The ability of spermatozoa to generate reactive oxygen species (ROS) has been appreciated since the 1940s. It is a universal property of mature spermatozoa from all mammalian species and a major contributor to the oxidative stress responsible for defective sperm function. The mechanisms by which oxidative stress limits the functional competence of mammalian spermatozoa involve the peroxidation of lipids, the induction of oxidative DNA damage, and the formation of protein adducts. ROS production in these cells involves electron leakage from the sperm mitochondria, triggered by a multitude of factors that impede electron flow along the electron transport chain. The net result of mitochondrial ROS generation is to damage these organelles and initiate an intrinsic apoptotic cascade, as a consequence of which spermatozoa lose their motility, DNA integrity, and vitality. This pathway of programmed senescence also results in the exteriorization of phosphatidylserine, which may facilitate the silent phagocytosis of these cells in the aftermath of insemination, in turn influencing the female tract immune response to sperm antigens and future fertility. Despite the vulnerability of sperm to oxidative stress, it is also clear that normal sperm function depends on low levels of ROS generation in order to promote the signal transduction pathways associated with capacitation. Modulators of ROS generation by spermatozoa may therefore have clinical utility in regulating the fertilizing capacity of these cells and preventing the development of antisperm immunity. Achievement of these objectives will require a systematic evaluation of pro-and antioxidant strategies in vivo and in vitro.

Document embargo till 13/10/2016.This paper reviews the state of the art in cyber security risk assessment of Supervisory Control and Data Acquisition (SCADA) systems. We select and in-detail examine twenty-four risk assessment methods developed for or applied in the context of a SCADA system. We describe the essence of the methods and then analyse them in terms of aim; application domain; the stages of risk management addressed; key risk management concepts covered; impact measurement; sources of probabilistic data; evaluation and tool support. Based on the analysis, we suggest an intuitive scheme for the categorisation of cyber security risk assessment methods for SCADA systems. We also outline five research challenges facing the domain and point out the approaches that might be taken.Peer reviewe

SummaryFully-grown mammalian oocytes maintain a prophase I, germinal-vesicle stage arrest in the ovary for extended periods before a mid-cycle luteinizing surge induces entry into the first meiotic division. Cdh1 is an activator of the Anaphase-Promoting Complex (APC), and APC cdh1 is normally restricted to late M -early G1 of the cell cycle. Here we find that APC cdh1 is active in mouse oocytes and is necessary to maintain prophase arrest.Fully-grown mammalian oocytes remain arrested at prophase I within antral follicles until stimulated to enter the first meiotic division by a mid-cycle surge in luteinising hormone. An oolemma receptor maintains this arrest by raising protein kinase A activity1 which inhibits Maturation-Promoting Factor (CDK1-cyclin B1) by affecting the phosphorylation status of CDK12. Oocytes can resume meiosis spontaneously, manifest by germinal vesicle breakdown (GVB), when released into culture media, but remain arrested if agents such as the phosphodiesterase inhibitor milrinone3, are added to maintain protein kinase A.Raising cyclin B1 levels in milrinone-arrested oocytes by microinjection of its cRNA coupled to GFP induced GVB. Spatially the cyclin B1-GFP expressed in oocytes mirrored the distribution reported in adult cells 4 ( Supplementary Information, Fig S1a). Cytoplasmic cyclin B1 entered the nucleus before GVB and became associated with chromatin afterwards. However, the GVB rate in these oocytes was <15% by 5 h (Fig 1a), and never exceeded 20%, even after 24 h. The proteasomal inhibitor MG132 had a mild stimulatory effect on GVB over 5 h, and when combined with cyclin B1 the rate GVB increased 2-3 fold compared to cyclin B1 alone (Fig 1a and see Supplementary Information, Table S1). The increased rate of GVB was likely caused by increased cyclin B1-GFP since levels doubled with MG132 (Fig 1b).Cyclin B1 degradation requires polyubiquitination by the Anaphase-Promoting Complex (APC) followed by proteasomal degradation5. In mitosis, the APC needs one of two essential co-activators, cdc20 and cdh1, which are both present in mouse eggs6. APC cdc20 and APC cdh1 both degrade substrates such as cyclin B1 that contain a Destruction-(D)-box. Therefore we repeated the above cyclin B1 experiment using Δ90-cyclin B1, an N-terminal truncation which removes the D-box 7. Δ90-cyclin B1 cRNA induced 70% GVB rates by 5 h (Fig 1a), and 80% by 24 h; rates that are 4-5 fold higher than cyclin B1-GFP and with the MG132 data are consistent with cyclin B1 being degraded in GV oocytes.Cyclin B1 degradation in oocytes, where MPF is low, is likely due to APC cdh1 because APC cdc20 requires high MPF levels for activity8. Oocytes do contain cdh1 (see Supplementary Information, Fig S1b) therefore to examine if APC cdh1 was active at this time, in addition to cyclin B1, we coupled two further APC cdh1 substrates to GFP, injected their cRNA and measured their stability following protein synthesis inhibition. We used cdc20 itself and a mutant form of securin (securin dm ) in which its D-Box has been mutated. Bo...

