Hamster sperm capacitation: role of pyruvate dehydrogenase A and dihydrolipoamide dehydrogenase

Recently, we demonstrated that pyruvate dehydrogenase A2 (PDHA2) is tyrosine phosphorylated in capacitated hamster spermatozoa. In this report, using bromopyruvate (BP), an inhibitor of PDHA, we demonstrated that hamster sperm hyperactivation was blocked regardless of whether PDHA was inhibited prior to or after the onset of hyperactivation, but the acrosome reaction was blocked only if PDHA was inhibited prior to the onset of the acrosome reaction. Further, inhibition of PDHA activity did not inhibit capacitation-associated protein tyrosine phosphorylation observed in hamster spermatozoa. It is demonstrated that the essentiality of PDHA for sperm capacitation is probably dependent on its ability to generate effectors of capacitation such as reactive oxygen species (ROS) and cAMP, which are significantly decreased in the presence of BP. MICA (5-methoxyindole-2-carboxylic acid, a specific inhibitor of dihydrolipoamide dehydrogenase [DLD]), another component of the pyruvate dehydrogenase complex (PDHc), also significantly inhibited ROS generation and cAMP levels thus implying that these enzymes of the PDHc are required for ROS and cAMP generation. Furthermore, dibutryl cyclic adenosine monophosphate could significantly reverse the inhibition of hyperactivation observed in the presence of BP and inhibition of acrosome reaction observed in the presence of BP or MICA. The calcium ionophore, A23187, could also significantly reverse the inhibitory effect of BP and MICA on sperm acrosome reaction. These results establish that PDHA is required for hamster sperm hyperactivation and acrosome reaction, and DLD is required for hamster acrosome reaction. This study also provides evidence that ROS, cAMP, and calcium are involved downstream to PDHA.     

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

How Pig Sperm Prepares to Fertilize: Stable Acrosome Docking to the Plasma Membrane

Background

Mammalian sperms are activated in the oviduct. This process, which involves extensive sperm surface remodelling, is required for fertilization and can be mimicked under in vitro fertilization conditions (IVF).

Methodology/Principal Findings

Here we demonstrate that such treatments caused stable docking and priming of the acrosome membrane to the apical sperm head surface without the emergence of exocytotic membrane fusion. The interacting membranes could be isolated as bilamellar membrane structures after cell disruption. These membrane structures as well as whole capacitated sperm contained stable ternary trans-SNARE complexes that were composed of VAMP 3 and syntaxin 1B from the plasma membrane and SNAP 23 from the acrosomal membrane. This trans-SNARE complex was not observed in control sperm.

Conclusions/Significance

We propose that this capacitation driven membrane docking and stability thereof is a preparative step prior to the multipoint membrane fusions characteristic for the acrosome reaction induced by sperm-zona binding. Thus, sperm can be considered a valuable model for studying exocytosis.     

L’irradiation des spermatozoïdes au Laser HeNe à faible énergie chez la souris et chez l’homme

Introduction

The effects of low energy He?Ne 630 laser (LEL) irradiation on various aspects of cell metabolism, including proliferation, respiration, ATP synthesis and Ca²⁺ uptake, have been recently recognized in somatic cells, animal spermatozoa and embryonic cells.

Objectives

1- To analyze thein vitro effects of LEL irradiation on the mouse IVF model. 2- To assess the effects of experimental LEL irradiation on human spermatozoa in the sperm-zona free hamster egg penetration (SPA) model.

Material & Methods

1- In a mouse IVF model, pooled oocytes were incubated for 24 h. with LEL-irradiated and non-irradiated epididymal mouse spermatozoa. The percentage of fertilized eggs, intracellular Ca²⁺ and Ca²⁺ uptake as well as the effects of reactive oxygen scavengers were compared in the two groups. 2- In the enhanced SPA model, with overnight TEST-Yolk preincubation, LEL-irradiated and non-irradiated ejaculated spermatozoa from 43 infertile men were incubated for 3 h with fresh zona-free hamster eggs. The percentage of eggs penetrated (SPA%) by irradiated or control sperm was compared. Acrosome reaction (AR) and Hemizona Assay (HZA) were also performed on the same ejaculates.

