Pascale Lybaert

Evidence for a clathrin-mediated recycling of albumin in human term placenta.

Abstract During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 μM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-β-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.

Expression of TMEM16A and SLC4A4 in human pancreatic islets.

Background/aims: Stimulation of insulin release by D-glucose is accompanied by Cl- and HCO3- efflux from pancreatic islet cells. The efflux of these anions may involve volume-regulated anion channels, including possibly TMEM16A, and the Na+-HCO3--cotransporter SLC4A4. The present study was designed to explore the expression of both TMEM16A and SLC4A4 in human pancreatic islets. Methods: Pancreases were obtained from human cadaveric donors. Immunodetection of TMEM16A and SLC4A4 was performed by immunohistochemistry on sections of fixed pancreas, while real-time PCR for the study of corresponding gene expression was performed on RNA extracted from both total pancreatic pieces and isolated pancreatic islets. Results: RT-PCR yielded lower levels of SLC4A4 in isolated islets than in the total pancreas, whilst a mirror image prevailed for TMEM16A mRNA. Immunohistochemistry of human pancreas, however, indicated comparable immunostaining of SLC4A4 in insulin-producing cells and exocrine pancreatic cells, whilst that of TMEM16A appeared less pronounced in insulin-producing cells than in exocrine cells. Conclusion: The present findings support the view that, in humans like in rodent, the regulation of anion fluxes in insulin-producing cells may involve both SLC4A4 and TMEM16A.

Team swimming in ant spermatozoa.

In species where females mate promiscuously, competition between ejaculates from different males to fertilize the ova is an important selective force shaping many aspects of male reproductive traits, such as sperm number, sperm length and sperm–sperm interactions. In eusocial Hymenoptera (bees, wasps and ants), males die shortly after mating and their reproductive success is ultimately limited by the amount of sperm stored in the queens spermatheca. Multiple mating by queens is expected to impose intense selective pressure on males to optimize the transfer of sperm to the storage organ. Here, we report a remarkable case of cooperation between spermatozoa in the desert ant Cataglyphis savignyi. Males ejaculate bundles of 50–100 spermatozoa. Sperm bundles swim on average 51% faster than solitary sperm cells. Team swimming is expected to increase the amount of sperm stored in the queen spermatheca and, ultimately, enhance male posthumous fitness.

A Genetic Variant of the Sperm-specific SLO3 K+ Channel has altered pH and Ca2+ sensitivities.

To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K+) channels and resultant K+ efflux. Sperm-specific SLO3 K+ channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pHi. However, in human sperm, the major K+ conductance is both Ca2+- and pHi-sensitive. It has been debated whether Ca2+-sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca2+, the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established.

KATP channel subunits are expressed in the epididymal epithelium in several mammalian species.

Abstract Adenosine triphosphate-sensitive K++ (KATP) channels are poorly characterized in the reproductive tract. The present study was designed to evaluate the putative expression of KATP channel subunits (Kir6.x and SURx) in the epididymis from different mammalian species. Immunohistochemical, Western blot, and RT-PCR techniques were used. A positive immunostaining for Kir6.2 (KCNJ11) and SUR2 (ABCC9) was observed by immunoenzymatic and immunofluorescent approaches in the principal epithelial cells throughout all regions of the rat and mouse epididymis. Double labeling with anti-aquaporin 9 (AQP9) and anti-Kir6.2 (KCNJ11) confirmed their colocalization in the principal cells. No immunostaining could be demonstrated for Kir6.1 (KCNJ8) and SUR1 (ABCC8) subunits. Under higher magnification, the immunostaining for Kir6.2 (KCNJ11) exhibited a cytoplasmic labeling that was more intense at the level of the Golgi apparatus along the whole epididymis. A similar pattern was observed for SUR2 (ABCC9), although in the latter case, the Golgi labeling appeared to be region specific. Spermatozoa in epididymal tubules from rodents also immunostained for Kir6.2 (KCNJ11) and SUR2 (ABCC9). Western blot analysis of epididymal total protein and crude membrane extracts from adult and prepubertal rats confirmed the presence of Kir6.2 (KCNJ11). SUR2 (ABCC9) protein expression was detected in adult epididymal extracts. Furthermore, RT-PCR established the presence of Kir6.2 (KCNJ11) and SUR2 (ABCC9) mRNA in prepubertal and adult mouse epididymis. Indirect immunofluorescence also documented the presence of Kir6.2 (KCNJ11) and SUR2 (ABCC9) in the epididymal epithelium, as well as in spermatozoa, of canine, feline, bovine, and human origin. These data demonstrate the presence of the KATP channel subunits, Kir6.2 (KCNJ11) and SUR2 (ABCC9), in epididymal epithelial cells and spermatozoa from several mammalian species. Although their physiological roles need to be fully characterized, it is tempting to propose that such types of K++ channels might be involved in protein secretion and fluid-electrolyte transport occurring along the epididymal epithelium, leading to spermatozoa maturation.

