Zona-binding Ability Research Articles

Novel Antioxidant Humanin Analogue Restores Bull Semen Cryosurvivability

The current study was intended to determine the effects of humanin analogue (HNG) supplementation on semen freezability and oxidative stress parameters of cryopreserved bull spermatozoa. A total of 24 semen ejaculates, from Frieswal breeding bulls, were selected for the experiment. Samples with ≥70% progressive motility, ≥500 million spermatozoa/mL, and >2 mL volume were utilized for further processing, supplementation, and cryopreservation. Each sample was divided into four equal groups and the experiment consisted of three treatment groups and one control group. In the control group, the spermatozoa were frozen in an extender without HNG, while in the other three groups, HNG supplementation was done at a concentration of 2, 10, and 20 μM, respectively. The fortification of HNG at a concentration of 10 and 20 µM significantly (p <0.05) improved the percent post-thaw motility and kinetic variables as assessed by CASA, viability, acrosome- and plasma membrane-integrity as compared to 2 µM and control group. The supplementation showed significant (p <0.05) controlled oxidative stress as exhibited by a reduction in lipid peroxidation and reactive oxygen species levels. The supplemented groups also exhibited significant (p <0.05) improvements in total antioxidant levels, increased mean percentage of spermatozoa exhibiting high mitochondrial membrane potential, DNA integrity, acrosome-intact noncapacitated spermatozoa, and zona binding ability. In conclusion, the supplementation of humanin analogue at 10 and 20 µM significantly improved the post-thaw semen quality, functional parameters, and zona binding ability and controlled the oxidative stress in cryopreserved spermatozoa. Thus, the supplementation of its analogue may potentially prove to be a valuable approach for the improvement of semen freezability in crossbred Frieswal bulls.

Does Better Post-Thaw Motility of Dog Sperm Frozen with CLC Mean Better Zona Pellucida Binding Ability?

Even though the search for methods improving cryopreservation of canine spermatozoa led to an improvement of post-thaw quality, fertilizing results after insemination with frozen-thawed semen are still not satisfying. In this study, we focused on modification of spermatozoa membrane fluidity and investigated whether kinematic parameters as assessed by computer-assisted semen analyzer (CASA) can be improved. The primary aim of our study was to investigate whether the use of cholesterol-loaded cyclodextrins (CLC; 0.5 mg, 1 mg, 2 mg) and 2-Hydroxypropyl-ß-cyclodextrin (HBCD; 1 mg) positively influence capacitation status as examined by tyrosinphosphorylation, cholesterol efflux and zona binding assay (ZBA) of spermatozoa. The use of 0.5 mg of CLC increased the percentage of motile, progressive and rapid spermatozoa compared to the control. Addition of HBCD decreased motility and progressive motility of spermatozoa and the population with rapid movement in comparison to the control. The percentage of live spermatozoa without efflux of cholesterol compared to the control was increased when extender with 0.5 mg of CLC was used. There was no change in capacitation status. The zona binding ability of spermatozoa was significantly lower in the group with 0.5 mg of CLC than in the control. In conclusion, these results suggest that improvement of kinematic parameters does not necessarily coincide with better zona pellucida binding ability of spermatozoa.

Freezability of Dog Semen after Collection in Field Conditions and Cooled Transport.

