Fluorescein Isothiocyanate-conjugated Peanut Agglutinin Research Articles

The sperm chromatin structure assay does not detect alterations in sperm chromatin structure induced by hydrogen peroxide

The objective of this study was to investigate the effects of hydrogen peroxide (H2O2) on the chromatin structure of sperm. For this purpose, 44 cryopreserved bovine ejaculates were analyzed immediately post-thaw (control sperm, CON S), after 1 h of post-thaw incubation (non-oxidized sperm, NOX S), and after 1 h of post-thaw incubation in different concentrations of H2O2 (OX S; 50, 100 µM, 1000 µM H2O2). Sperm motility was determined using computer-assisted sperm analysis. Sperm plasma membrane and acrosome integrity were assessed by flow cytometry after staining with propidium iodide and fluorescein isothiocyanate-conjugated peanut agglutinin. Chromatin damage was assessed using the sperm chromatin structure assay (SCSA), and DNA damage was evaluated using the single cell gel electrophoresis (Comet) assay. Sperm motility and plasma membrane and acrosome integrity decreased while chromatin damage and DNA damage increased after 1 h of incubation (P < 0.05). The addition of H2O2 adversely affected all parameters (P < 0.05) except for chromatin structure. The role of H2O2 in sperm chromatin damage was investigated by supplementation of 10 IU catalase, which reversed the damage (P < 0.05). Interestingly, the chromatin decondensation induced by 10 mM dithioreitol, evidenced by an increase in chromatin damage in the SCSA (P < 0.05), was reversed by H2O2. In conclusion, H2O2 causes alterations in sperm functional status and DNA integrity. However, our results show that high concentrations of H2O2 can induce chromatin condensation, and thus the use of the SCSA for the assessment of H2O2-induced chromatin damage should be carefully considered.

Asthenozoospermic stallions tend to have high acrosome reacted spermatozoa as evidenced by dual fluorescent staining assay

Acrosome intactness of spermatozoa is the critical factor for establishing sperm reservoir in oviduct and for fertilizing an oocyte. However, frozen thawed spermatozoa tend to show higher proportion of acrosome reacted spermatozoa thereby compromising the fertility. Conventional staining techniques identify only sperm acrosome integrity and not precisely the acrosome reaction status. In this context, the current study was conducted to assess the acrosome status of cryopreserved spermatozoa using Fluorescein isothiocyanate conjugated peanut agglutinin and propidium iodide (FITC-PNA+PI) in stallions with varying sperm motility. Stallions were classified into high- (≥45%) and low-motile group (≤30%) based on their post-thaw sperm motility. The proportion of live acrosome intact (LAI) spermatozoa was significantly higher in high-motile group as compared to low-motile group. A significant positive correlation was observed between LAI and post-thaw sperm motility. In conclusion, the present study showed that FITC-PNA+PI combination could be used for rapid and accurate assessment of acrosome reaction status of stallion spermatozoa, and the proportion of LAI population in cryopreserved stallion semen had a strong correlation with sperm motility.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&amp;gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&amp;gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

