Motile Human Spermatozoa Research Articles

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 × 10 8 cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 ± 0.5 × 10 4 to 575.0 ± 56.3 × 10 4 cells/mL; mean ± SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 ± 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 ± 4.8%) than those at 7.5 mm (53.1 ± 6.0%) or further (41.9 ± 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 ± 0.040) than both standard IVF and transient IVF (0.222 ± 0.028 and 0.189 ± 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 ± 2.3%) than in either standard or transient IVF (22.6 ± 1.4 and 33.7 ± 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.

IMSI : indication, résultats et réflexions

The presence of nuclear vacuoles in human sperm decreases pregnancy rates. Intracytoplasmic morphologically selected sperm injection (ISMI) increases pregnancy rate rather than ICSI after real time fine morphology of motile human sperm (MSOME). However, the exact indications of IMSI are on debate.

Identification of diabetes- and obesity-associated proteomic changes in human spermatozoa by difference gel electrophoresis

Difference gel electrophoresis (DIGE) of fluorescently labelled human sperm proteins was used to identify diabetes- and obesity-associated changes of the sperm proteome. Semen samples from type 1 diabetics, non-diabetic obese individuals and a reference group of clinically healthy fertile donors were evaluated in a comparative study. The adaptation of a general protein extraction procedure to the solubilization of proteins from isolated progressively motile human spermatozoa resulted in the detection of approximately 2700 fluorescent protein spots in the DIGE images. Comparison of the patients' sperm proteomes with those of the reference group allowed the identification of 20 spots containing proteins that were present in the sperm lysates at significantly increased or decreased concentrations. In detail, eight of these spots were apparently related to type 1 diabetes while 12 spots were apparently related to obesity. Tryptic digestion of the spot proteins and mass spectrometric analysis of the corresponding peptides identified seven sperm proteins apparently associated with type 1 diabetes and nine sperm proteins apparently associated with obesity, three of which existing in multiple molecular forms. The established proteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in monitoring any fertility-restoring therapy.

Monoklonale Antikörper Ki-Sp II-13 und Ki-Sp VI-2 erkennen Oberflächen-Antigene humaner Spermatozoen/Monoclonal Antibodies Ki-Sp II-13 and VI-2 react with the Surface Membrane of Human Spermatozoa

Immunizing Balb-c mice with washed motile human spermatozoa enabled the production of 2 monoclonal spermatozoal antibodies designated Ki-Sp II-13 and Ki-Sp VI-2. Immunohistochemically Ki-Sp VI-2 reacts only with mature spermatozoa. The monoclonal antibody Ki-Sp II-13 recognizes besides spermatozoa also T- and B-lymphocytes. Electronmicroscopically both antibodies react with the surface membrane of spermatozoa in the area of the head, the neck and the proximal part of the tail. Functional tests show a high sperm-agglutinating and sperm-immobilizing activity, using different test systems for Ki-Sp II-13. Both antibodies proved to be IgG. On immunoprecipitation Ki-Sp II-13 was identified as IgG-2a and Ki-Sp VI-2 as IgG1. Immunoprecipitates of SDS-electrophoretically separated and radiolabeled sperm surface antigens revealed a molecular weight of 18 KD for Ki-Sp II-13 and of 24 KD for Ki-Sp VI-2.

Isolation and storage of progressively motile human sperm.

Human sperm progressively motile and free of seminal debris can routinely be isolated, during a 1.5 hour process, from semen of normal and subfertile men. Serum albumin, in a vertical column, acts to filter sperm based on their swimming abilities of forward progression. The isolated sperm constitute a population that can be inseminated as an aid to male subfertility or, to enhance the chances of sperm survival upon deep-freeze preservation. The latter use resulted in twice the survival rate, based on sperm motility, or random populations of control sperm.

A motile human sperm head fixation method.

In a solution containing less than 5% seminal fluid, human sperm has a tendency to attach its head on the smooth surface of an object. Based on such a phenomenon, a simple method was developed to fix sperm head on the surface of glass slide. This method was demonstrated by studying caffeine effect on the tail movement of human sperm in phosphate buffered saline. By analysing the images recorded on a videocassette, caffeine, a well known stimulator of human sperm motility, was shown to induce an immediate increase of sperm TBF (tail beat frequency) and the amplitude of TBF increase was found to be larger in sperm with lower basal TBF value. This method can provide a flock of head-fixed motile sperm for a precise sperm tail movement assessment or for other purposes.

Acrosome reaction in the head-attached human sperm.

The physiological acrosome reaction of the sperm occurs on the surface of the oocyte. Although its occurrence on the zona pellucida is considered to be a specific response to zona compounds, whether the simple attachment itself could influence the reaction of motile spermatozoa to a fixed surface and promote onset of the reaction is questioned. A motile human sperm head fixation method was developed recently, through which a group of head-attached sperm can be established easily. These head-attached sperm were investigated for their acrosome reactions. After staining by fluorescein iso-thiocyanate conjugated peanut lectin (FITC-PNA), many head-attached spermatozoa (75.7%) were found to have undergone an acrosome reaction.