Simple SummaryDog semen freezing is gaining popularity, but it has to be performed in equipped facilities, which can be far from the place where the stud dog lives. To avoid animal movement, it seemed interesting to investigate whether freezing dog semen after 24 or 48 h of cooled transport to an equipped laboratory was possible when semen collection was performed in the field such as in local breeding kennels. The influence of two pre-freezing holding times (i.e., 24 or 48 h) and two holding diluents (solutions used to dilute semen before freezing) was evaluated. Post-thaw morphofunctional sperm features, such as motility, morphological integrity, and ability to bind female gametes, were assessed. No differences between times or diluents were observed, but motility tended to decrease in the samples frozen at 48 h. Since the insemination dose was based on the number of motile spermatozoa, a shorter pre-freezing time is advisable. Yet, considering that the rest of the morphofunctional parameters remained comparable between samples frozen after collection or after 24/48 h of transport, freezing after cooled transport is a good option for avoiding animal stress and for promoting a greater diffusion of semen cryopreservation.Dog semen freezing is gaining popularity, but it has to be performed in equipped facilities, which can be far from the place where the stud dog lives. The aim of this study was to evaluate whether freezing dog semen after 24 or 48 h of cooled transport to an equipped laboratory was possible when semen collection was performed in the field such as in local breeding kennels. Single ejaculates from different dogs (mixed breeds and ages) were collected. In Experiment I, 10 ejaculates were conventionally frozen using the Uppsala method or frozen after 24 or 48 h of storage in a Styrofoam transport box cooled by icepacks. In Experiment II, 10 ejaculates were used to assess the influence of two extenders (Uppsala chilling extender or freezing extender 1) used for semen dilution during the 24 or 48 h storage. Motility, morphology, membrane, and acrosome integrity were analyzed as well as spermatozoa zona-binding ability. No significant differences were observed among the frozen groups, regardless of freezing time (Experiment I) or extender (Experiment II). Motility at thawing, however, decreased in absolute value at 48 h. Freezing of freshly collected semen is the gold standard, but the results obtained in this study prompt the application of freezing after cooled transport for the long-term preservation of dog semen, especially if the transport can be organized in 24 h.

Supplementation of Mito TEMPO and acetovanillone in semen extender improves freezability of buffalo spermatozoa.

Oxidative stress is one of the leading factors responsible for poor post-thaw semen quality because of overproduction of reactive oxygen species (ROS) over neutralizing antioxidants present in semen. Mainly two ROS generation sites are present in spermatozoa, that is, mitochondria and plasma membrane. Therefore, the idea of targeting these specific sites for minimization of ROS production with the compounds having known mechanism of actions was built up as a core for this research. Present study was done to investigate the effects of Mito TEMPO and acetovanillone individually and in combination on freezability of buffalo spermatozoa. For the experiment, semen extender was supplemented with Mito TEMPO (50 μM), acetovanillone (50 μM), and a combination of Mito TEMPO + acetovanillone (50 μM+ 50 μM), designated as Group II, Group III, and Group IV, respectively. Control group without any supplementation was designated as Group I. A total of 24 ejaculates with individual progressive motility (IPM) of ≥70% were selected for the study. After final dilution, filling-sealing of straws, equilibration, and freezing were done as per the standard procedure. Semen samples were evaluated for IPM, plasma membrane integrity, lipid peroxidation, total antioxidant capacity (TAC), and cholesterol to phospholipids (C/P) ratio at both fresh and post-thaw stages. Evaluation of ROS, mitochondrial membrane potential (MMP), capacitation status (CTC assay), and in vitro fertility potential were conducted only on frozen-thawed samples. The addition of Mito TEMPO (50 μM) and acetovanillone (50 μM) individually and in combination significantly (p<0.05) improved post-thaw semen quality in terms of IPM, plasma membrane integrity, TAC, cholesterol content, C/P ratio, MMP, Chlortetracycline (CTC)-Full (F) pattern, and zona binding ability of buffalo spermatozoa, while significantly (p<0.05) reduced ROS production, lipid peroxidation, and capacitation like changes as compared to the control group. As Mito TEMPO acts as an SOD mimetic and also detoxifies ferrous iron at the mitochondria level, it aids in neutralization of excessive ROS production and minimizes oxidative stress-related damages that enhances the antioxidant potential of sperm mitochondria. Earlier studies also indicated improved post-thaw semen quality in 50 μM supplemented group. The improvement observed in acetovanillone (50 μM) group might be because of inhibition of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as this enzyme activation by various physical/chemical inducers during cryopreservation process leads to activation of CatSper channel resulting in calcium influx, premature capacitation, and acrosomal reaction like changes through activation of adenylate cyclase and cAMP/PKA-mediated tyrosine phosphorylation of sperm proteins. Acetovanillone also prevents NADPH oxidase-mediated inhibition of glutathione reductase activity, which has a vital role in protecting the structural and functional integrity of sperm plasma membrane. Results indicated beneficial effects of supplementation of Mito TEMPO and acetovanillone on sperm freezability and individual supplementation was as efficient as the combination group for sustaining post-thaw semen quality.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p<0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Using egg yolk in a TRIS-Equex STM paste extender for freezing of dog semen is superior to egg yolk plasma, also after addition of lecithin and catalase

