Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro

Aim: The effects of certain uropathogenic microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermidis and Mycobacterium tuberculosis) on human sperm motility characteristics were studied in vitro. Methods: In 10 healthy fertile men, ejaculates were aseptically obtained by masturbation and With a swim-up technique, a sperm suspension of high motility and purity was obtained. Several uropathogenic bacteria were obtained from outpatients with genitourinary tract infections. The sperm suspension was incubated with the pathogens at a bacteria: sperm ratio of 50:1 at 37℃. The sperm mobility parameters were estimated with a computerassisted sperm analyzer (CASA) provided with a multiple-exposure photography system (Madi Corp., Zhejiang, China). Measurements were carried out at 0, 2 and 4 hours of incubation. Results: Staphylococcus aureus significantly decreased the sperm motility and viability, but Staphylococcus epidermidis, Mycobacterium tuberculosis and Neisseria gonorrhoeae did not. Conclusion: Staphylococcus aureus has an inhibitory effect on human sperm motility in vitro.

The cytoplasmic droplet may be indicative of sperm motility and normal spermiogenesis

Although the cytoplasm of spermatids is removed at the end of spermiogenesis, a tiny portion is usually retained in the sperm flagellum, which is termed the cytoplasmic droplet （CD） in mammals. CDs are believed to play a role in sperm volume adaptation. However, we have noticed that epididymal spermatozoa that display initial （flagellation in situ） and progressive motility mostly possess CDs, whereas spermatozoa without CDs are rarely motile, suggesting that CDs have a role in motility development during sperm epididymal maturation. In the present study, we analyzed the relationship between the presence or absence of CDs, motility development and positional changes of CDs during sperm epididymal maturation in mice and monkeys. We also examined CDs on spermatozoa of three knockout mouse lines with late spermiogenic defects. Our data suggest that the CD is a normal organelle transiently present exclusively on epididymal spermatozoa, and normal CD morphology and location are associated with normal motility development during epididymal maturation of spermatozoa. Abnormal CD formation, e.g., a complete lack of CDs or ectopic CDs, is indicative of defective spermiogenesis. If CDs are essential for sperm motility development, then CDs may represent an ideal drug target for the development of non-hormonal male contraceptives.

Protein phosphatase PP1γ2 in sperm morphogenesis and epididymal initiation of sperm motility

The serine/threonine phosphatase （PP1） isoform PP1γ2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1γ2 catalytic activity holds motility in check in immature spermatozoa. Inhibition of PP1γ2 causes motility initiation in immature spermatozoa and motility stimulation and changes in flagellar beat parameters in mature spermatozoa. The PP1γ2 isoform is present in all mammalian spermatozoa studied： mouse, rat, hamster, bovine, non-human primate and man. We have now identified at least four of its regulatory proteins that regulate distinct pools of PP1γ2 within spermatozoa. Our studies provide new insights into biochemical mechanisms underlying development and regulation of sperm motility. We hypothesize that changes in sperm PP1γ2 activity as a result of phosphorylation and reversible binding of the regulatory proteins to the catalytic subunit are critical in the development and regulation of motility and the ability of sperm to fertilize eggs. Targeted disruption of the Ppplcc gene, which encodes the PP1γ1 or PP1γ2 isoforms, causes male infertility in mice as a result of impaired spermiogenesis. Our observations suggest that, in addition to motility, the protein phosphatase PP1γ2 might play an isoform-specific function in the development of specialized flagellar structures of mammalian spermatozoa. （Asian J Androl 2007 July; 9： 445--452）

Sperm motility inhibitory effect of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in langur monkey, Presbytis entellus entellus

