Single-cell Motility Analysis of Tethered Human Spermatozoa.

Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm's ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. This assay complements bulk measurements of sperm parameters using commercially-available systems for computer-assisted sperm analysis (CASA)

Role of Carbonic Anhydrase IV in the Bicarbonate-Mediated Activation of Murine and Human Sperm

HCO3− is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO3− containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO3− stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO3− enters spermatozoa either directly by anion transport or indirectly via diffusion of CO2 with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO2 with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV−/− mice we found a 32.13% reduction in total CA activity in the latter. The CA IV−/− sperm also have a reduced response to CO2. While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO2 application, beat frequency of CA IV−/− sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO3−, which is necessary for stimulation of sAC and hence early activation of spermatozoa

Legionella pneumophila - intracellular processing of LPS and association of the phagosome with the endoplasmic reticulum during intracellular replication within human monocytes

Da das LPS von L. pneumophila Sg 1 keine klassische Endotoxinfunktion aufweist und einen ungewöhnlichen strukturellen Aufbau hat, übernimmt diese Molekül mit großer Wahrscheinlichkeit andere Funktionen, wie beispielsweise Interaktion mit Wirtszellmembranen, die im Rahmen der vorliegenden Arbeit mittels Immunogoldmarkierungen herausgearbeitet werden sollten. Ein weiteres Ziel war es, die zeitlichen Abläufe, denen die Rekrutierung von endoplasmatischen Retikulum zu L. pneumophila-Phagosomen unterliegt, mit Hilfe konventioneller Elektronenmikroskopie genauer zu untersuchen, da hierfür bis dato nur spärliche Daten vorliegen. Zur Erfassung der LPS-Synthese dienten RT-PCR Studien mit acht daran beteiligten Genen. Es zeigte sich, dass sich das LPS von internalisierten Bakterien ablöste und an inneren Phagosomenmembranen ansammelte. Darüber hinaus war auch eine intrazelluläre Prozessierung dieses Moleküls in das Zytoplasma der Wirtszellen nachzuweisen. Weiterhin konnte gezeigt werden, dass die Rekrutierung des endoplasmatischen Retikulums zu L. pneumophila enthaltenden Phagosomen in zwei sich wiederholenden Zyklen verlief. Des Weiteren unterlag die LPS-Synthese in intrazellulären Legionellen einer Regulation, wohingegen die LPS-Synthese nicht reguliert war, wenn die Bakterien extrazellulär replizierten. Erste Versuche machten deutlich, dass dem LPS für das intrazelluläre Überleben wahrscheinlich eine essentielle Funktion zukommt, da Legionellen, die nur sehr geringe Mengen an LPS aufwiesen, nicht zur intrazellulären Replikation befähigt waren.In comparison to lipopolysaccharides of other gram-negative bacteria, the L. pneumophila-LPS has a different and unique structure. Due to high levels of long branched fatty acids and elevated levels of O- and N-acetyl groups, this LPS is highly hydrophobic. Additionally, this type of LPS does not function as a classical endotoxin. Therefore, other possible functions, such as integration into host cell membranes during intracellular replication remain to be investigated. Additionally, the kinetics regarding recruitment of endoplasmic reticulum towards L. pneumophila containing phagosomes were determined since such data is not complete yet. Following a complete infection cycle, we could elucidate by electron microscopy a biphasic traffick of endoplasmic reticulum towards the phagosomes. Performing immunogold labeling, we also observed remarkable intracellular traffic of L. pneumophila-LPS during intracellular multiplication within human monocytes as well as shedding of LPS from bacterial surfaces towards phagosomal membranes. Additionally, intracellular processing of LPS across the cytoplasm could be observed. Furthermore, time-dependent regulation of LPS content could be detected. To further elucidate the intracellular fate of L. pneumophila-LPS, genes coding for LPS synthesis and LPS transport were investigated on mRNA level by RT-PCR. These studies revealed intracellular regulation, whereas no regulation occurred in extracellularly broth cultivated Legionella bacteria. Since Legionella bacteria exposing low levels of LPS were not able to multiply intracellularly, the LPS is likely to be an essential factor to guarantee intracellular survival

Regulation of the sperm calcium channel CatSper by endogenous steroids and plant triterpenoids

The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that (i) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and (ii) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization

Glucose as energy substrate increases sperm beat frequency dramatically.

<p>Sperm were continuously perifused for 20 min with buffer HS containing different energy substrates (10 mM lactate, 10 mM pyruvate and 5 mM glucose) as indicated. Beat frequency of single sperm was analyzed at t  = 0 min (white bars), t  = 20 min (gray bars) and t  = 60 min (black bars). Shown are mean values±s.e. of 35 cells of 3 animals. With no energy substrate present, flagellar beat declines significantly compared to t  = 0 min (***p<0.001). With glucose as the mere substrate, beat acceleration is significant compared to t  = 20 min and 60 min (***p<0.001) of all other substrate compositions.</p

Glucose is a pH-Dependent Motor for Sperm Beat Frequency during Early Activation

<div><p>To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH_i) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO₃− is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH_i ≥7.1, whereas a pH_i ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO₂ hydration and thus the production of HCO₃− by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH_i-dependent way to produce HCO₃−_, thus enhancing sperm beat frequency and contributing to fertility.</p> </div

The glucose-mediated acceleration of sperm beat frequency involves mitochondria and can be mimicked by succinate.

<p><b>A</b>, After a 5 min pre-run of either 3 mM AOA (black plot) or 3 mM DCA (grey plot) in buffer HS containing 10 mM lactate, sperm were perifused with the respective inhibitor in the presence of 5 mM glucose for 15 min. AOA/DCA were washed out for a subsequent period of 60 min with HS buffer supplemented with 5 mM glucose. Compared to t  = 0 min, the frequency values significantly increased at t ≥30 min (***p<0.001). <b>B</b>, After initial perifusion with 10 mM lactate, sperm were first stimulated for 10 min with 5 mM succinate and then for another 10 min with 5 mM glucose (black plot). For control measurements (gray plot), sperm were stimulated for 20 min with only glucose. Compared to t  = 0 min, the frequency values significantly increased at t  = 5 min (**p<0.05) and at t ≥10 min (***p<0.001) in both approaches. <b>C</b>, Treatment with 10 mM lactate was followed by a 10-min perifusion with 10 mM pyruvate and 15 mM MA simultaneously present in the buffer and a subsequent stimulation with 5 mM glucose for further 20 min (black plot). During the control measurements (gray plot), sperm were treated the same way except for omitting MA. Co-application lead to a significant increase of flagellar beat at t  = 5 min with **p<0.05 and at t ≥10 min with ***p<0.001. In the control significance of ***p<0.001 was reaches at t ≥15 min. Shown are mean values±s.e. of 38 cells of 3 animals. <b>D</b>, Sperm were exposed for 100 s to 10 mM lactate before being stimulated for 150 s with 10 mM pyruvate +15 mM MA (dashed line) or 5 mM succinate (dotted line). Thereafter, cells were allowed to recover for 200 s to initial values. The plots of <b>A</b> to <b>D</b> represent mean values±s.e. of 25–33 cells of 3 animals. The graphs in <b>d</b> show mean values of 30–35 cells of 3 animals and mean slopes were analyzed as described in the penultimate figure legend.</p