Mannose-binding molecules of rat spermatozoa and sperm-egg interaction

We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.     

Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated.Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks.In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age.In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.     

Assessment of sperm functional competence and sperm-egg interaction

A precise understanding in the functional competence of mammalian sperm is essential to generate clinical advances for the treatment of infertility and novel contraceptive strategies. The fundamental knowledge on the controlling parameters for spermatozoal activation process will help in the identifying the causes in fertilization failure due to male factor as well as in developing male contraceptive methodologies. The defects in the sperm-egg interaction seem to be one of the controlling mechanisms, however, none of the presently available methods for the evaluation of the fertilizing ability of sperm precisely indicates the reason for the failure or the success of sperm entry into egg. Adequate number of motile spermatozoa with normal morphology and timely occurrence of acrosome reaction are presumably the major prerequisites for the penetration through the egg investments. The present communication briefly reviews some of the main features of mammalian sperm which control the success or the failure of fertilization and existing clinical methods indicating the lack of fundamental knowledge on the sub-cellular and molecular aspects of this unique and species-specific cell-cell interaction.     

Comet assay of fresh and cryopreserved bull spermatozoa

In this study, we describe DNA fragmentation of fresh and cryopreserved bull spermatozoa using the comet assay. Cryopreservation caused a significant but low (3.8%) decrease in the percentage of DNA in the comet head and an increase (5.3%) in the tail length. Our results suggest that in addition to motility and viability, low levels of DNA fragmentation after cryopreservation is a characteristic of bull spermatozoa and can be a part of remarkable cryoresistance of bull spermatozoa.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Functional changes of mouse spermatozoa stored in vitro

In suspensions of epididymal spermatozoa in vitro at +10°C and +37°C, all nuclei-containing and mitochondria-containing structures (normal spermatozoa, spermatozoa with the bent and coiled tails, complexes of head and neck) are with propidium iodide and rhodamine 123, respectively. Intracellular ATP concentration determined by a bioluminescent method in mitochondria-containing elements of suspension decreases (significantly faster at 37°C than at 10°C) up to a certain unchangeable level (2.5 × 10^?8 M/l at 37°C and to 1.6 × 10^?8 M/l at 10°C per 10⁶ of mitochondria-containing elements). Mechanisms of spermatozoa destruction are discussed.     

Mouse sperm-egg interaction in vitro in the presence of neem oil

In vitro evidence is presented showing toxicity of neem oil on sperm-egg interaction in mouse. Cumulus oophorus-enclosed ova, inseminated with capacitated spermatozoa, were cultured in 1 ml of in vitro fertilization (IVF) medium and overlayered by 1 ml of different concentrations of neem oil (1, 5, 10, 25, 50 and 100%) for IVF duration of 4h. At the end of incubation, ova were allowed to grow in neem oil-free culture medium and assessed for fertilization, first cleavage (2-cell formation) and blastocyst formation in vitro at 4–14h, 24h and 108h post-insemination respectively. The study showed that the presence of neem oil at concentrations of 10, 25 and 50% caused inhibition of IVF in a dose-dependent manner. The toxic effect of exposure of 25 and 50% neem oil was further carried over to the first cleavage of the resulting fertilized ova and the toxic effect of 5, 10, 25, and 50% was carried over to the blastocyst formation from the resulting fertilized ova when grown in neem-oil free culture medium. A total of 94.1% inhibition of 2-cell formation and 100% inhibition of blastocyst formation from the inseminated ova was observed in 50 and 25% neem oil-treated groups respectively. Neem oil at 100% concentration caused 100% degeneration of ova at 1h of sperm-ova coculture. The study showed a direct toxic effect of neem oil on sperm-egg interaction in vitro and encourages research investigations of this herbal product as a pre-coital contraceptive.     

Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress fluorescent staining and flow cytometry

A study was conducted to evaluate the integrity of turkey sperm plasma membrane subjected to various hypo-osmotic conditions, and to develop a test to determine the percentage of viable spermatozoa capable of withstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial was repeated 6 times. For Trial I, spermatozoa were subjected to varying osmotic solutions by suspension in 100, 80, 60, 40, 20 or 0% PBS in distilled water (297 to 19 mosm/kg H₂O) and stained to assess membrane integrity with Calcein-AM (CAL) and propidium iodide (PI). The CAL detected viable spermatozoa (green fluorescence) while the PI stained dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determined by flow cytometry, was not different from that in 100% PBS (76.4 ± 3.8) to 20% PBS (74.1 ± 3.5). Fewer viable spermatozoa, however, were detected in 0% PBS (61.1 ± 4.8, P < 0.05). The percentages of swollen tails observed for viable (green stained) spermatozoa were 0, 4.5, 6.5, 24.3, 50.5 and 100% for 100, 80, 60, 40, 20 and 0% PBS, respectively. Semen was also evaluated fresh or after 24 h in vitro storage at 5 °C in PBS or H₂O (Trial II). The percentage of viable spermatozoa was not different for fresh or in vitro-stored spermatozoa in PBS. For spermatozoa stored 24 h in vitro, the percentage of viable cells was lower in H₂O (48.0 ± 5.1) than in PBS (66.1 ± 5.6, P < 0.05). Subjecting in vitro-stored sperm cells to hypo-osmotic stress before fluorescent staining resulted in detection of labile spermatozoa not accounted for by staining alone, indicating that the turkey sperm membrane is more susceptible to damage after cold storage.     

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Atlhough isolated IPVL can be stored for up to 24 h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean ± SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0 ± 17.7 holes/mm² IPVL and 159.5 ± 17.7 holes/mm² IPVL, respectively, P > 0.05; n = 123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.     

Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa

Prostatic fluid is unsuitable for preserving dog semen at 4 degrees C and exerts harmful effects upon the spermatozoa during the freezing process. Centrifugation immediately after sperm collection is a common method to remove prostatic admixture. In the present study, dog semen, diluted to 25 x 10(6)/ml, was exposed for 5 min to four different centrifugation speeds (180 x g, 720 x g, 1620 x g and 2880 x g) to determine subsequent sperm losses in the supernatant and to assess sperm survival over time. Using 180 x g as centrifugation speed, 8.9% of the sperm cells was lost upon supematant removal. Using 720 x g, 1620 x g or 2880 x g, sperm losses were lower, 2.3, 0.4 and 0.006%, respectively. After centrifugation, the sperm pellet was rediluted in egg-yolk-Tris extender, cooled and stored for 3 days at 4 degrees C. Motility, progressive motility, membrane integrity and sperm morphology were assessed daily. Acrosomal status was assessed after 3 days of storage. The only functional parameter which was influenced by centrifugation speed was membrane integrity as evaluated by means of SYBR14-PI staining: significantly more dead and moribund sperm cells were found after centrifugation at 1620 x g and 2880 x g after 48 and 72 h of storage at 4 degrees C. When higher initial sperm concentrations (50 x 10(6), 75 x 10(6) or 100 x 10(6)/ml) were evaluated for sperm losses, less than 2.3% of the initial total sperm cells was lost at lower centrifugation speeds. We conclude that centrifuging dog sperm for 5 min at 720 x g is the best strategy to remove prostatic fluid because the loss of sperm cells is acceptable and the functional parameters of the spermatozoa are well preserved, even after 3 days of storage.     

Carp ovarian cystatin binds and agglutinates spermatozoa via electrostatic interaction

A sperm-agglutinating factor was purified from ovulated carp eggs and the conditioned medium (CM) of cortical-reacted eggs. It was identified to be the carp ovarian cystatin. Three cystatin isoforms were found. The cystatin isolated from the CM had a higher sperm-agglutinating activity than that isolated from eggs, although the cystatins have identical N-terminal amino acid sequences, masses, and positive charges. Differences in sperm-agglutinating activity between the cystatins of the CM and eggs may be caused by the different conformations because they differed in circular dichroism spectrum and tryptic map. Cystatin was discharged from cortical granules to the perivitelline space after fertilization and is abundant in the perivitelline fluid (PVF) of early stage embryos. Cystatin rapidly agglutinated spermatozoa via an electrostatic interaction. Other basic proteins also agglutinated carp spermatozoa. Their activities were inhibited by salt and high pH. Cystatin bound to the entire surface of carp spermatozoa. The PVF of early embryos agglutinated carp spermatozoa. The activity was related to the cystatin content and influenced by ionic strength and pH. Therefore, cystatin is the major sperm-agglutinating factor of PVF. Owing to the rapid action of cystatin on spermatozoa agglutination and the presence of a high concentration of cystatin in PVF, cystatin is considered important for preventing polyspermy in carp eggs.     