Aim： To assess the contraceptive efficacy of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in langur monkeys. Methods： The test substance was given p.o. to five monkeys at 50 mg/kg body weight/day for 360 days. Control animals （n = 3） received olive oil as vehicle. Sperm parameters as per World Health Organization standards, sperm functional tests, morphology of testis and epididymis, haematology, clinical biochemistry, serum testosterone and libido were evaluated. Following completion of 360 days treatment the animals were withdrawn from the treatment and the recovery pattern was assessed by semen analysis and sperm functional tests. Results： Total inhibition of sperm motility was observed following 60 days of treatment that continued until 360 days study period. Sperm count, percent viability and percent normal spermatozoa showed a drastic decline following 30 days of treatment. Sperm morphology showed predominant mid piece abnormalities. Sperm functional tests scored in sterile range. Histology and ultrastructure of testis revealed vacuolization in the Sertoli cells and germ cells. Loss of cytoplasmic organelles was evident in spermatocytes and round spermatids. Histology and ultrastructure of epididymis of treated animals were comparable to those of control animals. Hematological and serum clinical parameters and testosterone levels fluctuated within the control range throughout the study period. Recovery was evident following 60--120 days of treatment withdrawal. Conclusion- The results suggest that the benzene chro- matographic fraction of the chloroform extract of the seeds of Carica papaya shows contraceptive efficacy without adverse toxicity, mediated through inhibition of sperm motility. （Asian JAndro12008 Mar; 10： 298-306）

Effect of vitamin E on human sperm motility and lipid peroxidation in vitro

Aim: To assess the protective efficacy of vitamin E to counteract the reactive oxygen species (ROS) mediated damage onsperm motility, viability and lipid peroxidation. Methods: Human semen samples were obtained from the local hospi-tal. The split seminal fractions freed of seminal plasma were reconstituted in Ringer-Tyrode and subjected to varied vita-min E concentrations (0.1 - 2 mmol/L). Results: Dose-dependent improvement in both motility and viability accom-panied by concomitant decrease in malondialdehyde (MDA--an end product of lipid peroxidation) following vitamin Esupplementation was noticed. Conclusion: Vitamin E protects against the ROS mediated damage on spermatozoa.Vitamin E supplementation could be of clinical importance for prolonged spermatozoal storage whenever needed. (AsianJ Androl 1999 Sep; 1: 151 - 154 )

Studies on the Relationship between Urokinase Plasminogen Activator(uPA)and Human Sperm Motility

To clarify the role of urokinase plasminogen activator(uPA) in the mechanisms of regulating sperm motility and the ability of fertilizing, we investigated the quantities and activities of uPA in human seminal Plasma and on the membrane of spermatozoa.Semens were harvested from 22 infertile patients with asthenospermia and 20 healthy fertile men according to WHO standards. To quantify the membrane-bound uPA in the samples, polyclonal antibodies against human urokinase were employed by means of a sandwich ELISA. The uPA activities in seminal plasma and on the surface of spermatozoa were determined using Agarose-Fibrine-Plate method and the experiment of immunological identification with polyclonal antibodies against urokinase. In lysates of spermatozoa, significantly lower levels of uPA(23. 1±7.35 mu/106 cells ) and uPA activity (5.13±3.85 mu/106 cells) were found in patient group as compared to healthy fertile men exhibiting normospermia (29. 89±9. 40 mu/105 cells and 10. 17±6. 18 mu/106 cells). In seminal plasma, uPA activity in patient group (2134±1581. 3 IU/L)was also found significantly lower than that of normal group (3365±1859. 5 IU/L). Positive correlations were observed between sperm motility and uPA quantities (r=0. 48, P＜0. 005), as well as with uPA activities (r= 0. 45,P＜0. 005).Thus, it is inferred that membrane associated uPA on human spermatozoa may be related directly to sperm motility and fertility.

Optimal analysis conditions for sperm motility parameters with a CASA system in a passerine bird, Passer montanus