79 Progesterone-induced acrosome reaction and fertilization rates with bovine intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been a valuable tool in many species because of its ability to overcome male factor infertility problems and eliminate risk of polyspermy; more recently, it been used to improve genome editing technologies. However, limited success with bovine ICSI has hindered these applications in cattle. Numerous treatments have been used to increase the success rate, with marginal improvement. Replicating events synonymous with fertilization, such as the acrosome reaction, may improve fertilization with bovine ICSI. Progesterone (P4) is naturally found in both the cumulus cells surrounding the oocyte and follicular fluid released at ovulation and activates a physiological pathway within sperm to induce an acrosome reaction, a crucial process in fertilization. Progesterone induction of the acrosome reaction as a sperm pretreatment for ICSI has not yet been evaluated in cattle. In this study, frozen–thawed bovine sperm was used. Sperm were first thawed, washed, and separated via gradient to obtain the motile population before capacitation with heparin over 4h before treatment with or without P4 (10 μM) for 15min. To measure the acrosome reacting population, sperm were stained with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) and observed over 2h using cytometric flow analysis and fluorescent microscopy to measure the population undergoing and completing an acrosome reaction. For ICSI, a total of 220 oocytes were used, and both treated and nontreated sperm were incubated for 1h before injection to allow for completion of an acrosome reaction. Fertilization rates were measured by observing pronuclear formation and an absence of sperm 18h after ICSI. Means of acrosome reacting population gathered by flow cytometry and microscopy were analysed by ANOVA. Differences in fertilization rates between groups post ICSI were analysed using a Yates’ corrected chi-squared test. A significantly higher proportion of P4 treated versus control sperm initiated an acrosome reaction at hour 1 (80.2%±4.2 vs. 19%±9.1), which increased to (89.3%±3.9 vs. 29%±8.3) after 2h. P4 also increased the percentage of sperm that completed an acrosome reaction, from 50% (±5.1) at hour 1 and 62.5% (±7.4) at hour 2. Only 14.2% (±3.6) completed acrosome reactions in sperm not treated with P4 by hour 2. Motile sperm in both groups did not decrease over the 2-h incubation time period (P&amp;lt;0.05). Progesterone treatment increased the percentage of fertilized embryos after ICSI, with 38.1% fertilized compared with 10.1% with heparin-treated control injections (P&amp;lt;0.001). These results show that P4 has effects on bovine sperm that allow for higher rates of fertilization after ICSI by utilising the sperm’s physiological response to progesterone. Further embryo development using ICSI with P4-treated sperm, or additional physiologically similar treatments, should continue to be assessed.

112 The effect of biological extenders on invitro induction of the acrosome reaction in bovine spermatozoa

Biological extenders in semen are used to protect the integrity of the sperm cells during cryopreservation. The two most common extenders are egg yolk and milk, and initial experiments describing heparin capacitation for IVF were conducted with semen frozen in egg yolk extender. However, semen of many bulls used for IVF is frozen in milk-based extender, and it is not clear whether this affects the response to heparin capacitation. This study aims to assess whether the use of milk- or egg-based extenders in frozen-thawed bovine semen affects sperm's ability to undergo the acrosome reaction, utilising either the endogenous progesterone pathway or calcium ionophore A23187 to induce the acrosome reaction following heparin capacitation. Frozen semen from 12 bulls (6 using egg yolk citrate-based extender containing 20% egg yolk, and 6 using milk-based extender) was used for no less than 3 replicates each. All semen was commercially processed and cryopreserved using the same methodology. Semen was thawed and separated using a Percoll gradient and centrifugation at 400×g for 10min, then incubated for 4h in a capacitating medium containing heparin (10μgmL−1) at a concentration of 1×106 spermmL−1. Capacitated sperm were then introduced to progesterone (10μM) or A23187 (10 μM) for acrosome induction and stained with fluorescein isothiocyanate-conjugated peanut agglutinin (5μgmL−1). After treatment and staining, the sperm were analysed using flow cytometry to determine the percentage of populations with fluorescent acrosomes, indicating an exposed acrosome and an ongoing acrosome reaction. Fluorescent microscopy was also used to confirm fluorescence in acrosome-reacting samples. Results were analysed as a 2×3 factorial design by ANOVA with extender and acrosome induction treatment as factors. For egg yolk-extended semen, the mean percent of acrosome-reacted sperm was 83.7 (±6.8), 81.8 (±7), and 15.0 (±7.5) for sperm treated with progesterone, A23187, and heparin only, respectively. Compared to milk-extended semen, for which the mean percent acrosome-reacted was 15.0 (±8.2), 14.8 (±7.4), and 12.0 (±6.8) for progesterone, A23187, and heparin only, respectively. Significant differences were observed between extenders, with egg yolk-extended semen having a significantly greater response to acrosome reaction-inducing agents than semen extended with milk-based extenders (P&amp;lt;0.05). Also, it was observed that progesterone induced a similar percentage of acrosome reactions as A23187. Variation between bulls in the response to heparin capacitation has been observed and cannot be eliminated in this study. The difference observed between extenders warrants further study with a split ejaculate design. The possibility that the semen extender used during cryopreservation affects the response to heparin capacitation suggests that this should be accounted for in IVF protocols to increase the efficiency of this technology.

Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa.

This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1–3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0–2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.