219 APPLICATION OF A MICROFLUIDIC SPERM SORTER TO THE IN VITRO PRODUCTION OF PORCINE EMBRYOS

In porcine in vitro fertilization (IVF), polyspermy is a persistent obstacle to the efficient production of normal embryos. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) was developed to isolate motile human spermatozoa (from diluted semen by two laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. We previously reported that the monospermy rate was higher when oocytes were co-cultured with isolated spermatozoa in an MFSS for 5 min than when spermatozoa were co-cultured traditionally in drops for 8 h (P &lt; 0.05; Reprod. Fertil. Dev. 20, 187 abst). The present study was undertaken to compare the effect of oocyte location within the MFSS chamber on early development after IVF in the MFSS. A sperm-rich fraction from Berkshire boars was diluted at 1 × 108 cells mL–1 with modified Modena solution containing 20% seminal fluid. In the first experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine (mm199-caf) for 5 min at room temperature. Before flowing, porcine IVM oocytes were positioned in a part of the MFSS chamber where motile spermatozoa would accumulate with mm199-caf. After flowing for 5 min, those oocytes were cultured in caffeine-free mm199 for 8 h and then in a chemically defined medium (PZM-5) for 7 days. In the second experiment, denuded oocytes were co-cultured with isolated spermatozoa at several locations in the MFSS chamber, and the penetration and monospermy rates were estimated. The concentration of motile spermatozoa was also measured at each place in the MFSS chamber after isolation for 5 min. Statistical analyses of results based on 4 to 5 replicates were carried out by ANOVA and Fisher’s PLSD post hoc test (significance, P &lt; 0.05). When IVM oocytes were co-cultured with spermatozoa gradually accumulated in the chamber of the MFSS for 5 min, the cleavage rate (83.7 ± 6.3% of 121 oocytes) was not different from that of control oocytes co-cultured with spermatozoa (5.7 × 105 cells mL–1) in 100-μL drops for 5 min (84.6 ± 6.6% of 126 oocytes). However, the blastocyst formation rate (38.2 ± 3.3%) was higher than for the controls (20.6 ± 6.8%; P &lt; 0.05). After flowing for 5 min, the distance from the inflow opening of the MFSS chamber to the location of the oocytes did not affect the sperm penetration rate, but did affect the monospermy rate (14.0 ± 4.0% of 48 oocytes at the nearest position to 50.0 ± 5.6% of 43 oocytes at the furthest position; P &lt; 0.05). After flowing for 5 min, the concentration of motile spermatozoa was also different at each location (57.5 ± 5.6 × 104 cells mL–1 at the nearest position to 0.8 ± 0.5 × 104 cells mL–1 at the furthest position; P &lt; 0.05). These observations demonstrate that co-culturing oocytes with spermatozoa that gradually accumulated in the MFSS chamber improved the efficiency of blastocyst formation in the pig, whereas efficiency was affected by the position where oocytes were located in the chamber.

How to improve the rate of good quality blastocysts after ICSI in a new approach for classification of motile human spermatozoa

Inclusion criteria for patients which may benefit intracytoplasmic morphologically selected sperm injection (IMSI) are currently under debate. Consequently, it is mandatory to analyse the presence of spermatozoa abnormality defects, not identified in ICSI at 400X, which has a negative impact on the outcome of IVF.

215 APPLICATION OFA MICROFLUIDIC SPERM SORTER TO THE IN VITRO FERTILIZATION OF PORCINE OOCYTES

In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 � 108 cells mL–1 with modified Modena containing 20% seminal fluid, cooled to 15�C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 � 105 cells mL–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 � 105 cells mL–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

To detect the expression of hepatitis B virus (HBV) genes (HB S and C genes) in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization (IVF) technique. Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction (PCR) was used to detect HB S and pre-Core/Core (pre-C/C) coding genes both in one- and two-cell embryos. Reverse transcription-PCR (RT-PCR) analysis was used to study the expression of the two genes. Fluorescence in situ hybridization (FISH) analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Role of the Na+/Ca2+ exchanger in calcium homeostasis and human sperm motility regulation

A number of cell functions, such as flagellar beating, swimming velocity, acrosome reaction, etc., are triggered by a Ca2+ influx across the cell membrane. For appropriate physiological functions, the motile human sperm maintains the intracellular free calcium concentration ([Ca2+]i) at a submicromolar level. The objective of this study was to determine the role of the Na+/Ca2+ exchanger (NCX) in the maintenance of [Ca2+]i in human spermatozoa. Spermatozoa maintained in extracellular medium containing>or=1 microM Ca2+ exhibited motility similar to that of the control. In addition to several calcium transport mechanisms described earlier, we provide evidence that the NCX plays a crucial role in the maintenance of [Ca2+]i. Three chemically unrelated inhibitors of the NCX (bepridil, DCB (3',4'-dichlorobenzamil hydrochloride), and KB-R7943) all blocked human sperm motility in a dose and incubation time dependent manner. The IC50 values for bepridil, DCB, and KB-R7943 were 16.2, 9.8, and 5.3 microM, respectively. The treatment with the above-mentioned blockers resulted in an elevated [Ca2+]i and a decreased [Na+]i. The store-operated calcium channel (SOCC) inhibitor SKF 96365 also blocked the sperm motility (IC50=2.44 microM). The presence of the NCX antigen in the human spermatozoa was proven by flow cytometry, confocal laser scanning microscopy, and immunoblotting techniques. Calcium homeostasis of human spermatozoa is maintained by several transport proteins among which the SOCC and the NCX may play a major role.