Background:Sperm motility parameters,which can be measured objectively and repeatedly by a computer-assisted sperm analysis(CASA)system,are important indicators of sperm quality.However,the sperm motility parameters assessed by a CASA system can be affected by various factors,including instrument components and settings,sperm preparation or analysis procedures.To date,no standardized protocol is available that would permit to assess sperm kinetic characteristics in passerine birds and this lack precludes any comparison of sperm swimming ability and sperm quality across species.Methods:In this study,we chose the Tree Sparrow(Passer montanus)as the object to evaluate sperm motility parameters,including sperm motility,sperm velocity and sperm movement trajectory,at different analysis time,temperatures and pH using the WLJY-9000 CASA system.Results:Sperm motility parameters remained statistically unchanged at 1‒9 min.Progressive motility was similar at 38℃ and 40℃,but a greater percentage of slow progressive sperm was detected at 38℃ compared to 40℃ and 42℃.Additionally,progressive motility was lower and immotility was higher at 42℃than 38℃and/or 40℃(close to the body temperature of the Tree Sparrow).The percentages of rapid progressive sperm,progressive sperm and immotile sperm were statistically similar at pH 7.0,7.5 and 8.0 with the exception of lower percentage of progressive sperm at pH 7.0 compared to pH 7.5.In addition,slower sperm velocity and worse sperm movement trajectory were found at pH 6.0 and 9.0 than those at pH 8.0,7.5 or 7.0.Conclusions:Our study indicates that the ideal conditions for sperm motility parameters assessment in Tree Sparrow are obtained between 1 and 9 min after dilution,an environment at body temperature(40℃)and a pH around 7.5-8.0.The results of this study provide a reference for the evaluation of sperm characteristics and sperm quality using a CASA system in passerine birds.

Influences of dibutyryl cyclic adenosine monophosphate and forskolin on human sperm motility in vitro

Aim: To study the influences of dibutyryl cyclic adenosine monophosphate (dbcAMP) and forskolin on human sperm motility in vitro. Methods: Semen samples, aseptically obtained by masturbation and prepared by swim-up technique from 20 fertile men, were incubated with different concentrations of dbcAMP and forskolin at 37 癈. Measurements were carried out after 10 min, 20 min, 30 min and 60 min incubation. Motility parameters were estimated by using an automatic analyzing system. Results: Treatment with dbcAMP or forskolin resulted in a significant increase in sperm motility and progressive motility. The larger the concentrations of dbcAMP or forskolin, the greater the effect appeared. The straight linear velocity and curvilinear velocity were not affected by both agents. Conclusion: dbcAMP and forskolin increase the motility and progressive motility of human sperm in vitro.

Human sperm motility stimulating activity of a sulfono glycolipid isolated from Sri Lankan marine red alga Gelidiella acerosa

Aim: To evaluate the sperm motility stimulating activity of a sulfono glycolipid (S-ACT-1) isolated from Gelidiellaacerosa, a Sfi Lankan marine red algae. Methods: S-ACT-I, a white amorphous powder was separated from morepolar fractions of the hexane soluble of 1:1 CH_2Cl_2/MeOH extract and subjected to ~1H, ^(13)C NMR and IR Spectroscopyafter reverse phase HPLC for identification. Effects of S-ACT-1 on human sperm motility was assessed in vitro at 10,100 and 1000μg/mL concentrations at 37℃ for 0, 5, 15, 30 and 60 min. Results: S-ACT-1 was identified as aglycolipid sulfate. The lower dose increased the sperm motility slightly, whilst the medium dose significantly increasedthe motility (P < 0.05) from 5 min of incubation reaching a peak at 15 min and the stimulant effect was sustainedthroughout the experimental period. Furthermore, the medium dose rendered 80% of the immotile viable sperm motile.In contrast, the highest dose impaired the sperm motility. The sperm stimulating activity of S-ACT-1 was dose-depen-dent and had a bell-shaped dose response curve for all the 5 incubation periods. Conclusion: S-ACT-1 of Gelidiellaacerosa is a Sulfono glycolipid. S-ACT-1 has a potent sperm motility stimulating activity in vitro and has the potentialto be developed into a sperm stimulant. (Asian J Androl 2001 Mar; 3: 27-31)

Inhibition of sperm motility does not affect live-dead separation of bull sperm by glass beads

