The Testicular and Epididymal Expression Profile of PLCζ in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides

Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22–54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ–related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyt

Effects of Ionomycin on Egg Activation and Early Development in Starfish

Ionomycin is a Ca2+-selective ionophore that is widely used to increase intracellular Ca2+ levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca2+ levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca2+ increase. The ionomycin-induced Ca2+ rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca2+ response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation

The clinical relevance of oliguria in the critically ill patient : Analysis of a large observational database

Funding Information: Marc Leone reports receiving consulting fees from Amomed and Aguettant; lecture fees from MSD, Pfizer, Octapharma, 3 M, Aspen, Orion; travel support from LFB; and grant support from PHRC IR and his institution. JLV is the Editor-in-Chief of Critical Care. The other authors declare that they have no relevant financial interests. Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Urine output is widely used as one of the criteria for the diagnosis and staging of acute renal failure, but few studies have specifically assessed the role of oliguria as a marker of acute renal failure or outcomes in general intensive care unit (ICU) patients. Using a large multinational database, we therefore evaluated the occurrence of oliguria (defined as a urine output 16 years) patients in the ICON audit who had a urine output measurement on the day of admission were included. To investigate the association between oliguria and mortality, we used a multilevel analysis. Results: Of the 8292 patients included, 2050 (24.7%) were oliguric during the first 24 h of admission. Patients with oliguria on admission who had at least one additional 24-h urine output recorded during their ICU stay (n = 1349) were divided into three groups: transient - oliguria resolved within 48 h after the admission day (n = 390 [28.9%]), prolonged - oliguria resolved > 48 h after the admission day (n = 141 [10.5%]), and permanent - oliguria persisting for the whole ICU stay or again present at the end of the ICU stay (n = 818 [60.6%]). ICU and hospital mortality rates were higher in patients with oliguria than in those without, except for patients with transient oliguria who had significantly lower mortality rates than non-oliguric patients. In multilevel analysis, the need for RRT was associated with a significantly higher risk of death (OR = 1.51 [95% CI 1.19-1.91], p = 0.001), but the presence of oliguria on admission was not (OR = 1.14 [95% CI 0.97-1.34], p = 0.103). Conclusions: Oliguria is common in ICU patients and may have a relatively benign nature if only transient. The duration of oliguria and need for RRT are associated with worse outcome.publishersversionPeer reviewe

Temporal changes in the epidemiology, management, and outcome from acute respiratory distress syndrome in European intensive care units: a comparison of two large cohorts

Background: Mortality rates for patients with ARDS remain high. We assessed temporal changes in the epidemiology and management of ARDS patients requiring invasive mechanical ventilation in European ICUs. We also investigated the association between ventilatory settings and outcome in these patients. Methods: This was a post hoc analysis of two cohorts of adult ICU patients admitted between May 1–15, 2002 (SOAP study, n = 3147), and May 8–18, 2012 (ICON audit, n = 4601 admitted to ICUs in the same 24 countries as the SOAP study). ARDS was defined retrospectively using the Berlin definitions. Values of tidal volume, PEEP, plateau pressure, and FiO2 corresponding to the most abnormal value of arterial PO2 were recorded prospectively every 24 h. In both studies, patients were followed for outcome until death, hospital discharge or for 60 days. Results: The frequency of ARDS requiring mechanical ventilation during the ICU stay was similar in SOAP and ICON (327[10.4%] vs. 494[10.7%], p = 0.793). The diagnosis of ARDS was established at a median of 3 (IQ: 1–7) days after admission in SOAP and 2 (1–6) days in ICON. Within 24 h of diagnosis, ARDS was mild in 244 (29.7%), moderate in 388 (47.3%), and severe in 189 (23.0%) patients. In patients with ARDS, tidal volumes were lower in the later (ICON) than in the earlier (SOAP) cohort. Plateau and driving pressures were also lower in ICON than in SOAP. ICU (134[41.1%] vs 179[36.9%]) and hospital (151[46.2%] vs 212[44.4%]) mortality rates in patients with ARDS were similar in SOAP and ICON. High plateau pressure (> 29 cmH2O) and driving pressure (> 14 cmH2O) on the first day of mechanical ventilation but not tidal volume (> 8 ml/kg predicted body weight [PBW]) were independently associated with a higher risk of in-hospital death. Conclusion: The frequency of and outcome from ARDS remained relatively stable between 2002 and 2012. Plateau pressure > 29 cmH2O and driving pressure > 14 cmH2O on the first day of mechanical ventilation but not tidal volume > 8 ml/kg PBW were independently associated with a higher risk of death. These data highlight the continued burden of ARDS and provide hypothesis-generating data for the design of future studies

IMPAIRED DNA REPAIR AS THE ROOT CAUSE OF OOCYTE AGING

Annual Meeting of the American-Society-for-Reproductive-Medicine -- OCT 15-19, 2011 -- Orlando, FLWOS: 000294452701051…Amer Soc Reprod Me