophosphate (cAMP) rose and fell sharply on initiation of motility and rose again after motility had declined. While salmonid spermatozoa can be mobilized by dilition with saline alone, the effectiveness of MIX in reactivating “spent” spermatozoa supports the assumption that cAMP plays a role in the initiation of sperm motility.     

Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes

The contents of the oviducts of ewes were recovered by flushing with small volumes of culture medium, 22½–24¼ hr after mating. The ampulla was flushed separately from the uterotubal junction and isthmus. Among the motile spermatozoa recovered, a proportion exhibited “hyperactivated” motility, also known as “activated”, or “whiplash” motility. This was characterized by increased flexion of the neck, increased amplitude of the flagellar waves, and marked asymmetry of beat. Two types of hyperactivation appeared: in the first, spermatozoa swam in a repetitive, nonprogressive circling pattern and appeared to have intact acrosome caps; in the second, the spermatozoa showed a propensity to stick to glass by the equatorial segment and most had modified or missing acrosome caps. The proportions of motile spermatozoa exhibiting hyperactivation were greatest in the ampullae, as were the proportions with modified or absent acrosomes. Hyperactivation is a capacitation-associated phenomenon that has now been reported for one or more species from seven orders of eutherian mammals. It may well be a universal aspect of the prefertilization behavior of mammalian spermatozoa and is probably of advantage to the fertilizing spermatozoon within the oviduct.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Quantitative comparison of the passage of homologous and heterologous spermatozoa through the uterotubal junction of the golden hamster

A quantitative method was used to determine whether the spermatozoa of foreign species could pass through the uterotubal junction (UTJ) of the hamster as efficiently as homologous (hamster) spermatozoa. Estrous female hamsters were artificially inseminated with epididymal spermatozoa of homologous and heterologous (foreign) species. The number and distribution of spermatozoa in the oviduct were determined several hours after insemination (shortly before ovulation). The passage of immotile (dead) hamster spermatozoa through the UTJ was also examined. It was found that the spermatozoa of all foreign species tested (rat, mouse, guinea pig, and rabbit), as well as immotile hamster spermatozoa, could pass through the UTJ but did so in much smaller numbers compared to live hamster spermatozoa. This was not specifically due to poor survival of foreign spermatozoa in the hamster uterus, as the viability of all inseminated spermatozoa (including hamster spermatozoa) was considerably reduced by 1 h after insemination. While a large number of live hamster spermatozoa were distributed throughout the caudal isthmus at the time of examination, none or only a very few foreign spermatozoa had advanced this far. The few foreign and immotile spermatozoa that reached the caudal isthmus were confined to the first ascending loop of this segment. Some possible causes for the small number and retarded advance of foreign spermatozoa in the hamster oviduct were discussed.     

Targeted disruption of Slc2a8 (GLUT8) reduces motility and mitochondrial potential of spermatozoa

GLUT8 is a class 3 sugar transport facilitator which is predominantly expressed in testis and also detected in brain, heart, skeletal muscle, adipose tissue, adrenal gland, and liver. Since its physiological function in these tissues is unknown, we generated a Slc2a8 null mouse and characterized its phenotype. Slc2a8 knockout mice appeared healthy and exhibited normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role for maintenance of whole body glucose homeostasis. However, analysis of the offspring distribution of heterozygous mating indicated a lower number of Slc2a8 knockout offspring (30.5:47.3:22.1%, Slc2a8^+/+, Slc2a8^+/?, and Slc2a8^?/? mice, respectively) resulting in a deviation (p=0.0024) from the expected Mendelian distribution. This difference was associated with lower ATP levels, a reduced mitochondrial membrane potential and a significant reduction of sperm motility of the Slc2a8 knockout in comparison to wild-type spermatozoa. In contrast, number and survival rate of spermatozoa were not altered. These data indicate that GLUT8 plays an important role in the energy metabolism of sperm cells.     

Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility

The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondiaidehyde dctermint-d by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 10⁸ spermatozoa after 1 hour of incubation) and 1) initial motility r = ?0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.     

Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments

Mechanisms of mammalian sperm migration through the female reproductive tract and ovum vestments are described. The perspective is biophysical as well as biochemical and morphological, and the focus is upon the role of sperm motility in these processes. Sperm forward progression is characterized as an interactive process between the the cell and its environment, and the mediation of flagellar bend propagation by the physical properties of its surroundings is described. These properties, together with flagellar beat kinematics, sperm morphology, and surface properties, determine the magnitude of the forces generated by sperm and their consequent rate of progression. Sperm interactions with the cervical mucus, the cumulus oophorus, and the zona pellucida are described. The poorly understood affinity of the sperm surface for the macromolecules of the mucus, cumulus, and zona is stressed, as is the viscoelastic structural mechanical resistance of these biopolymers to sperm motion. The kinematics and consequences of hyperactivated sperm motion are presented, with emphasis on objective characterization of such motion (as a biomarker), along with analysis of the mechanical advantage that such motion may confer on spermatozoa during egg–vestment interaction.     