Aim: This study was designed to explore factors which influence binding of dead versus live sperm to glass filters.Methods: Multiple semen collections from bulls were used to explore selective filtration of bull sperm as influencedby nonlethal inhibition of sperm motility with fluoride, killing of sperm by quick-freezing, alteration of the glass sur-face with silicone, and different intervals of sexual rest between semen collections. Results: A comparison of glassspheres 100, 200 and 390μm in diameter indicated that 200 μm spheres were optimal for selective filtration. Quantita-tive separation of live from dead sperm was demonstrated with a correlation between the percentage of motile sperm andretention of sperm by the filter of r = -0.87 (P < 0.05). Up to 0.02 mol/L NaFl did not alter the proportion ofsperm retained by the filter despite inhibiting sperm motility during filtration, an inhibition which was reversible. Pro-portions of live-dead sperm, based upon eosin staining, were unaffected by fluoride. Coating the glass spheres with sil-icone greatly reduced selective filtration. Dead sperm adherence to glass was reduced and resistance to NaFl inhibitionwas increased by daily ejaculation versus one-week intervals of sexual rest. Conclusion; These studies indicate thatthe adherence of sperm to glass is primarily due to some form of physico-chemical change accompanying death of thesperm cell independent of active sperm motility. This attraction between the sperm plasma membrane and glass is modi-fied by the age of the ejaculated sperm. This information is useful in evaluating different clinical procedures used forsperm separation. (Asian J Androl 2001 Sep; 3: 193-198)

Sensitivity of sperm motility assay for detecting endotoxin effect on human sperm in vitro

Objective: To evaluate the sensitivity of sperm motility assay for detecting the endotoxin effect on human sperm in vitro. Methods: Motile human sperm were separately incubated for up to 24 hours with different concentrations of endotoxin (0.5, 1, 10, 1000, 10 000 and 50 000 ng/mL). Then the sperm motility was determined. The effect of endotoxin on the sperm motility in media without albumin was also determined. In addition, at the endotoxin concentrations of 0.5, 1 and 10 ng/mL, the sensitivity of the assay was compared to those of 1-cell and 2-cell mouse embryo bioassays. Results: At levels of 0.5-1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility after 24 h of incubation (P>0.05), while it was significantly inhibited at endotoxin levels of 10 000 and 50 000 ng/mL. In media without albumin, endotoxin levels of 50 000 and 1 000 ng/mL, markedly inhibited the sperm motility after 2 or 8 h of incubation (P<0.01). With media containing 0.5 and 1 ng/mL endotoxin, there was a significant reduction in the development rate at all developmental stages with 1-cell and 2-cell mouse embryo assays and at the level of 10 ng/mL, the embryo development was completely arrested. Conclusion: The sperm motility assay could detect high levels of endotoxin effect in vitro, but its sensitivity is low as compared with the 1-cell or 2-cell mouse embryo bioassay.

Evaluation of effects of 1,3-dinitrobenzene on sperm motility of hamster using computer assisted semen analysis (CASA)

Aim: To evaluate the effects of 1,3-dinitrobenznene (mDNB) on sperm motility of hamster and to correlate the re-sults with the fertility. Methods: Adult male hamsters were gavaged with one of the 3 dose regimes of mDNB(1.5 mg daily for 4 weeks ,1.5 mg one day a week for 4 weeks and 1.0 mg 3 days a week for 4 weeks). Computerassisted semen analysis (CASA) was used to analyse the sperm motility parameters, curvilinear velocity (VCL) andstraight line velocity (VSL) of sperm in distal corpus epididymides and distal cauda epididymides. In vitro fertilisationwas carried out only for 1.5 mg mDNB daily group to determine the sperm fertilising capacity. Results: There wasa significant reduction in sperm velocity parameters at weeks 3 and 4 after treatment, which was correlated with a de-cline in sperm fertility. Conclusion; Sperm velocity parameters may be used to determine the effect of a toxic insulton the sperm function.

Involvement of Ca^(2+)-activated K^+ Channels in Receptor-Regulated Sperm Motility in Rats