We compared the results of using egg yolk plasma (EYP) instead of egg yolk (EY) in a TRIS-based Equex STM Paste freezing extender system for dog semen [25]. We also tested whether the addition of lecithin and catalase to the EYP extenders would improve results. Fractionated semen collection was done in 17 stud dogs and the sperm rich fraction diluted with different extenders in 2 steps: (I) TRIS-fructose-citric acid extender (TRIS) containing 20% egg yolk (EY) and 3% glycerol [25], (II) TRIS containing 20% egg yolk plasma (EYP) and 3% glycerol, and (III) TRIS containing 20% EYP and 0.8% lecithin (EYP-L) and 3% glycerol. After equilibration the second dilution step was done: samples with (I) were diluted with TRIS-EY with 7% glycerol and 1% Equex STM paste [25]; samples with (II) and (III) were divided in 2 aliquots each, and one part diluted with TRIS-EYP or TRIS-EYP-L, both containing 7% glycerol and 1% Equex STM paste, and the other one part with the same extenders containing additionally 300 I.U./mL catalase. After freezing and thawing, samples were analyzed by CASA and sperm chromatin structure assay (SCSA); reactive oxygen species (ROS), degree of apoptosis and zona binding ability were determined. Semen samples with TRIS-EY with a final concentration of 5% glycerol and 0.5% Equex STM paste [25] showed best post thaw progressive motility (P), most intact cells, lowest percentage of ROS, acrosome damages, dead and apoptotic cells. Curvilinear velocity (VCL), DNA fragmentation, morphological abnormalities and zona binding ability did not differ between groups. Replacement of egg yolk by EYP increased the ROS and late apoptotic cells. Addition of lecithin and catalase to EYP containing extenders decreased motility and increased complete apoptosis. We conclude that egg yolk is superior to EYP in the here investigated extenders. The TRIS-based extender [25] with EYP could not be improved by addition of lecithin and catalase; however, in-vivo fertilization capacity of the here examined extenders remains to be investigated.

Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes’ zona pellucida

Dog sperm cryopreservation is gaining importance both in breeding dogs for commercial purposes and for pet animals. Anyway, cryopreservation of mammalian spermatozoa, including dog ones, induces some negative effect on sperm fertility, leading to a lower use of this technique and limiting its widespread use. Therefore, studies to improve the quality of canine semen after cryopreservation could have a relevant impact on both the scientific advancement and the clinical practice. The aim of the present work was to investigate the putative ameliorative effect of Epigallochatechin-3-gallate (EGCG) addition to post thawing medium on dog sperm motility, mitochondrial activity, acrosome integrity and on zona-binding ability (zona binding assay). Spermatozoa were thawed in Tris-fructose-citrate medium supplemented with EGCG (0, 25 and 50 μM) and sperm motility, mitochondrial activity and acrosome integrity were assayed at 0.5, 1.5, 3 and 6 h after post thawing incubation at 37 °C. An aliquot of semen from each treatment group after 1.5 h post thawing incubation was washed and used to perform heterologous (using porcine oocytes) or homologous zona binding assay. The results obtained showed that no significant effect is exerted by EGCG on sperm parameters analysed neither at 0.5, 1.5, 3 or 6 h after thawing excepting for the reduction of the percentage of live cells with active mitochondria at the higher dose at 6 h; furthermore, both homologous or heterologous zona binding ability, was not influenced by EGCG. In conclusion, EGCG supplementation to thawing medium does not improve dog sperm quality or zona binding capacity.

Administration of slow release exogenous melatonin modulates oxidative stress profiles and in vitro fertilizing ability of the cryopreserved mithun (Bos frontalis) spermatozoa