A scanning electron microscopic study of rabbit spermatozoa in the female reproductive tract following coitus

The surface morphology of rabbit spermatozoa, fixed in situ (female reproductive tract) and prepared for scanning electron microscopy by critical point drying, was studied for as many as 36 hours post coitum. The findings demonstrate that 1) spermatozoa in the reproductive tract following coitus exist as a heterogenous, morphological population and 2) with time, shifts within this population from one predominant morphology to another take place. In the fresh ejaculate, most spermatozoa have intact surfaces free of membranous disruptions. With time, a process of labilization (denudation) of the membranes covering the acrosomal region occurs in a progressively larger proportion of spermatozoa. The labilization originates by a process of vesiculation and/or vacuolation and leads to the appearance of a series of small fenestrations of perforations of the surface membranes. The perforations coalesce, and gradually larger areas of the surface membranes are eroded such that by 15 hours post coitum, the outer acrosomal membrane, as well as other acrosomal areas, are to varying degrees, directly exposed to the uterine milieu. Secretory granules, picked up by cilia and transferred to the spermatozoa become localized over the acrosomal region shortly after coitus. The possible significance of these time-dependent morphological events with the phenomena of capacitation and the "true" and "false" acrosome reactions are discussed.     

Effects of transglutaminase substrates and inhibitors on the motility of demembranated reactivated spermatozoa

The effects of transglutaminase (TGase) substrates putrescine, dansylcadaverine, spermine, etc., and the TGase inhibitor cystamine were tested on the motility of demembranated mammalian spermatozoa. These products blocked within a few seconds the motility of demembranated reactivated spermatozoa at concentrations ranging from 0.25 to 5 mM. These minimal inhibitory concentrations could be decreased 5–150-fold when TGase substrates and inhibitor were incubated with demembranated spermatozoa for 15 min prior to the addition of Mg·ATP. The inhibition was reversed by higher concentrations of Mg·ATP but none of these TGase substrates or inhibitor could inhibit bull sperm dynein ATPase. TGase activities, as measured by the incorporation of ³H-putrescine into TCA-precipitable proteins, were present in both sperm Triton-soluble and -insoluble fractions. On the other hand, amine acceptor protein substrates for the TGase-catalyzed reaction were present only in the insoluble fraction. The Triton-soluble TGase was similar to the known “tissue” TGases; the Triton-insoluble TGase activity was calcium independent. The same TGase substrates and inhibitor that blocked the motility of reactivated spermatozoa also blocked TGase activities. Linear relationships were observed between the concentrations of these substances required to block sperm motility and those to block TGase activities. These data suggest the involvement of a TGase activity in sperm motility.     

Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembranated cells

Hamster spermatozoa from the caput and cauda epididymides were demembranated with 0.04% Triton X-100 and reactivated with 1 mM ATP. Motility parameters were analysed by video recording and stroboscopic photography. In the absence of added cAMP, reactivated cauda sperm showed percentage motility and forward swimming patterns similar to those of intact cells, but velocities were lower. When 2 or 20 μM cAMP was present, the velocities were increased but there was no effect on beat frequencies or percentage of forward progressing sperm. Cyclic AMP also markedly increased the percentage of cauda sperm which at first displayed nonprogressive “looping” movement. Addition of cAMP to the reactivation medium greatly improved the otherwise feeble and irregular motility of the demembranated caput sperm by increasing the percentage motility and beat frequencies of nonprogressive cells. It also induced forward motility with beat frequencies and velocities similar to cauda sperm reactivated in the absence of cAMP, but looping was never seen, indicating a change in the flagellar apparatus with maturation. The time required for the exhibition of the cAMP effects was reduced when caput sperm were reactivated in extracts of another previously maximally reactivated caput sperm preparation. The results suggest the production of some potent compound(s) by the axonemes for the manifestation of the cAMP effects.     

Fine structure and motility of spermatozoa and composition of the seminal plasma in the perch