Results

1- In mice, brief LEL irradiation enhanced intracellular Ca²⁺ influx and increased thein vitro fertilization capacity of spermatozoa. Mitochondrial Ca²⁺ transport mechanisms and H₂O₂ appeared to be involved in these LEL effects. 2- In the 43 male patients, AR and SPA were correlated, while SPA and HZA were poorly correlated. In the whole group, the mean SPA% of LEL-irradiated and control sperm were not significantly different: 49.8±39.9 and 51.0±34.9, respectively. No significant change was observed in 50% of cases. Following irradiation, SPA% increased in 25% of cases and decreased in 25% of cases. However, classifying all cases according to the SPA cut-off point of 30% as poor and good sperm in this enhanced SPA model, showed that LEL irradiation significantly increased SPA% only in the poor sperm subgroup (n=16) from 5.5.±8.9 to 23.1±25.2 (p<0.0001). This increase was observed in 50% of these patients. In the good sperm category (n=27), no improvement of SPA% was observed. On the contrary, LEL irradiation decreased SPA% from 76.1±24.4 to 67.6±28.

Conclusion

In a mouse IVF model, LEL irradiation of spermatozoa improved intracellular Ca²⁺influx and egg fertilization. H₂O₂ seems to participate in the biochemical cascade transforming light signals into a biological response. In man, using the SPA model, LEL sperm irradiation might improve the penetration capacity only in the case of poor quality sperm, by increasing sperm egg penetration in one half of cases, otherwise LEL is ineffective. Further studies are required to confirm these results, but LEL should not be considered for clinical use until its safety has been proven. (Supported by grants from the Chief Scientist Office, Ministry of Health)     

Estrous sheep serum as a potent agent for ovine IVF: effect on cholesterol efflux from spermatozoa and the acrosome reaction

The aim of the present study was to evaluate the effect of heat-inactivated estrous sheep serum (ESS) on sheep IVF. When the capacitation and the fertilization media contained 20% ESS, a fertilization rate of 85% was achieved. The beneficial effect of ESS on sheep IVF was further demonstrated since the fertilization rate was null when ESS was omitted during sperm capacitation and fertilization. Estrous sheep serum supported both sperm capacitation and fertilization as shown by the results of experiments in which it was omitted during one of these steps: sperm capacitation in serum-free medium resulted in delayed sperm-oocyte penetration, while fertilization in serum-free medium significantly decreased the percentage of fertilized oocytes. To investigate the influence of serum on sperm ability to undergo the acrosome reaction, salt-stored follicular sheep oocytes were inseminated, and the acrosomal status of spermatozoa attached to zonae was examined by electron microscopy after a 4-h period of coincubation. Quantitative analysis on thin sections demonstrated that fewer acrosome-reacted spermatozoa were observed when the capacitation and insemination steps were carried out in DM-H medium without serum than in DM-H-SS supplemented with 20% ESS (0.08, [0; 0.34], (median, range)/100mum zona vs 1.32, [0.90; 2.28]/100mum zona; P < 0.01). Since a higher number of spermatozoa attached to the zona surface in DM-H medium, the proportion of acrosome-reacted spermatozoa was much lower (0.7%, [0%; 2.2%], (median, range) vs 54%, [25%; 100%]; P < 0.01) in the absence of serum. These results indicate that in our IVF system the development of the acrosome reaction depended on serum. Sperm cholesterol efflux during in vitro capacitation was measured on [(3)H] cholesterol labeled spermatozoa resuspended in DM-H or DM-H-SS medium. A time-dependent cholesterol removal was observed in the presence of serum (60 +/- 5%, mean +/- SD, after 5 h), whereas it was limited to 14 +/- 3 % in DM-H medium; hence addition of serum to the capacitation medium efficiently supports cholesterol efflux, which is thought to be a key-event in the capacitation process.     

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa

Background

Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.

Result

We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.

Conclusion

The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.     

Epididymal maturation and ejaculation are key events for further in vitro capacitation of boar spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation. Epididymal spermatozoa showed low membrane fluidity and intracellular calcium content; ejaculation led to an increased calcium content, while membrane fluidity showed no changes. Acrosome integrity remained constant throughout the epididymal duct and after ejaculation and in vitro capacitation. The frequency of viable spermatozoa with intact mitochondrial sheath was higher in caput and ejaculated samples than in corpus and cauda samples, whereas the frequency of spermatozoa with high membrane potential was significantly lower in cauda samples. In vitro capacitation resulted in a decreased frequency of viable spermatozoa with intact mitochondrial sheath and an increased frequency of spermatozoa with high membrane potential in ejaculated samples. These results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable of undergoing the set of changes leading to capacitation.     

Relationship between Semenogelins bound to human sperm and other semen parameters and pregnancy outcomes

Background

Semenogelins (SEMGs) are major components of human seminal vesicle secretions. Due to SEMG’s sperm-motility inhibitor, a significant negative correlation between sperm motility and the proportion of SEMG-bound spermatozoa (SEMG+) was found in asthenozoospermic patients. SEMGs also show intrinsic inhibitory capability for sperm capacitation; however, studies on actual clinical specimens have not been conducted.