Mithun (Bos frontalis) is a unique domestic free range bovine species of North Eastern hilly regions of India. The present study was designed to assess the seasonal effect of slow release exogenous melatonin (MT) implant on semen quality parameters (SQP) and in vitro zona binding ability (IVZ) of spermatozoa. The experimental animals were divided into Gr I: Control (n = 5) and Gr II: Treatment (n = 5; melatonin implant @ 18mg/50 kg bwt). A total of 20 semen samples/group in winter, spring, autumn and summer seasons (n = 160), twice per week were collected. Following cryopreservation, samples were evaluated for motility parameters (forward progressive, mobility & velocity by computer assisted sperm analyser (CASA), viability, acrosome integrity, plasma membrane and nuclear abnormality, functional status of mitochondria, enzymatic, antioxidant and oxidative profiles, and IVZ. The study revealed significant (p < 0.05) improvement in total motility, viability, acrosome-, plasma membrane-, and nuclear-integrity, and antioxidant profiles; with highest values in spring and lowest in summer season in the fresh semen in Gr II than the Control. A significant (p < 0.05) improvement in motility parameters, membrane potential of mitochondria, antioxidant profiles and reduction in sperm and nuclear abnormalities, leakage of intracellular enzymes and oxidative stress and IVZ index & binding percentage in post-thaw semen samples in melatonin supplemented than in un-supplemented control group was observed. It can be concluded from the study that slow-release melatonin supplementation can be effectively utilized to improve the antioxidant profiles and reduction of oxidative stress, with cascading beneficial effects on semen quality parameters and fertility status of the mithun bull.

Freezability of mithun (Bos frontalis) semen

The present study was conducted to assess the freezability of the mithun semen. Post-thaw physicomorphological parameters (PMPs) such as post-thaw motility, viability, total sperm abnormality, integrity of acrosome, plasma membrane and nucleus and vanguard distance travelled in bovine cervical mucus by sperm (BCMPT) of ejaculates, biochemical attributes such as, aspartate amino transaminase (AST), alanine amino transaminase (ALT) and lactate dehydrogenase (LDH) and in vitro zona binding ability of spermatozoa with heterologous (buffalo) oocyte were studied in freezable and nonfreezable ejaculates. Ejaculates (50) were collected from healthy matured mithun bulls and grouped based on the post-thaw motility into freezable and nonfreezable. These selected ejaculates were extended with standard Tris egg yolk citrate glycerol extender and stored at ultra low temperature (–196°C). Post-thaw PMPs, binding index (BI) and binding % (BP) and biochemical profiles were examined with standard protocols. The results revealed that the PMPs, biochemical profiles, BI and BP differed significantly between freezable and nonfreezable ejaculates. The post-thaw motility was positively correlated with livability, intactness of acrosome, plasma membrane, nucleus and vanguard distance travelled by sperm and negatively correlated with post-thaw total sperm abnormality. It was concluded that the post-thaw PMPs and BI and BP were significantly higher, biochemical intracellular enzymatic profiles were significantly lower in freezable than in nonfreezable ejaculates indicates freezable sperm has functionally stable sperm structures and higher intactness of plasma and acrosomal membrane to motile faster and in forward direction to reach the fertilization site and successful conception.

Nuclear DNA fragmentation negatively affects zona binding competence of Y bearing mouse spermatozoa

To investigate the influence of sperm DNA integrity on the zona binding ability of mouse spermatozoa in relation to their sex chromosomal constitution. In this prospective experimental study, the sperm DNA fragmentation was induced by exposing testicular area of Swiss Albino mice (Mus musculus) to different doses of γ-radiation (0, 2.5, 5.0 and 10.0Gy). Sperm DNA fragmentation was quantified by single cell gel electrophoresis (comet assay). In vitro sperm zona binding assay was performed and the numbers of zona bound X and Y bearing spermatozoa were determined using fluorescence in situ hybridization (FISH). The assessment of zona pellucida bound X and Y-bearing spermatozoa using fluorescence in situ hybridization has revealed a unique binding pattern. The number of zona bound Y-spermatozoa declined significantly (P < 0.01 to 0.0001) with increase in the DNA damage. The skewed binding pattern of X and Y-bearing sperm was strongly correlated with the extent of sperm DNA damage. The zona pellucida may have a role in preventing DNA damaged mouse sperm binding especially towards Y-bearing sperm. However, the exact mechanism behind this observation needs to be elucidated further.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.