SUMMARYHomologous chromosome segregation errors during meiosis I are common and generate aneuploid embryos. Here, we provide a reason for this susceptibility to mis-segregation by live cell imaging of mouse oocytes. Our results show that stable kinetochoremicrotubule attachments form in mid-prometaphase, 3-4 hours before anaphase. This coincided with the loss of Mad2 from kinetochores and with the start of anaphase-promoting complex/cyclosome (APC/C)-mediated cyclin B1 destruction. Therefore, the spindle assembly checkpoint (SAC) ceased to inhibit the APC/C from mid-prometaphase. This timing did not coincide with bivalent congression in one-third of all oocytes examined. Non-aligned bivalents were weakly positive for Mad2, under less tension than congressed bivalents and, by live-cell imaging, appeared to be in the process of establishing correct bi-orientation. The time from when the APC/C became active until anaphase onset was affected by the rate of loss of CDK1 activity, rather than by these non-aligned bivalents, which occasionally persisted until anaphase, resulting in homolog non-disjunction. We conclude that, in oocytes, a few erroneous attachments of bivalent kinetochores to microtubules do not generate a sufficient SAC 'wait anaphase' signal to inhibit the APC/C.

SummaryMammalian oocytes are particularly error prone in segregating their chromosomes during their two meiotic divisions. This results in the creation of an embryo that has inherited the wrong number of chromosomes: it is aneuploid. The incidence of aneuploidy rises significantly with maternal age and so there is much interest in understanding this association and the underlying causes of aneuploidy. The spindle assembly checkpoint, a surveillance mechanism that operates in all cells to prevent chromosome mis-segregation, and the cohesive ties that hold those chromosomes together, have thus both been the subject of intensive investigation in oocytes. It is possible that a lowered sensitivity of the spindle assembly checkpoint to certain types of chromosome attachment error may endow oocytes with an innate susceptibility to aneuploidy, which is made worse by an age-related loss in the factors that hold the chromosomes together.

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1.Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.

Mammalian oocytes begin meiosis in the fetal ovary, but only complete it when fertilized in the adult reproductive tract. This review examines the cell biology of this protracted process: from entry of primordial germ cells into meiosis to conception. The defining feature of meiosis is two consecutive cell divisions (meiosis I and II) and two cell cycle arrests: at the germinal vesicle (GV), dictyate stage of prophase I and at metaphase II. These arrests are spanned by three key events, the focus of this review: (i) passage from mitosis to GV arrest during fetal life, regulated by retinoic acid; (ii) passage through meiosis I and (iii) completion of meiosis II following fertilization, both meiotic divisions being regulated by cyclin-dependent kinase (CDK1) activity. Meiosis I in human oocytes is associated with an age-related high rate of chromosomal mis-segregation, such as trisomy 21 (Down's syndrome), resulting in aneuploid conceptuses. Although aneuploidy is likely to be multifactorial, oocytes from older women may be predisposed to be becoming aneuploid as a consequence of an age-long decline in the cohesive ties holding chromosomes together. Such loss goes undetected by the oocyte during meiosis I either because its ability to respond and block division also deteriorates with age, or as a consequence of being inherently unable to respond to the types of segregation defects induced by cohesion loss.

Fertilization of mature mouse oocytes triggered highly repetitive Ca2+ oscillations lasting 2-3 h. However, immature oocytes generated only two or three oscillations, which ceased within 1 h. Development of repetitive Ca2+ transients to sperm occurred late in oocyte maturation and was dependent on cytoplasmic modifications that were independent of cell cycle progression from metaphase I to metaphase II. Immature oocytes released significantly less Ca2+ from stores than mature oocytes in response to ionomycin and thapsigargin. Ryanodine had no effect on intracellular Ca2+ in maturing oocytes but stimulated an increase in Ca2+ in mature oocytes. The ability of ryanodine to increase Ca2+ levels was, however, strain-dependent. Preincubation of oocytes with thapsigargin or ryanodine significantly attenuated the normal fertilization Ca2+ response, causing a decrease in the number and the rate of rise of the transients. The inhibition of sperm-induced Ca2+ transients by ryanodine was independent of its ability to cause an immediate Ca2+ increase. Low concentrations of ryanodine had no effect on resting Ca2+ levels but inhibited Ca2+ oscillations at fertilization. Similarly Ca2+ oscillations were blocked in oocytes from a strain of mouse that showed no immediate Ca2+ increase with ryanodine. These results suggest that modifications in Ca2+ stores and ryanodine-sensitive Ca2+ release mechanisms during oocyte maturation play an important role in Ca2+ oscillations at fertilization.