Methods

To reveal the relationship between SEMGs and the fertilizing capacity of sperm from male infertile patients who are not restricted to asthenozoospermia, we measured the proportion of SEMG+ in the spermatozoa of 142 male infertile patients. The pregnancy outcomes in partners of these patients were retrospectively analyzed using questionnaires.

Results

Among examined semen parameters, only the total SEMG-unbound sperm count showed a tendency to be different between the spontaneous pregnancy or intra-uterine-insemination-pregnancy groups and in-vitro-fertilization- or intracytoplasmic-sperm-injection-pregnancy groups. It was elevated in the former group, which includes patients who used in vivo fertilization.

Conclusions

The total SEMG-unbound sperm count would be a relevant parameter for in vivo fertilization. This result suggests that SEMGs inhibit ectopic capacitation before sperm reach the fertilization site and that the number of total SEMG-unbound sperm is a parameter directly linked to the possibility of in vivo fertilization.

     

Effect of exogenous nitric oxide on sperm motility in vitro

Background

Nitric oxide (NO) has been shown to be important in sperm function, and the concentration of NO appears to determine these effects. Studies have demonstrated both positive and negative effects of NO on sperm function, but have not been able to provide a clear link between NO concentration and the extent of exposure to NO. To study the relationship between nitric oxide and sperm capacitation in vitro, and to provide a theoretical basis for the use of NO-related preparations in improving sperm motility for in vitro fertilization, we investigated the effects of NO concentration and time duration at these concentrations on in vitro sperm capacitation in both normal and abnormal sperm groups. We manipulated NO concentrations and the time duration of these concentrations using sodium nitroprusside (an NO donor) and NG-monomethyl-L-argenine (an NO synthase inhibitor).

Results

Compared to the normal sperm group, the abnormal sperm group had a longer basal time to reach the appropriate concentration of NO (p < 0.001), and the duration of time at this concentration was longer for the abnormal sperm group (p < 0.001). Both the basal time and the duration of time were significantly correlated with sperm viability and percentage of progressive sperm (p < 0.001). The experimental group had a significantly higher percentage of progressive sperm than the control group (p < 0.001).

Conclusions

We hypothesize that there is a certain regularity to both NO concentration and its duration of time in regards to sperm capacitation, and that an adequate duration of time at the appropriate NO concentration is beneficial to sperm motility.     

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.     

Mouse Uterine 24p3 Protein as a Suppressor of Sperm Acrosome Reaction

Under in vitro conditions, incubation with 0.3% bovine serum albumin (BSA) and 1.8 mM CaCl₂ induces mouse sperm capacitation and increases the consequential acrosome-reaction. The effect of mouse uterine 24p3 protein on such stimulated sperm has been investigated to understand the biological function of the 24p3 protein. Variations in the intracellular pH (pHi), calcium concentration, cAMP levels and tyrosine phosphorylation in cytosol were determined and on in vitro mouse fertilization was evaluated. The presence of 24p3 protein reduced the response of sperm to BSA and calcium by suppressing the elevation of intracellular pH, calcium uptake, cAMP accumulation and protein tyrosine phosphorylation of BSA/calcium-stimulated sperm and showed inhibitory effect on mouse in vitro fertilization. The results indicated the inhibition of the BSA-stimulated sperm acrosome reaction by 24p3 protein then suppressed sperm fertilization. We suggested that the 24p3 protein acts as an in vitro inhibitor of the acrosome reaction in BSA stimulated sperm and this might be an anti-fertilization factor in vitro.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

Medium effects on capacitation and sperm penetration through the zona pellucida in inbred BALB/c spermatozoa