Previous voltage clamp studies have demonstrated the modulation of sperm Ca 2+ activated K + (KCa) channels expressed in Xenopus oocytes by angiotensin II (Ang II) and extracellular ATP via AT 1 receptor and P 2U receptor, respectively. In the present study, we investigated the involvement of KCa channels in receptor regulated sperm motility of the rat using a computer aided sperm analysis system, HTM IVOS, in conjunction with Ca 2+ mobilizing agents, receptor agonists/antagonists and KCa channels blockers. The percentage of motile sperm was increased by ionomycin (0.5 μmol/L), which could be inhibited by K + channel blockers, tetraethylammonium (TEA 1 μmol/L ) or charybdotoxin (ChTX, 300 nmol/L) indicating the presence of KCa channels. Ang II, at low concentration, 10 nmol/L, was found to increase motility, however, at higher concentration, 1 μmol/L, percentage of motility was found to be suppressed. Both stimulatory and inhibitory effects of Ang II could be reversed by losartan, a specific antagonist of AT 1 receptors, but not AT 2 antagonist PD123177, indicating the involvement of AT 1 but not AT2 receptor in mediating both effects. ChTX also abolished both stimulatory and inhibitory effects of Ang II, suggesting the involvement of KCa channels. The percentage of motility was also enhanced by extracellular ATP, a factor known to be involved in sperm activation. The ATP enhanced sperm motility was mimicked by UTP, and inhibited by ChTX and reactive blue, an antagonist of P 2 receptor, indicating the involvement of both P 2U and KCa channels. RT PCR study was also conducted to confirm the expression of KCa channels, AT 1 receptors and P 2U receptor, but not AT 2 receptor, in rat caudal epididymal sperm. The present findings suggest an important role of KCa channels in the regulation of sperm motility by AT 1 and P 2U receptors.

Effects of Seminal Plasma Relaxin on Human Sperm Motility

To clarify the role of endogenous relaxin on sperm motility, relaxin in semen was neutralized by anti relaxin antibody in vitro. 22 semen samples were collected from infertility clinic and tested with Hamilton Thorn 2000 Motility Analyzer to detect sperm motility (%), progressive motility (%), path velocity (micro/sec) and velocity (0～4 grade) at the time of 0, 15, 30 and 60 min respectively. The results showed that sperm motility declined significantly after being incubated with anti relaxin serum. Sperm progressive motility declined more obviously. This experiment revealed that endogenous relaxin could play an important role in the physiological process of maintaining sperm motility, especially progressive motility.

Association of Lifestyle Factors and Sperm Motility in Adults from an Ethnic Minority Region of Southwest China

Objectives: To understand sperm motility in adults and its association with lifestyle in an ethnic minority area in Southwest China. Methods: A hospital-based cross-sectional study to assess sperm motility in male adults was conducted at the Reproductive Health Center from January 2018 to May 2019. The data was collected with a questionnaire and semen quality was analyzed with Computer-Aided Sperm Analysis system (CASA). Analysis of covariance (ANCOVA) was used to measure the relationship between lifestyle factors and sperm motility. Results: A total of 349 people were recruited. Dietary celery intake was significantly related to the increase of sperm progressive motility and total motility (β = 7.00, 95% CI: 1.59, 12.42 and β = 7.26, 95% CI: 1.45, 13.07, respectively). Cola consumption was associated with increased sperm progressive motility (β = 9.71, 95% CI: 1.46, 17.96). Frequent use of plastic bags for meat food storage (β = -5.56, 95% CI: -10.61, -0.51), industry work (β = -5.64, 95% CI: -11.21, -0.07), organic disease (β = -6.14, 95% CI: -11.00, -1.28) and sedentary lifestyle (β = -5.92, 95% CI: -10.66, -1.17 for 3-5 h/d and β = -6.04, 95% CI: -11.60, -0.47 for ≥5 h/d, respectively) were related with the decreased sperm progressive motility. Meanwhile, using plastic bags for meat food storage (β = -6.37, 95% CI: -11.79, -0.95), industry work (β = -7.96, 95% CI: -13.94, -1.98) and sedentary lifestyle (β = -5.51, 95% CI: -10.60, -0.42 for 3-5 h/d and β = -6.03, 95% CI: -12.01, -0.06 for ≥5 h/d, respectively) were also risk factors for total motility. Conclusions: Some modifiable lifestyle factors such as job title, cola consumption, dietary celery intake, plastic bags for meat food storage, and sedentary hours were linked to male sperm motility, indicating that changing these lifestyles may improve it.

