Preincubation Of Sperm Research Articles

Control of sturgeon sperm motility: Antagonism between K+ ions concentration and osmolality

Spermatozoa are stored in a quiescent state in the male reproductive tract and motility is induced in response to various environmental stimuli, such as change of osmolality (general case) and a decrease of extracellular K+ in fish from Acipenseridae family. This study was aimed to investigate the relationship between osmolality and extracellular K+ concentration in controlling sperm motility in sturgeon. Pre-incubation of sturgeon sperm for 5s in hypertonic solutions of glycerol, NaCl, or sucrose (each of 335mOsm/kg osmolality) prepares sturgeon spermatozoa to become fully motile in presence of high concentration of K+ ions (15mM), which has previously been demonstrated to fully repress motility. Furthermore, presence of 0.5mM KCl during the high osmolality pre-incubation exposure completely prevented subsequent spermatozoa activation in a K+-rich media. Manipulating the transport of K+ ions by the presence of K+ ionophore (valinomycin), it was concluded that once an efflux of K+ ions, the precursor of sturgeon sperm motility activation, is taking place, spermatozoa then become insensitive to a large extracellular K+ concentration.

Methyl-β-Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida-Binding Ability of Frozen/Thawed Bovine Spermatozoa.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein-free media. However, the additive capacitating effect of methyl-β-cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen-thawed bovine spermatozoa in a BSA-containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose-dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre-incubation of spermatozoa in MβCD-supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)-binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen-thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.

Rescue &lt;i&gt;In Vitro&lt;/i&gt; Fertilization Method for Legacy Stock of Frozen Mouse Sperm

Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.

Insulin affects sperm capacity in pig through nitric oxide

Insulin (Ins) has recently been demonstrated to have the ability to induce the capacitation process in pig spermatozoa. In various mammalian species, capacitation has been linked to the nitric oxide (NO) signalling; therefore, this study investigated NO production in Ins-treated pig spermatozoa by fluorescence-activated cell sorting. For the same samples, sperm capacitation was evaluated by chlortetracycline staining, protein tyrosine phosphorylation pattern and acrosomal status. A significant increase of the intrasperm NO level and the activation of three capacitation indices were detected in response to Ins treatment. Conversely, sperm preincubation with an NO synthase inhibitor (N-nitro-L-arginine methyl ester) or with the anti-Ins receptor β (IRβ) antibody reversed all of the Ins-related effects. These results suggest that Ins has the capacity to enhance intracellular NO concentrations in pig spermatozoa and indicate a possible NO implication upon Ins promotion of capacitation.

MARCKS Protein Is Phosphorylated and Regulates Calcium Mobilization during Human Acrosomal Exocytosis

Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering PIP2, and that it is phosphorylated during acrosomal exocytosis.

Progesterone utilizes the PI3K-AKT pathway in human spermatozoa to regulate motility and hyperactivation but not acrosome reaction

Progesterone is a physiologic regulator of sperm hyperactivation and acrosome reaction and it does so by activating a range of kinases present in the spermatozoa. In the present study, the involvement of the AKT- phosphatidylinositol 3-kinase (PI3K) signaling pathway in mediating progesterone response in human spermatozoa was investigated. In capacitated spermatozoa, progesterone transiently and concentration dependently lead to phosphorylation of AKT at both Thr 308 and Ser 473 in the tail region. This phosphorylation was inhibited by the PI3K inhibitor wortmannin, suggesting that progesterone leads to activation of PI3K-AKT pathway. The activation of AKT in response to progesterone is calcium dependent and the CatSper channel inhibitor mibefradil significantly reduced progesterone mediated AKT phosphorylation. Preincubation of spermatozoa with wortmannin inhibited the progesterone mediated increase in tyrosine phosphorylation and also attenuated the increase in number of motile, progressively motile and hyperactive spermatozoa but not the number of acrosome reacted spermatozoa. These observations imply that progesterone via CatSper activates the PI3K-AKT pathway required for motility and hyperactivation but not for acrosome reaction.