Enhancement of Human Sperm Motility by Inclusion of Acetyl-L-carnitine in Processing Media

Various methods are employed in assisted reproductive technologies for separation of highly motile human spermatozoa for insemination. The swim-up method’ is the one of the most commonly used due to its simplicity and cost effectiveness. Unfortunately, the yield of highly motile sperm fraction by this method varies widely, depending on initial sample quality. Research has demonstrated the importance of L-carnitine and acetyl-L-carnitine (ALC) to sperm metabolism and their benefits to sperm development and maturation.

Effect of Cryoprotective Additives-Reduced Glutathione, Acetyl-L-Carnitine on Sperm Membrane Lipid Peroxidation, DNA Integrity and Recovery of Motile Human Sperm

Cryopreservation induces lethal and sub-lethal sperm damage that lead to loss of viability, acrosome loss, DNA fragmentation and poor fertilization. Reactive oxygen species, lipid peroxidation and loss of antioxidant activity such as reduced glutathione (GSH). are considered as mediators of sub-lethal cryodamage to human sperm during cryopreservation. Improvements in semen parameters have been reported by in vivo use of carnitine and acetyl-L-carnitine (ALC), possibly due to its key role in energy metabolism and antioxidant activity.

Optimizing incubation conditions for the preservation of sperm motility in processed semen samples

To determine the optimal conditions to maintain sperm motility and viability for prolonged periods, semen specimens were processed and cultured in different temperature and conditions. We conclude that processing of semen and incubation of sperm in protein-supplemented N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) human tubal fluid medium at room temperature best preserved viability and motility of sperm.

Native specific activity of glutathione peroxidase (GPx‐1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo‐ and hypomotile human sperm samples

Native specific activity of glutathione peroxidase (GPx‐1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo‐ and hypomotile human sperm samples

Percoll Gradient-Centrifuged Capacitated Mouse Sperm Have Increased Fertilizing Ability and Higher Contents of Sulfogalactosylglycerolipid and Docosahexaenoic Acid-Containing Phosphatidylcholine Compared to Washed Capacitated Mouse Sperm1

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.

Detection and expression of hepatitis B virus X gene in one and two‐cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying HBV DNA

The main objectives of this study were to introduce motile human sperm carrying Hepatitis B virus (HBV) DNA to golden hamster oocytes in a co-culture environment and to detect the replication and expression of the HBx gene in early embryonic cells. Zona-free hamster oocytes were inseminated with human sperm carrying pBR322-HBV DNA plasmid using the in vitro fertilization (IVF) technique. Both the one- and two-cell stages of early embryonic development were studied. PCR, RT-PCR, and Dot hybridization were performed to observe the HBx gene and its expression in these stages. "Fluorescence in situ hybridization" (FISH) was carried out to confirm the integration of HBV into the pronucleus, nucleus, and the chromosomes of embryos. The results showed that we have the ability to obtain a fertilization rate of 80%. RT-PCR showed that the HBx gene could be expressed in both one- and two-cell stages of embryonic development. The data suggested the possibility of sperm as a vector for the vertical transmission of HBV DNA to the next generation.

Studies on the membrane integrity of human sperm treated with a new injectable male contraceptive.

The aim of this study was to evaluate the integrity of sperm surface characteristics in the presence of a new male contraceptive, RISUG [1 mg styrene maleic anhydride (SMA)/100 microl dimethylsulphoxide (DMSO) in 1 ml sperm solution]. Progressively motile human sperm were treated in vitro with RISUG. The cells were analysed for the release of 5'-nucleotidase (5'-NT) (a plasma membrane marker) using 3 mmol/l 5'-AMP and 3 mmol/l beta-glycerophosphate as substrates. Hyaluronidase (an acrosomal membrane marker) was analysed using hyaluronic acid as a substrate. The contents of free and total acrosin, and % proacrosin (all acrosome markers) were assayed using 0.5 mmol/l alpha-N-benzoyl-L-arginine ethylester (BAEE). RISUG caused almost complete disintegration of the plasma membrane leading to significant (P < 0.0001) release of 5'-NT into the surrounding media. Complete dissolution of the acrosome with concomitant vesiculation of the membrane system, as judged from the loss of hyaluronidase, was observed. Total acrosin content in the sperm was also reduced to almost 10%, and proacrosin dropped to 13.2% in the presence of RISUG in comparison to 90.2% in control (P < 0.0001), indicating dispersion of acrosomal contents. Under in vitro conditions, RISUG, at a concentration of 1 mg SMA dissolved in 100 microl of DMSO, caused significant damage to the acrosome and its contents, indicating loss of functional ability of sperm.

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media.

The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.