Pyruvate kinase M in germ cells is essential for sperm motility and male fertility but not spermatogenesis

Male germ cells employ specific metabolic pathways throughout their developmental stages.In a previous study,we discovered heightened expression of pyruvate kinase M(PKM),a pivotal glycolytic enzyme,in spermatogonia and spermatids.To gain deeper insights into PKM's roles in spermatogenesis,sperm function,and male fertility,we engineered a conditional-knockout mouse model(Pkm-vkO mice)to selectively disrupt the Pkm gene within germ cells.Despite maintaining regular testicular histology and sperm morphology,the male Pkm-vko mice were infertility,characterized by significant impairments in sperm motility and adenosine triphosphate(ATP)generation.In addition,Pkm-null spermatozoa exhibited similar deficits in protein tyrosine phosphorylation linked to capacitation,as well as compromised performance in in vitro fertilization experiments.To conclude,PKM's presence is not obligatory for the entirety of spermatogenesis in male germ cells;however,it emerges as a critical factor influencing sperm motility and overall male fertility.

Phosphodiesterase 10A inhibitor PF-2545920 as a prospective agent for the clinical promotion of sperm motility

Phosphodiesterase(PDE)inhibitors can improve sperm motility in patients with asthenozoospermia.However,the most commonly reported nonselective PDE inhibitor pentoxifylline and PDE5 inhibitor sildenafil have the disadvantages of requiring a high concentration and destroying sperm integrity.We examined the PDE10A inhibitor PF-2545920 to compare its ability to promote sperm motility with that of pentoxifylline and sildenafil.After seminal plasma was discarded,several semen samples were subjected to four treatments(control,PF-2545920,pentoxifylline,and sildenafil)to evaluate their ability to affect motility,viability,and spontaneous acrosome reactions.Intracellular calcium and adenosine triphosphate(ATP),mitochondrial membrane potential,and penetration through viscous medium were assessed by flow cytometry,luciferase,and hyaluronic acid after treatment with PF-2545920.Statistical analyses were performed using the analysis of variance statistical test.PF-2545920 elevated the percentage of motile spermatozoa compared to the control,pentoxifylline,and sildenafil groups at 10μmol l^(-1)(P<0.01).It is less toxic to GC-2spd mouse spermatocytes cells and spermatozoa and causes fewer spontaneous acrosomal reactions(P<0.05).PF-2545920 also increased mitochondrial membrane potential(P<0.001)and altered intracellular calcium(P<0.05)in a dose-dependent manner,including increasing sperm hyaluronic acid penetrating ability(P<0.05).Therefore,PF-2545920 might be an excellent choiceforstimulatingthe spermmotility.

Major regulatory mechanisms involved in sperm motility

The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.

AMP-activated kinase in human spermatozoa： identification, intracellular localization, and key function in the reRulation of sperm motility

AMP-activated kinase （AMPK）, a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work＇s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System （ISAS） in the presence or absence of the AMPK inhibitor compound C （CC）. AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed： percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

Prohibitin(PHB)interacts with AKT in mitochondria to coordinately modulate sperm motility

Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in spermatocytes results in impaired mitochondrial function.In addition,PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels,but a positive one with mitochondrial membrane potential and sperm motility.These results suggest that mitochondrial PHB expression plays a role in sperm motility.However,the mechanism of PHB-mediated regulation of sperm motility remai ns unk nown.Here,we dem on strate for the first time that PHB interacts with protei n kinase B(AKT)and exists in a complex with phospho-PHB(pT258)and phospho-AKT in the mitochondrial sheath of murine sperm,as determined using colocalization and coimmunoprecipitation assays.After blocking AKT activity using wortmannin(a phosphatidylinositol 3-kinase[PI3K]inhibitor),murine sperm have significantly(P<0.05)decreased levels of phospho-PHB(pT258)and the total and progressive motility.Furthermore,significantly(P<0.05)lower levels of phospho-PI3K P85 subunit a+γ(pY199 and pY46)and phospho-AKT(pS473;pT308)are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with no rmospermic subjects,which suggest a reduced activity of the PI3K/AKT pathway in these in fertile subjects.Importantly,these sperm from infertile subjects also have a significantly(P<0.05)lower level of phospho-PHB(pT258).Collectively,our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility,where PHB phosphorylation(pT258)level and PI3K/AKT activity are key regulatory factors.