A Case Report Concerning Male Gametes Rescued from a Siamese Eld’s Deer (&lt;i&gt;Rucervus eldii siamensis&lt;/i&gt;): Post-Thawed Testicular and Epididymal Sperm Quality and Heterologous Zona Pellucida Binding Ability

In the present study, the quality of frozen-thawed epididymal and testicular sperm recovered from a Siamese Eld's deer was examined. The epididymal sperm quality was assessed in fresh, cold-stored at 4°C and frozen-thawed samples. Zona binding ability of the frozen-thawed epididymal samples with Burmese Eld's deer oocytes was also evaluated. Testicular sperm extracted from tissues frozen at -80 or -196°C for one month were examined for membrane and DNA integrity. Epididymal sperm retained their quality for up to 24 hr of cold storage at 4°C. The percentages of sperm motility, intact membrane, intact acrosome and intact DNA were 30, 46.5, 27 and 89.5% in the frozen and thawed epididymal sperm, and the average ability to bind with oocytes was 92.5 ± 64 sperm/oocytes. Around 70% of the sperm extracted from testicular tissues cryopreserved at -196 and -80°C for one month showed an intact membrane. In conclusion, epididymal and testicular sperm survived for more than 13 hr post-mortem. Furthermore, cold storage at 4°C and cryopreservation at -196 and -80°C maintain the quality of epididymal and testicular sperm. This study represents a model for male gamete rescue in endangered Eld's deer.

Mechanisms of Fertilization—A View From the Study of Gene‐Manipulated Mice

Recent gene knockout approaches have revealed that many of the factors previously considered to be important were largely dispensable in gene-knockout animals, and that previously unknown factors are emerging. Unexpectedly, sperm from 5 different gene-disrupted mouse lines (calmegin (Clgn), Adam1a, Adam2, Adam3, and Ace) all have defective zona-binding ability and oviduct-migrating ability. We have disrupted a new testis-specific molecular chaperone, calsperin (Calr3), which became the sixth gene sharing the same infertile phenotype. The relationships among these 6 factors are discussed. After zona penetration, sperm needs to fuse with eggs. We reported that sperm require IZUMO1 and eggs require CD9 for sperm-egg fusion. However, the distribution of IZUMO1 is not limited to the equatorial segment where it is believed the fusion takes place. Therefore, we produced a mouse line that lacks an equatorial segment-specific antigen, Spesp1. The Spesp1 +/- and -/- sperm showed a decreased fusing ability compared with wild-type sperm, but the cause of the impaired fusion may not directly relate to the mechanism involving IZUMO1. In order to study the mechanism of fertilization, the visualization of sperm in vivo provides a powerful tool. We made a new transgenic mouse line that produces sperm with green fluorescent protein-tagged acrosome and DSRed2-tagged mitochondria. Studies of fertilization using gene-manipulated animals are described in the present review.

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.

Association between the extent of DNA damage in the spermatozoa, fertilization and developmental competence in preimplantation stage embryos

To examine the fertilizing ability and DNA damage response of preimplantation stage embryos derived from the γ-irradiated mouse sperm carrying varying amounts of DNA strand-breaks. The DNA damage in the sperm was induced by exposing the testicular area to different doses of γ-radiation. After mating with healthy female mice, sperm zona binding, fertilizing ability of DNA damaged sperm and developmental competence of embryos derived from the DNA damaged sperm were assessed. The in vivo zona binding ability and fertilizing ability of DNA damaged sperm was significantly affected in the 5.0 and 10.0 Gy sperm irradiation groups. Although the development of the embryos derived from the DNA damaged sperm was not significantly affected until day 2.5 post-coitus, further development was significantly altered, as evidenced by the total cell number in the embryos. The sperm carrying DNA strand breaks still has the ability to fertilize the oocyte normally. However, the events like zona-binding and successful fertilization depend on the extent of sperm DNA fragmentation. The study has also showed a great heterogeneity in embryonic development at peri-implantation period with respect to the degree of sperm DNA damage.

The mechanism of sperm–egg interaction and the involvement of IZUMO1 in fusion

An average human ejaculate contains over 100 million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilization. There might be an ingenious molecular mechanism to ensure that the very best sperm fertilize an egg. However, recent gene disruption experiments in mice have revealed that many factors previously described as important for fertilization are largely dispensable. One could argue that the fertilization mechanism is made robust against gene disruptions. However, this is not likely, as there are already six different gene-disrupted mouse lines (Calmegin, Adam1a, Adam2, Adam3, Ace and Pgap1), all of which result in male sterility. The sperm from these animals are known to have defective zona-binding ability and at the same time lose oviduct-migrating ability. Concerning sperm-zona binding, the widely accepted involvement of sugar moiety on zona pellucida 3 (ZP3) is indicated to be dispensable by gene disruption experiments. Thus, the landscape of the mechanism of fertilization is revolving considerably. In the sperm-egg fusion process, CD9 on egg and IZUMO1 on sperm have emerged as essential factors. This review focuses on the mechanism of fertilization elucidated by gene-manipulated animals.