Inbred BALB/c mice are one of the most difficult inbred strains to fertilize in vitro. In this study we examined the abilities of various media used for mouse in vitro fertilization (IVF) to support capacitation and sperm penetration through the zona pellucida (ZP) of inbred BALB/c spermatozoa. Media examined were TYH, M16, CZB, mWhitten medium, T6, modified Tyrode's solution (mTyrode's), mKSOM, MEM and TCM199. Modified human tubal fluid (mHTF) was used as a control medium. When sperm were capacitated and inseminated in the same medium, mHTF showed the best fertilization (approximately 80%) scored by male pronuclear formation (<26%) at 5h post-insemination (PI). When sperm were capacitated in various media and inseminated in mHTF, sperm capacitated in TYH solution (93%) but no other media (<45%) showed a significantly higher level of sperm nuclear decondensation (SND) than mHTF at 2 h PI (approximately 65%). When sperm were capacitated in mHTF and inseminated in various media, only mTyrode's (52%) was not significantly lower than mHTF (66%) in terms of SND at 2h PI (<49%). Sperm capacitation also was examined by chlortetracycline (CTC) staining. Sperm capacitated in TYH solution showed a significantly higher percentage of capacitation (46%) than those treated in HTF (28%) and other media (<24%). These results indicate that the best approach for IVF in the BALB/c strain is capacitation in TYH and insemination in mHTF. Poor fertilization of BALB/c may result from suboptimal conditions of sperm capacitation and insemination, and overall IVF success may differ depending on strains used.     

Nutlin-3a Decreases Male Fertility via UQCRC2

Ubiquinol-cytochrome-c reductase core protein 2 (UQCRC2) is a component of ubiquinol-cytochrome c reductase complex that is known to correlate with male fertility via spermatogenesis. Simultaneously, nutlin-3a is a small molecule antagonist of mouse double minute 2 repressor (MDM2), activate p53 and induce apoptosis responsible for spermatogenesis. To date, however there are no known effects of nutlin-3a on reproduction. Therefore, present study was designed to investigate the effect of nutlin-3a on male fertility via UQCRC2. In this in vitro trial with mice spermatozoa, we utilized CASA, CTC staining, ATP assay, western blotting, and IVF to measure the main study outcome. The short-term exposure of spermatozoa in nutlin-3a decreases sperm motion kinematics, intracellular ATP production, capacitation, the acrosome reaction, UQCRC2, and tyrosine phosphorylation (TYP) of sperm proteins in a dose-dependent manner. Notably, the decreased UQCRC2 and TYP were associated with reduced sperm kinematics, ATP production, and capacitation, which ultimately led to adverse effects on male fertility such as poor fertilization rates and embryo development. Thus, nutlin-3a may be considered as a potential male contraceptive agent due to its ability to decrease fertility secondary to changes in overall sperm physiology and embryonic development. However, the results of this preliminary study have to be confirmed by additional independent trial.     

Reproductive Effects of Two Neonicotinoid Insecticides on Mouse Sperm Function and Early Embryonic Development In Vitro

Acetamiprid (ACE) and imidacloprid (IMI) are two major members in the family of neonicotinoid pesticides, which are synthesized with a higher selectivity to insects. The present study determined and compared in vitro effects of ACE, IMI and nicotine on mammalian reproduction by using an integrated testing strategy for reproductive toxicology, which covered sperm quality, sperm penetration into oocytes and preimplantation embryonic development. Direct chemical exposure (500 µM or 5 mM) on spermatozoa during capacitation was performed, and in vitro fertilization (IVF) process, zygotes and 2-cell embryos were respectively incubated with chemical-supplemented medium until blastocyst formation to evaluate the reproductive toxicity of these chemicals and monitor the stages mainly affected. Generally, treatment of 500 µM or 5 mM chemicals for 30 min did not change sperm motility and DNA integrity significantly but the fertilization ability in in vitro fertilization (IVF) process, indicating that IVF process could detect and distinguish subtle effect of spermatozoa exposed to different chemicals. Culture experiment in the presence of chemicals in medium showed that fertilization process and zygotes are adversely affected by direct exposure of chemicals (P<0.05), in an order of nicotine>IMI>ACE, whereas developmental progression of 2-cell stage embryos was similar to controls (P>0.05). These findings unveiled the hazardous effects of neonicotinoid pesticides exposure on mammalian sperm fertilization ability as well as embryonic development, raising the concerns that neonicotinoid pesticides may pose reproductive risks on human reproductive health, especially in professional populations.     

Intracytoplasmic Sperm Injection (ICSI) in Extreme Cases of Male Infertility

Introduction

Severely compromised spermatogenesis typical of men with virtual azoospermia or non-obstructive azoospermia requires an extreme search for spermatozoa. Our goal was to evaluate the usefulness of a meticulous search carried out in ejaculated or surgically retrieved specimens in achieving pre- and post-implantation embryo development.

Patients and Methods

In a retrospective cohort study carried out in an academic institution, intracytoplasmic sperm injection (ICSI) outcomes were reviewed as a function of length of microscopic sperm search in ejaculated and surgically retrieved specimens. Couples whose male partner presented with either virtual or non-obstructive azoospermia were treated by ICSI and categorized according to the time spent in identifying and retrieving enough spermatozoa to inject all the oocyte cohort. Semen parameter, fertilization, pregnancies, deliveries, and child welfare in relation to increasing search time were analyzed and compared.