Intracytoplasmic sperm injection (ICSI) of prepubertal goat oocytes using fresh and frozen-thawed semen

The aim of this study was to test the effect of fresh and frozen-thawed semen on embryo development of prepubertal goat oocytes fertilized by intracytoplasmic sperm injection (ICSI). Oocytes were in vitro matured in BO-IVM medium for 26 h. Fresh and frozen-thawed semen were selected by centrifugation in BoviPure® commercial gradient. Before injection, spermatozoa were assessed for: capacitation through the detection of the tyrosine phosphorylation, viability through propidium iodide stain, and acrosomal status through fluorescein isothiocyanate-conjugated peanut agglutinin stain. Metaphase II oocytes were injected with a spermatozoon selected by hyaluronic-acid binding medium (SpermSlow ™). Results of frozen-thawed spermatozoa showed significantly higher level of capacitation than fresh spermatozoa (64.92 vs 34.67%, respectively). Moreover fresh semen group showed significantly higher (p < 0.02) number of live acrosome reacted sperm (8.84%) than frozen-thawed group (6.42%). At 17 h post ICSI, zygotes (2 pronuclei) were evaluated but no differences were found using fresh and frozen-thawed semen (39.06 vs 37.50% respectively). Moreover, no differences were obtained comparing the percentage of blastocysts produced at day 9 post injection (18.46 vs 12.12%, respectively). In conclusion, either fresh or frozen-thawed semen did not have an effect on the embryo production of prepubertal goat oocytes fertilized by ICSI, despite the differences on viability, acrosomal status and sperm capacitation.

Effect of Lepidium meyenii on in vitro fertilization via improvement in acrosome reaction and motility of mouse and human sperm.

PurposeThe direct effects of Lepidium meyenii (Maca) on sperm remain unclear. Herein, we examined the direct effect of Maca on in vitro fertilization.MethodsWe examined the fertilization rate in a mouse model and the rate of acrosome reaction in sperm from transgenic mice expressing enhanced green fluorescent protein (EGFP) in a Maca extract‐containing human tubal fluid (HTF) medium. Using human sperm, we assessed acrosome status via fluorescein isothiocyanate‐conjugated peanut agglutinin (FITC‐PNA) staining and performed detailed analysis using a sperm motility analysis system (SMAS).ResultsIn the mouse model, the fertilization rate in the Maca extract‐containing HTF was significantly higher than that in the control medium. The acrosome reaction rate in sperm from transgenic mice expressing EGFP was also significantly higher in the Maca extract‐containing HTF than that in the control medium. Similarly, a high acrosome reaction rate, identified via FITC‐PNA staining of human sperm samples, was found in the Maca extract‐containing HTF compared with that in the control medium. Human sperm motility in the Maca extract‐containing HTF was also increased compared with that in the control medium as measured using an SMAS.ConclusionsMaca improved in vitro fertilization rates by inducing an acrosome reaction and increasing sperm motility.

On-chip cryopreservation: a novel method for ultra-rapid cryoprotectant-free cryopreservation of small amounts of human spermatozoa.

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.

Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders

The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r2&gt;0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P&lt;0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage.

Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation

This study examines (1) the effectiveness of transrectal, ultrasound-guided massage of the accessory sex glands (TUMASG) combined with electroejaculation for obtaining aoudad (Ammotragus lervia sahariensis) sperm samples for cryopreservation, and (2) the effectiveness of a Tris-citric acid–glucose-based medium (TCG; usually used for freezing ibex sperm) and a TES-Tris–glucose-based medium (TTG; typically used in the cryopreservation of mouflon sperm) as sperm extenders. After TUMASG, just one to three electrical pulses were required for ejaculation to occur in five of the six animals studied; one ejaculated after TUMASG alone. Transrectal, ultrasound-guided massage of the accessory sex glands would therefore appear to be useful in obtaining sperm samples from this species, requiring few subsequent electrical electroejaculation stimuli and sometimes none at all. After thawing, the membrane integrity (assessed by nigrosin-eosin staining) of sperm extended with TTG was greater than that of sperm extended with TCG (P < 0.05). The total percentage of sperm showing an intact acrosome, as assessed by fluorescein isothiocyanate-conjugated peanut (Arachis hypogea) agglutinin, was also higher in the TTG-extended sperm (P < 0.05), and the percentage of dead sperm with a damaged acrosome was lower (P < 0.05). No differences were seen between TCG and TTG in terms of apoptotic manifestations (DNA damage, caspase activity, mitochondrial membrane potential, and plasmalemma stability). Therefore, TTG appears to be a better extender than TCG for cryopreserving aoudad sperm.