The role of carbohydrates in sperm-egg interaction in rats

The first step in fertilization is the interaction of the capacitated sperm with the zona pellucida. It has been proposed that the initial interaction, as in other types of cell adherence, is due to complementary interacting sites on the opposing surfaces of the gametes. This work intended to investigate the role of carbohydrates in sperm-egg binding in rats. Ejaculated sperm was collected from uterine horns of mated females. The sperm was suspended in Rat Fertilization Medium at final concentrations of 3-7 times 10(5) sperm/ml. After 5 1/2 h of sperm incubation, eggs were added to the sperm suspensions concomitantly with various carbohydrates to achieve a final concentration of up to 50 mM. The eggs were separated after 30 min, and the number of sperm bound to the zona pellucida was counted. Among a variety of monosaccharides tested at 50 mM concentration, it was found that alpha-methyl-mannoside was the most potent inhibitor (producing 80% inhibition); less potent was D-mannose and even less, L-fucose. A combination of alpha-methyl-mannoside and L-fucose showed a synergistic effect. Mannan was not more effective as an inhibitor than the monosugar mannose, while fucoidin was extremely potent, causing over 90% inhibition of binding at 0.1%. We assume the presence of macromolecules containing sugars on the zona pellucida because inhibition of sperm binding to this layer was observed: a) after preincubation of mannan or fucoidin with sperm, but not with the eggs; and b) after pretreatment of the egg with specific enzymes. The results obtained in this study in the rat are consistent with the hypothesis that carbohydrates are critical for the sperm-egg interaction.     

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing–thawing.     

Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C

The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.     

Assessment of goat semen freezability according to the spermatozoa characteristics from fresh and frozen samples

A total of 110 ejaculates were assessed in order to determine the influence of the physical parameters of goat semen on post-thaw motility and acrosome integrity. Sperm ejaculate characteristics, sperm motility, morphology and acrosome integrity were assessed in fresh and frozen samples by the sperm class analyzer (SCA) and Spermac staining technique. A decrease in acrosome integrity and sperm motility was found after thawing (P<0.01). Six semen parameters assessed before freezing were identified as predictors of sperm freezability (P<0.01). The percentage of morphological abnormalities (R=0.856) and motile sperm cells (R=0.655) in fresh semen are the best predictors to know the total post-thaw variability parameters.     

Effects of dietary maize solubles and minerals on albumen quality of fresh and stored brown-shelled eggs

Two hundred and ten Warren SSL laying hens (producing brown eggs) were used to test the effects on Haugh Units (HU) and on egg-white foaming capacity of seven dietary treatments: control (maize, wheat, soya bean meal); 25 and 100 g kg^?1 of maize solubles (DDGS); chromium (10 mg kg^?1); chromium + zinc (10 + 1000 mg kg^?1); magnesium (4 g kg^?1); and current trace mineral supplementation. Several chemical parameters of egg-white (proteins, lysozyme, sialic acid as an indicator of ovomucin, Ca, Mg, Na and K levels) were also recorded. These analyses were performed on frozen albumen from fresh (1-day) and stored (21-day) eggs.Egg production, feed consumption and egg weight were reduced only by Mg supplementation. The HU value of fresh eggs also remained constant except in the case of Cr + Zn supplementation, which caused a small decrease.The foaming capacity of egg white was hardly affected by the dietary treatments; a slight fall in foam stability occurred only after adding trace minerals. Egg storage caused a lightening of foam and increased its stability.Lysozyme activity was decreased and the sodium content of the egg white increased by the lowest level of DDGS, Cr and Mg treatments. The Ca and Mg contents of egg-white dry matter decreased during egg storage.These results are discussed in relation to the dietary use of DDGS or Mg supplementation, the effects of egg storage and the interrelationships between the composition and the functional properties of egg-white.