Gamete Fusion and Sperm Protein IZUMO1

Abstract: An average human ejaculate contains over one hundred million sperm, but only a few succeed in accomplishing the journey to an egg by migration through the female reproductive tract. Among these few sperm, only one participates in fertilizing an egg. Surely there must be an ingenious molecular mechanism to ensure that the very best sperm succeed in fertilization. Recent gene manipulation approaches in mice have revealed that many factors previously described as important for fertilization are largely dispensable; however, novel factors are also emerging. For example, sperm from six different gene-disrupted mouse lines (Calmegin, Adam1a, Adam2, Adam3, Ace and Pgap1) are all known to have defective zona binding ability and at the same time lose oviduct migrating ability. Concerning gamete fusion, we found essential factors, IZUMO1 on sperm and CD9 on the egg, in genedisruption experiments. More recently, the structure, localization, and interacting proteins of IZUMO1 are gradually being elucidated....

Disruption of ADAM3 Impairs the Migration of Sperm into Oviduct in Mouse1

Sperm from four different gene-disrupted mouse lines (calmegin [Clgn], Adam1a, Adam2, and Ace) are known to have defective zona-binding ability. Moreover, it is also reported that the sperm from all of these mouse lines exhibit another common phenotype of impaired migration into oviduct despite the large number of sperm found in uterus after coitus. On the other hand, the sperm from the Adam3-disrupted mouse line was reported to have defects in binding ability to zona, but were able to move into the oviduct. In order to clarify the difference, we investigated the migration of ADAM3-null sperm into oviduct precisely by visualizing the sperm by using acrosin-green fluorescent protein as a tag. As a result, in contrast to previous observations, it was demonstrated that the Adam3-disrupted sperm were unable to migrate into the oviduct after coitus. It was ultimately shown that, in five out of five different gene-disrupted mouse lines, the phenotype of impaired sperm binding to zona pellucida was accompanied by the loss of ability of sperm to migrate into the oviduct. This indicates a close relationship between the two phenomena, and also that sperm migration into the oviduct is a crucial step for fertilization.

Infertility in a Dog due to Proximal Cytoplasmic Droplets in the Ejaculate: Investigation of the Significance for Sperm Functionality In Vitro

A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 x 10(6)), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35-40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5 degrees C and 5% CO(2) in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 microl droplets of CCM were incubated for 1 h. Afterwards, 5 microl of CCM containing 25,000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.

Porcine sperm zona binding ability as an indicator of fertility

The escalated use of artificial insemination in swine has increased the importance of determining fertility of a semen sample before it is used. Multiple laboratory assays have been developed to assess fertilizing potential but they have yielded inconsistent results. This experiment sought to determine the relationship between in vitro competitive zona binding ability and in vivo fertility based on heterospermic inseminations and paternity testing. The zona pellucida binding ability and fertility of sperm from 15 boars was assessed by comparing sperm from one boar with sperm from other individual boars in a pairwise fashion using four ejaculates. The relationship of zona binding ability to the mean number of piglets sired per litter for each boar as well as historic fertility data (litter size and farrowing rate) was assessed. The in vitro competition assay consisted of labeling sperm from each boar of the pair with a different fluorophore and incubating an equal number of sperm from each boar in the same droplet with porcine oocytes. The competitive assay was highly effective in ranking boars by zona binding ability ( R 2 = 0.94). Paternity testing using microsatellite markers was used to determine the mean number of piglets sired per litter for each boar during heterospermic inseminations. The pairwise heterospermic insemination assay was effective in ranking boar fertility ( R 2 = 0.59). Using historical data from these boars, average litter size and farrowing rate were correlated ( r = 0.81, p < 0.001). However, zona binding ability was not significantly correlated with historic farrowing rate data or historic average litter size. Boar sperm zona binding ability was also not correlated significantly with the mean number of piglets sired per litter following heterospermic insemination. But the number of piglets sired by each boar was related to a combination of zona binding ability, sperm motility, normal morphology, acrosomal integrity, and the presence of distal droplets ( R 2 = 0.70). These results suggest that zona binding ability is not an accurate predictor of fertilizing ability when used alone; however, when coupled with other sperm assessments, fertility may be predicted successfully.