Mechanisms underlying the inhibition of murine sperm capacitation by the seminal protein, SPINKL

SPINKL, a serine protease inhibitor kazal-type-like protein initially found in mouse seminal vesicle secretions, possesses structurally conserved six-cysteine residues of the kazal-type serine protease inhibitor family. However, it has no inhibitory activity against serine proteases. Previously, it was found to have the ability to suppress murine sperm capacitation in vitro. Herein, we investigated the mechanisms underlying the suppressive effect of SPINKL on sperm capacitation. Three in vitro capacitation-enhancing agents, including bovine serum albumin (BSA), methyl-beta-cyclodextrin (MBCD), and dibutyryl cyclic AMP (dbcAMP), coupled with 3-isobutyl-1-methylxanthine (IBMX), were used to evaluate the influence of SPINKL on capacitation signaling. Preincubation of sperm with SPINKL suppressed BSA- and MBCD-induced sperm capacitation by blocking three upstream signals of capacitation that is the cholesterol efflux from sperm plasma membranes, extracellular calcium ion influx into sperm, and increases in intracellular cAMP. Moreover, SPINKL also inhibited downstream signal transduction of capacitation since it suppressed dbcAMP/IBMX and N(6) -phenyl cAMP (6-Phe-cAMP)-activated cAMP-dependent protein kinase-associated protein tyrosine phosphorylation. Such inhibition is probably mediated by attenuation of SRC tyrosine kinase activity. Furthermore, SPINKL could not reverse capacitation once sperm had been capacitated by capacitation-enhancing agents or capacitated in vivo in the oviduct. SPINKL bound to sperm existed in the uterus but had disappeared from sperm in the oviduct during the sperm's transit through the female reproductive tract. Therefore, SPINKL may serve as an uncapacitation factor in the uterus to prevent sperm from precocious capacitation and the subsequent acrosome reaction and thus preserve the fertilization ability of sperm.

Differential Attachment of Bovine Y Chromosome-Bearing Sperm to the Zona Pellucida

The theoretical rate of Y chromosome-bearing sperm (Y-sperm) and X chromosome-bearing (X-sperm) in ejaculate is 50:50 in mammals, therefore the sex ratio of the embryos following fertilization is expected to be 50:50. The sex ratio of embryos produced in vitro, however, is skewed towards either males or females. The primary aim of the present study was to investigate the factors of in vitro fertilization (IVF) that affect hte attachment or binding rate of bovine Y chromosome-bearing sperm (Y sperm rate) to the zona pellucidae (ZPe). Oocytes collected from bovine ovaries were cultured for 21 h in vitro under various conditions. ZPe were collected from the oocytes. After frozen-thawed bovine semen and ZPe were co-incubated, the ZPe were mounted on glass slides. Then, sperm attached or bound to the ZPe were subjected to in situ hybridization with a Y chromosome-specific probe to determine the Y-sperm rate. In a preliminary experiment, the Y-sperm rate of frozen-thawed sperm was 50.2% without deviation from the theoretical rate (50%), and the Y sperm rate in Y sperm sorted semen was 93.4% as expected. In Experiment 1, the effect of the sperm-ZP coincubation period on the Y-sperm rate was investigated. Short coincubation periods (5 min and 8 min) deviated the Y-sperm rate (55.1% and 54.9%) from the theoretical rate, but long coincubation periods (300 min) did not affect the Y-sperm rate (49.2%). In Experiment 2, a combination of preincubation of sperm for 3 h prior to sperm-ZP coincubation and extension of the maturation period to 36 h did not change the skewed Y-sperm rate of coincubation of untreated sperm with ZPe derived from 21-h-matured COCs (53.0%, 53.9%, and 54.5%, respectively). In conclusion, the ability of frozen-thawed sperm to attach to the ZPe during the first 5 min or 8 min of coincubation was slightly higher in Y sperm than X sperm although neither sperm preincubation nor extension of the maturation period affected the Y-sperm rate.

Testicular sperm preincubation and ICSI outcome

Testicular sperm preincubation and ICSI outcome

Reduced Glutathione Enhances Fertility of Frozen/Thawed C57BL/6 Mouse Sperm after Exposure to Methyl-Beta-Cyclodextrin1

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.