The fine structure and motility of spermatozoa and the composition of the seminal plasma of the perch Perca fluviatilis are investigated by electron microscopy, computer assisted cell motility analysis (CMA) and biochemical methods. The spermatozoon is asymmetrical as the flagellum inserts mediolateral on the nucleus. It lacks an acrosome, has an ovoid head and a small midpiece with one mitochondrion. Sperm motility–initiated in distilled water (10° C)–is characterized as follows: 85·0 ± 2·7% of the spermatozoa are motile, the main swimming type (10 ± 1 s after motility initiation) is the linear motion (61·4 ± 24·4%) and the average swimming velocity is 122·4 ± 21·9 μm s^–1. When motility is initiated with NaCl, glucose or sucrose solutions of 100 mosmol kg^–1 the percentage of motile spermatozoa and the swimming types are similar as in water, but the swimming velocity (174·0 ± 22·3 μm s^–1) is significantly higher. Motility is inhibited by high osmolality of the diluent: when increasing the osmolality of the saline solutions to 350 mosmol kg^–1 sperm motility is totally suppressed while potassium (10–40 mmol 1^–1) does not affect motility parameters. pH optimum for sperm motility is between pH 7·0 and 8·5. The seminal fluid contains 124·01 ± 21·68 mmol 1^–1 sodium, 10·22 ± 1·11 mmol 1^–1 potassium and 0·72 ± 0·26 mmol 1^–1 calcium. pH is 8·25 ± 0·09, and osmolality 283·90 ± 37·19 mosmol kg^–1. The following organic components were determined: monosaccharides (glucose 63 ± 19 μmol 1^–1, fructose 54 ± 28 μmol 1^–1, galactose 59 ± 25 μmol 1^–1), lipids (cholesterol 5·51 ± 6·42 μmol 1^–1, triglycerides 72 ± l00 μmol l^–1, cholesteryloleate 15–150 μmol 1^–1, phosphatidylcholine 26 · 31 μmol 1^–1, glycolipids 1–10 mg 100 m1^–1), lactate 108 ± 99 μmol 1^–1, hydroxybutyrate 102 ± 99 nmol 1^–1, choline 59 ± 159 μmol 1^–1, protein 344·75 ± 59·06 mg 100m1^–1, enzymes (β-d -glucuronidase l.4 ± 0.7 μmol h^–1 100 ml^–1, protease (caseolytic activity) 1·0 ± 0·6 μmol h^–1 100 ml^–1, alkaline phosphatase 2520·0 ± 861·0 μmol h^–1 100 ml^–1, acid phosphatase 44.0 ± 16.0 μmol h^–1 100 ml^–1, glucose-6-phosphate dehydrogenase 38·9 ± 86·9 μmol h^–1 100 ml^–1, lactate dehydrogenase 134·4 ± 69·6 μmol h^–1 100 ml^–1, butyrylcholine esterase 0·014 ± 0·010 μmol h^–1 100 ml^–1, adenosine triphosphatase 562·8 ± 665·4 μmol h ^–1 100 ml^–1).     

Immunolocalization of proacrosin/acrosin in rabbit sperm during acrosome reaction and in spermatozoa recovered from the perivitelline space

The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization—by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin—of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.     

Ecto-cyclic AMP-receptor in goat epididymal intact spermatozoa and its change in activity during forward motility

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8-3H]cyclic AMP. The ecto-cyclic AMP-receptor activity was not due to contamination of broken or "leaky" cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP-receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 microM) of the unlabelled nucleotide. The ecto-receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 X 10(-8) M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 micrograms/ml). Sonication caused a nearly 450% increase of the ecto-receptor activity. The specific activity of the ecto-cyclic AMP-receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the "composite" cells, suggesting that the ecto-receptors may have a role in modulating flagellar motility.     

The effect of anoxia on fowl spermatozoa: Recovery of fertilizing ability,motility, and cellular concentrations of potassium and ATP

Fowl semen when diluted in a glutamate-based medium without glucose, gradually lost its fertilizing ability during 4 hr of anaerobic incubation at 30°C. This incubation regime offered a system by which various in-vitro tests of spermatozoal viability could be assessed for their usefulness as monitors of fertilizing ability. Widely used tests such as spermatozoal enzyme leakage, dye exclusion, and morphology as assessed by light microscopy showed no change in spermatozoal status as the fertilizing ability declined. However the ability of sperm, during a short aerobic incubation to restore their motility and ATP and K⁺ concentrations, declined as did their fertilizing ability. When glucose was added to this re-aeration medium, spermatozoal motility, K⁺ and ATP concentrations, and fertilizing ability were restored to optimal levels. Thus the fertilizing ability of fowl sperm, following anaerobic storage at 30°C, appeared to be related to their ability to restore ATP and K⁺ concentrations and motility. An initial event in the loss of fertilizing ability was a loss in the ability of sperm to oxidise endogenous substrates. This could be restored by the addition of glucose.     

The distribution of diploid rabbit spermatozoa in the female tract after artificial insemination

The distribution of diploid rabbit spermatozoa in the female tract following artificial insemination is generally similar to that after natural mating, except that the incidence of diploid spermatozoa in the female tract is generally greater after A.I. than after coitus, despite a lower proportion of diploids in the semen used for A.I. Therefore the chances of the formation of triploid zygotes due to fertilization of normal eggs by diploid spermatozoa may be increased following A.I., although the frequency of such zygotes would still be very low.