Ubiquitin‐activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm–egg coat penetration during porcine fertilization

Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation-induced modification of those proteins was altered by PYR-41. In summary, it appears that de novo protein ubiquitination involving UBA1 contributes to sperm capacitation and acrosomal function during fertilization.

English

&nbsp; Soybean lecithin has been attracted increasing attention and has been used to replace egg yolk in the cryopreservation of domestic animal semen. However, its effects on freezing boar spermatozoa have never been evaluated. In the present study, semen was collected from five Duroc boars and frozen-thawed in extender with different concentrations of soybean lecithin (3, 6, 9 and 12%) and 20% egg yolk. Semen parameters including sperm motion characteristics (the percentage of total motile sperm and motility), plasma membrane integrity and acrosome integrity were assessed with a computer-aided semen analysis (CASA) system, hypoosmotic swelling test (HOST) and&nbsp;fluorescein isothiocyanate-conjugated peanut agglutinin&nbsp;(FITC-PNA) method, respectively. The result best result was obtained for the extender supplemented with 6% soybean lecithin, with values of 59.7% for the percentage of total motile sperm (TM%), 44.3% for motility, 45.3% for plasma membrane integrity and 61.9% for acrosome integrity. TM%, motility, acrosome integrity and plasma membrane integrity in the extender containing 6% soybean lecithin were significantly higher than that of other concentrations of soybean lecithin and 20% egg yolk (P&nbsp;&lt; 0.05). However, the percentages of TM, acrosome integrity and plasma membrane integrity decreased with the increasing concentration of soybean lecithin in extender. In summary, the effect of soybean lecithin on spermatozoa quality was superior and the effective concentration of soybean lecithin in extender was 6% (w/v). Soybean lecithin might replace egg yolk in extender in the cryopreservation of boar semen. &nbsp; Key words:&nbsp; Boar semen, cryopreservation, soybean lecithin, motility, acrosome integrity.

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of α2β1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with beta3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that alpha2beta1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of beta1 integrin, leading to decreased levels of the alpha2beta1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to beta1 and alpha2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.

Assessment of viability and acrosomal status of Asian elephant (Elephas maximus) sperm after treatment with calcium ionophore and heparin

Knowledge about the acrosomal status of Asian elephant (Elephas maximus) sperm is extremely limited. The objective of this study was to evaluate the viability and acrosomal status of Asian elephant sperm following induction by calcium ionophore and heparin using propidium iodide (PI) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA). Semen samples were collected from elephant bulls by manual stimulation. Semen was diluted with extender, cooled to 4 degrees C and transported to a laboratory for the experiment. Sperm cells were incubated in modified Tyrode's medium containing either 1 mM calcium ionophore or 10 mg/ml heparin for 5 h at 39 degrees C. Sperm recovered at the onset (0 h), 1, 2, 3, 4 and 5 h of incubation were simultaneously assessed for the viability and acrosomal status using dual staining of FITC-PNA and PI. Results were confirmed by transmission electron microscopy. A progressive increase in the proportion of live-acrosome reacted sperm was observed within 3 h of incubation in both treatment groups which slightly decreased at 4 to 5 h of incubation. At 1 to 3 h of incubation, the percentage of live-acrosome reacted sperm induced by calcium ionophore was higher (P < 0.05) than those induced by heparin and the control. However, there were no statistical differences at 4 to 5 h of incubation. A progressive reduction of the percentage of motile sperm was observed in the control as well as both treatment groups. Sperm motility decreased sharply when they were incubated in calcium ionophore compared with incubation in heparin and control groups. These results indicate that the occurrence of live-acrosome reacted sperm in the Asian elephant was induced by calcium ionophore at a rate higher than that induced by heparin.

Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.