Combined Application of Methyl-Beta-Cyclodextrin and Reduced Glutathione to the In Vitro Fertilization of Cryopreserved C57BL/6 Mouse Sperm.

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. The benefits of sperm cryopreservation include the saving of space and costs relating to the upkeep of each mouse lineage, the potential avoidance of a genetic drift during the storage term, and the ease of transporting the mice between research facilities. However, embryos need to be obtained using in vitro fertilization (IVF) in order to reproduce live animals from frozen/thawed sperm. In general, the fertility of frozen/thawed sperm taken from C57BL/6 mice, which is mainly used as a base for genetically engineered mice, is severely lower than that of fresh sperm. Previously, we reported that methyl-β-cyclodextrin (MBCD) dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. MBCD effectively induces sperm capacitation by facilitating a cholesterol efflux from the cells. Recently, an interesting report was published on the topic of the supportive effects of reduced glutathione (GSH) on improving the fertility of mouse sperm. However, the action mechanism of GSH during the fertilization process is not fully understood. In this study, we examined the most efficient timing of GSH treatment and the combined effects of MBCD and GSH on the rate of fertilization for IVF using frozen/thawed C57BL/6 mouse sperm. As a result, we found that the presence of GSH in the fertilization medium increased the fertilization rate whereas GSH had no effects in the medium of sperm preincubation, and that the elevation of fertilization rates started at 0.5 mM GSH and reached a peak at 1.0 mM GSH (0 mM: 66%, 0.5 mM: 79% and 1.0 mM: 92%). We also discovered that the MBCD and GSH independently increased the rate of fertilization, while a combination of MBCD and GSH exhibited the highest fertilization rate in each group (control: 14%, MBCD: 65%, GSH: 36% and MBCD + GSH: 87%). In summary, we have demonstrated a novel IVF method using the combined application of MBCD for sperm preincubation and GSH for IVF, in order to achieve a high and stable fertilization rate in frozen/thawed C57BL/6 mouse sperm. (poster)

The effects of methyl-β-cyclodextrin on in vitro fertilization and the subsequent development of bovine oocytes

In this study, we investigated the effects of methyl-β-cyclodextrin (MBCD) on in vitro fertilization and the subsequent development of bovine oocytes. Bovine oocytes matured in serum-free medium were inseminated with frozen-thawed sperm pre-incubated in protein-free modified Brackett and Oliphant medium (BO) containing various concentrations of MBCD for various periods. MBCD decreased the frequency of live sperm, however enhanced the capacitation and acrosome reaction of the live sperm. Pre-incubation of sperm with 0.5, 1.0 and 1.5 mM MBCD for 2 and 4 h increased the frequency of normal fertilization. Embryos derived from oocytes fertilized with spermatozoa pre-incubated with MBCD developed normally to the blastocyst stage and term. There were individual differences and similar tendencies in four different sires in terms of the effects of MBCD upon fertilization. These results indicate that the pre-incubation of bovine sperm with MBCD affects viability and capacitation status of the sperm and promotes fertilization in vitro. Embryos derived from oocytes fertilized with sperm pre-incubated with MBCD developed normally to the blastocyst stage and term.

284 FERTILIZING CAPACITY OF BUFFALO (BUBALUS BUBALIS) SPERM CO-CULTURED WITH OVIDUCT EPITHELIAL CELLS

The overall in vitro embryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106 sperm mL-1). In vitro-matured bovine oocytes (n = 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2 in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P &lt; 0.01) in group B (79.3%) compared with group A (69.6%), with intermediate values in groups C (75.2%) and D (76.0%). Interestingly, the percentage of advanced embryos (&gt;4 cells) was higher (P &lt; 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies.

Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase‐3A serine 21 phosphorylation in boar spermatozoa

The cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) pathways control most relevant functions in male germ cells including motility. Recently we demonstrated that phosphorylation state of glycogen synthase kinase-3alpha (GSK3A) is also a key event in the control of boar spermatozoa motility. However, the upstream regulators of GSK3A serine phosphorylation (inhibition) in male germ cells remain largely unknown. This work investigates the involvement of PKA, PKC and PI3K pathways in GSK3A phosphorylation in boar spermatozoa. A capacitating medium (TCM) or the phosphodiesterase-resistant cell permeable cAMP analogue 8Br-cAMP cause a significant increase in Ser21 GSK3A phosphorylation associated with a simultaneous significant increase in boar spermatozoa motility. These effects are blocked after preincubation of spermatozoa with PKA inhibitor H89 or PKC inhibitor Ro-32-0432. The PI3K inhibitor LY294002 increases both spermatozoa motility parameters and the basal GSK3A phosphorylation, but does not affect either TCM- or 8Br-cAMP-stimulated GSK3A phosphorylation. PI3K inhibition effects are mediated by an increase in intracellular cAMP levels in boar spermatozoa and are suppressed by PKA inhibitor H89. In summary, we demonstrate that PKA, PKC and PI3K pathways crosstalk in porcine male germ cells to crucially regulate GSK3A phosphorylation which subsequently controls cell motility. In addition, our results suggest that PI3K is upstream of PKA which lies upstream of PKC in this regulatory cascade(s). Our findings contribute to elucidate the molecular mechanisms underlying the regulation of one of the most relevant male germ cell functions, motility.

Involvement of signaling pathways in bovine sperm motility, and effect of ergot alkaloids

There is evidence that ergot alkaloids can directly interact with mammalian spermatozoa affecting sperm functions. Ergot alkaloids exert their toxic or pharmaceutical effects through membrane receptor-mediated activities. This study investigated the signaling pathways involved in the in vitro inhibitory effects of both ergotamine (ET) and dihydroergotamine (DEHT) on the relative motility of bovine spermatozoa using specific inhibitors. Motile bovine spermatozoa were prepared using a Percoll gradient and incubated with ergot alkaloids with and without signaling pathway inhibitors. Co-incubation of ET or DHET with 100 microM prazosin (alpha 1-adrenergic receptor inhibitor) decreased (p < 0.05) relative motility of spermatozoa when compared with controls. In addition, preincubation of spermatozoa with 10 or 20 microM prazosin and DHET also reduced (p < 0.05) the number of motile spermatozoa. Relative sperm motility (motility of treated spermatozoa normalized to control sperm motility) was increased (p < 0.05) when co-incubations included ET and yohimbine (alpha 2-adrenergic receptor inhibitor); conversely, co-incubation of yohimbine (100 microM) and DHET decreased (p < 0.05) the percentage of motile spermatozoa when compared with controls. Pertussis toxin and cholera toxin (effectors of inhibitory and stimulatory G-proteins, respectively) altered (p < 0.05) relative sperm motility in a concentration dependent manner; however, co-incubation of pertussis or cholera toxin with ergot alkaloids had no interactive (p = 0.83) effects on the relative motility of spermatozoa. Co-incubation of Rp-cAMP (a membrane-permeable cAMP inhibitor) with 50 microM DHET had no effect (p > 0.05) on relative sperm motility; whereas, the co-incubation of 22.4 or 44.8 microM Rp-cAMP with 50 microM ET increased (p < 0.05) the percentage of motile spermatozoa when compared with 0 or 224 microM Rp-cAMP (49%, 65%, 59%, and 54%, respectively, for 0, 22.4, 44.8, and 224 microM of Rp-cAMP. An interaction between BAPTA-AM (a chelator of intracellular calcium) and alkaloids also impacted (p < 0.05) relative sperm motility. Generally, co-incubating spermatozoa with BAPTA-AM and ET increased the percentage of motile spermatozoa; however, co-incubation with DHET decreased relative sperm motility except with 41 microM BAPTA-AM. Collectively, these observations suggest that ET and DHET decreased the percentage of motile bovine spermatozoa via alpha adrenergic receptors. However, the second messenger systems involved with ergot alkaloid inhibition of relative motility of bovine spermatozoa remain to be elucidated.