Detection of KATP channels subunits in human term placental explants and evaluation of their implication in human placental lactogen (hPL) and human chorionic gonadotropin (hCG) release.

INTRODUCTION ATP-sensitive potassium channels (KATP channels) have been identified in a variety of tissues. Nevertheless, the presence and role of such metabolism-sensitive K+ channels still remain to be unraveled in the reproductive system. METHODS The study evaluates the presence of KATP channel subunits in human term placental explants by immunohistochemistry, proximity ligation assay, Western blot and RT-PCR techniques. The potential involvement of KATP channels in human placental lactogen (hPL) and human chorionic gonadotrophin (hCG) release has been assessed radioimmunologically from human term placental explants incubated in the presence of different KATP channel modulators. RESULTS Immunolocalization of the KATP channel subunits documented both the Kir6.2 and SUR2 subunits in the syncytiotrophoblast of human term placenta. Their colocalization was demonstrated by proximity ligation assay and their presence was further confirmed by immunoblotting and RT-PCR. Kir6.1 subunit was immunolocalized in blood vessels media. SUR1 was not expressed at the mRNA level. Incubation of human term placental explants in the presence of increasing concentrations of modulators of KATP channels such as glibenclamide, tolbutamide, pinacidil or diazoxide did not affect the measured hCG and hPL secretory rates. DISCUSSION Our study reports, for the first time, the presence of the KATP channel subunits Kir6.2 and SUR2 in the human term syncytiotrophoblast. However, under the present experimental conditions, the activation or inhibition of these putative KATP channels by different pharmacological agents did not affect the hPL and hCG secretory rate of human term placental explants. CONCLUSION The present findings suggest that the human term syncytiotrophoblast might be endowed with KATP channels. Further studies should clarify their implication in the syncytiotrophoblast ionic homeostasis and hormone regulation.

Characterization and Potential Roles of Calretinin in Rodent Spermatozoa

Calretinin has been detected in various excitable cells but the presence and putative roles of such a calcium-binding protein has never been characterized in sperm. Epididymal spermatozoa were collected from C57Bl6 (wild-type, WT) or calretinin knockout (CR-/-) mice and Wistar rats. A specific staining for calretinin was detected by immunofluorescence in the principal piece of the flagellum, both in WT mouse and rat spermatozoa. Western blots confirmed the expression of calretinin in rat and WT spermatozoa as well as its absence in CR-/- mice. No significant difference was observed in the spontaneous acrosome reaction between WT and CR-/- sperm. The addition of the calcium-ionophore A-23187, Thapsigargin or Progesterone to WT or CR-/- incubated spermatozoa induced increases in the acrosome reaction but the stimulatory effects were identical in both genotypes. Motility measurements assessed by computer-assisted sperm analysis indicated that, under basal non-stimulatory conditions, CR-/- sperm exhibited a lower curvilinear velocity and a smaller lateral head movement amplitude, although no difference was observed for the beat cross frequency. After incubation with 25 mM NH4Cl, the curvilinear velocity, the amplitude of the lateral head movement and the hyperactivation were increased, while the beat cross frequency was decreased, in both genotypes. Evaluation of the in vivo fertility potential indicated that the CR-/- litter sizes were clearly reduced compared to the WT litter sizes. Our study describes, for the first time, the expression of calretinin in sperm. These data extend the potential implication of calcium-binding proteins in the sperm calcium-signaling cascade and bring new insights into the understanding of sperm physiology.