SINGLE UV EXCITATION OF HOECHST 33342 AND PROPIDIUM IODIDE FOR VIABILITY ASSESSMENT OF RHESUS MONKEY SPERMATOZOA USING FLOW CYTOMETRY

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better preservation of plasma and acrosome integrity than TEST. Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Birth of piglets by in vitro fertilization of zona-free porcine oocytes

The present experiments were conducted to optimize in vitro fertilization conditions for zona pellucida-free (ZP-free) oocytes and their subsequent development. The results demonstrated that: (1) maximal fertilization efficiency was achieved at 200 spermatozoa per ZP-free oocyte. At this sperm dose, there were no significant differences in penetration rates and polyspermy rates from controls (zona-intact oocytes with 1000 spermatozoa/oocyte), indicating that ZPs of in vitro matured pig oocytes failed to block polyspermy during in vitro fertilization. (2) In vitro development of zygotes from ZP-free oocytes showed that there was no difference in cleavage rates. The blastocyst rate was slightly lower in the ZP-free group than the control. However, there was no difference in cell number per blastocyst between the control and the ZP-free group. (3) Examination of acrosome status by a specific fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining procedure revealed that frozen–thawed pig spermatozoa could undergo acrosome reaction and penetrate oocytes without induction by ZP. These data suggested that there are alternative mechanistic pathways for acrosome reaction induction during the fertilization process than the widely accepted sperm–zona receptor models. Finally, the viability of ZP-free derived embryos was demonstrated by full-term development and the delivery of healthy piglets following embryo transfer. In conclusion, the present experiments showed for the first time in farm animals, that normal embryos could be produced by in vitro fertilization of ZP-free oocytes in optimized conditions and that they could develop normally to full-term.

Effects of the supplementation of trehalose extender containing egg yolk with sodium dodecyl sulfate on the freezability of goat spermatozoa

Two experiments were conducted to determine the effect of sodium dodecyl sulfate (SDS) added to a trehalose-egg yolk extender on the cryopreservation of goat spermatozoa. In Experiment 1, semen from four goats was frozen in trehalose extender (osmolality=370, pH=7) containing 4 and 20% (v/v) glycerol and egg yolk, respectively, and 0.035–0.2% SDS. After thawing, sperm motility and acrosome integrity were assessed using a computer-assisted sperm analysis (CASA) system and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Both motility and progressive motility were improved ( P<0.05) by increasing the concentration of SDS in the trehalose-egg yolk extender, with the best results obtained with SDS at 0.1% (80.0±1.5% and 65.0±1.7%, respectively). There were no significant differences in path velocity when spermatozoa were frozen in a diluent containing 0.035, 0.05, 0.075, or 0.1% SDS, but path velocity decreased significantly with 0.2% SDS. The percentage of acrosome-intact sperm were highest ( P<0.05) when 0.05% (74.0±1.1) and 0.075% (70.0±1.2) SDS were used. In Experiment 2, the effect of diluent storage time (6, 24, or 48 h) before freezing on the cryoprotective effect of SDS was investigated. Prolonged storage of the diluent had slight cryoprotective effects when 0.2% SDS is used, while motility and the acrosome integrity of the cryopreserved spermatozoa improved slightly when the extender was stored for 48 h at 5 °C before use. In conclusion, goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration of SDS.

Fate of the acrosome in ooplasm in pigs after IVF and ICSI.

ICSI bypasses the sperm-oolemma interactions that, in normal fertilization, depend on completion of the acrosome reaction. Morphological changes in the acrosomes of sperm in the ooplasm were therefore examined following IVF and ICSI using pig gametes. In-vitro-matured porcine oocytes were used for ICSI or IVF. Oocytes were then stained with fluorescein isothiocyanate-conjugated peanut agglutinin lectin (FITC-PNA), which specifically labels the outer acrosomal membrane of boar sperm and the cortical granules (CG) in porcine oocytes. This was followed by observation under a confocal laser scanning microscope. In ICSI, PNA showed the presence of disintegrated acrosomes that classified into four categories. Heterogeneous chromatin decondensation was observed in the sperm with intact/disintegrated acrosome, whereas acrosomes were barely detected in oocytes which had formed a male pronucleus. Both in ICSI and IVF, PNA-positive tails were concomitantly observed with one type of disintegrated acrosome, which was considered to be acrosome-reacted. The disappearance of CG in activated oocytes after ICSI was similar to that after IVF. The PNA-binding properties of sperm head components introduced into the ooplasm during ICSI are different from those after IVF. The delay of sperm chromatin decondensation is associated with that of acrosomal disassembly. Acrosomes appear to disintegrate in the ooplasm whether or not the acrosome reaction has taken place. Oocytes undergoing ICSI appear normally activated in terms of meiotic resumption and CG exocytosis.