Lycopene supplementation in vitro can protect human sperm deoxyribonucleic acid from oxidative damage

To examine the effects of lycopene on human sperm motility and DNA damage. Prospective study. Andrology research laboratory. Twelve fertile donors. Preincubation of washed sperm suspensions with or without lycopene (2 or 5 micromol/L) followed by a 2-hour incubation with or without hydrogen peroxide (H2O2, 50 micromol/L). Assessments of sperm motility (percentage) and DNA fragmentation index (percent DNA fragmentation index) before and after incubation. Sperm motility (percentage) and sperm percent DNA fragmentation index. Incubation of spermatozoa with H2O2 resulted in a significant decline in mean (+/-SD) percent sperm motility (28%+/-13% vs. 73%+/-4%, respectively) and a significant increase in percent DNA fragmentation index compared with samples incubated without H2O2 (29.8%+/-39.4% vs. 11.1%+/-14.6%, respectively). Pretreatment of samples with 5 micromol/L lycopene resulted in a significantly lower percent DNA fragmentation index than samples incubated without lycopene (8.0%+/-7.9% vs. 29.8%+/-39.4%, respectively). However, lycopene did not protect spermatozoa from the decline in motility after H2O2 treatment. The data suggest that preincubation of spermatozoa with lycopene offers protection against oxidative DNA damage in vitro. These data also highlight the differential protective effects of lycopene on sperm motility and sperm DNA integrity.

Protective effect of fallopian tubal fluid against activated leucocyte-induced sperm DNA fragmentation: preliminary results

The integrity of the paternal genome is of paramount importance in the initiation and maintenance of a viable pregnancy. Oxygen radicals (ROS) have been identified as one of the main factors responsible for the induction of sperm DNA damage. Spermatozoa are mainly protected against ROS-induced damage by seminal plasma. However, this protective effect disappears once spermatozoa enter the female genital tract. The fallopian tube mucosa may play a protective role against ROS-induced sperm damage. The main objective of this study was to determine whether human tubal explants and tubal fluid exert a protective effect on ROS-induced sperm DNA damage. Spermatozoa were exposed to tubal explants and/or tubal fluid in the presence of phorbol myristate acetate (PMA)-activated polymorphonuclear leucocytes or control medium and sperm DNA fragmentation was measured using the TdT-mediated dUTP-biotin nick end labelling (TUNEL) test. Exposure of human spermatozoa to PMA-activated leucocytes resulted in a 2-fold increase in sperm DNA fragmentation. Co-incubation of spermatozoa with tubal explants did not reduce this damage. However, pre-incubation of spermatozoa with tubal fluid resulted in a statistically significant reduction in sperm DNA fragmentation levels, comparable to those observed in control. In conclusion, tubal fluid appears to protect against activated leucocyte-induced sperm DNA fragmentation, thus preserving the integrity of the paternal genome.

Effect of milk-yolk on the fertilizing capacity of spermatozoa

Pre-incubation of human sperm in egg yolk based medium (TEST-yolk) as well as milk have been shown to improve sperm fertilizing capacity. The purpose of this study was to compare a combination of these two media, called milk-yolk, against the two established media, in order to test for a possible synergistic effect(s) on sperm functions as measured by sperm motility, acrosome reaction and penetration assay. Statistically, no differences have been observed among the three media, except for SPA outcome in milk-yolk, being significantly lower than the corresponding values for the other two media. In conclusion the beneficial effects of egg yolk and milk appear to be eliminated once both media are combined, resulting in an antagonistic effect on the fertilizing capacity of the spermatozoa.

Marked Improvement of Fertility of Cryopreserved C57BL/6J Mouse Sperm by Depletion of Ca2+ in Medium

Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca(2+)), phosphate (PO(4)(3-)) and lactate. In all media containing no Ca(2+), including medium lacking Ca(2+), lacking Ca(2+) and PO(4)(3-), lacking Ca(2+) and lactate and lacking Ca(2+), PO(4)(3-) and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca(2+) were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca(2+). In conclusion, preincubation of thawed sperm in medium containing no Ca(2+) markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca(2+) is practical for use in cryopreserved C57BL/6J sperm.