Result(s)

The maternal and paternal ages were comparable in both ejaculated and testicular sperm extraction (TESE) groups along with the oocytes retrieved. The fertilization rates for both ejaculated and TESE progressively decreased with increasing time (P<0.0001). Clinical pregnancies in the ejaculated cohort remained satifactory. In the TESE cohort, there was a decrease in pregnancy rate with increasing time, from 44% to 23%. In a limited number of cases, offspring health was evaluated in both semen sources and appeared reassuring.

Conclusion(s)

An extensive and at time exhaustive sperm quest yields kinetically and morphologically impaired spermatozoa without apparent impact on embryo developmental competence. Retrieval of spermatozoa from the seminiferous tubules provided more consistent fertilization and pregnancy outcomes than those retrieved from the ejaculate. A trend indicated that pregnancy rate decreased as search time increased in the TESE group. The utilization of the scarce and unselected spermatozoa did not obviously impair embryo development or cause post-implantation errors.     

Vasopressin Effectively Suppresses Male Fertility

Arginine vasopressin (VP) is neurohypophysial hormone has been implicated in stimulating contractile activity of the male reproductive tract in the testis. Higher levels of VP decrease sperm count and motility. However, very little is known about the involvement of VP in controlling mammalian reproductive process. The goal of this study was to confirm that effect of VP receptor (AVPR2) on sperm function in capacitation condition. Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist that operates only on AVPR2, was used. Also, Mouse spermatozoa were incubated with various concentrations of dDAVP (10⁻¹¹–10⁻⁵ M) and sperm motility, capacitation status, Protein Kinase A activity (PKA), tyrosine phosphorylation, fertilization, and embryo development were assessed using computer-assisted sperm analysis, Combined Hoechst 33258/chlortetracycline fluorescence, Western blotting, and in vitro fertilization, respectively. AVPR2 was placed on the acrosome region and mid-piece in cauda epididymal spermatozoa, but the caput epididymal spermatozoa was mid-piece only. The high dDAVP treatment (10⁻⁸ and 10⁻⁵ M) significantly decreased sperm motility, intracellular pH and PKA substrates (approximately 55 and 22 kDa) and increased Ca²⁺ concentration. The highest concentration treatment significantly decreased PKA substrate (approximately 23 kDa) and tyrosine phosphorylation (approximately 30 kDa). VP detrimentally affected capacitation, acrosome reaction, and embryo development. Treatment with the lowest concentration (10⁻¹¹ M) was not significantly different. Our data have shown that VP stimulates ion transport across sperm membrane through interactions with AVPR2. VP has a detrimental effect in sperm function, fertilization, and embryonic development, suggesting its critical role in the acquisition of fertilizing ability of mouse spermatozoa. These research findings will enable further study to determine molecular mechanism associated with fertility in capacitation and fertilization. It is also an important pivotal precondition to the progress of diagnostic test to identify infertility and to apply male contraception.     

Promotion of capacitation of guinea pig spermatozoa by the membrane mobility agent,A2C,and inhibition by the disulfide-reducing agent,DTT

The membrane mobility agent A₂C accelerates the onset of the acrosome reaction of guinea pig spermatozoa by promoting capacitation. Spermatozoa incubated in a suspension of A₂C particles in Ca²⁺-free medium for one hour undergo a synchronous, rapid acrosome reaction upon the addition of Ca²⁺. These acrosome-reacted spermatozoa are capable of fertilization as assessed by their ability to penetrate (fuse with) zona-free hamster eggs. The disulfide-reducing agent, dithiothreitol (DTT) inhibits A₂C-mediated capacitation. It also blocks fertilization of zone-free eggs by acrosome-reacted spermatozoa by preventing attachment of the spermatozoa to the egg plasma membrane. The mode of A₂C action on spermatozoa is compared to that of A₂C-induced fusion in somatic cells. The similarity of the molecular events in the sperm membrane during capacitation and the acrosome reaction to these in other fusion events is pointed out. Inhibition of capacitation by DTT points to the importance of membrane and/or submembrane proteins and thiol groups in this process. Oxidation of sperm membrane SH groups may play an important role in in vivo capacitation.     

Relationship between calcium,cyclic AMP,ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility

Capacitation of hamster caudal spermatozoa at a density of 1 × 10⁶/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation. Treatment of spermatozoa at a density of 1 × 10⁶/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility. To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors. These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species.