Sperm PLCζ: From structure to Ca2+ oscillations, egg activation and therapeutic potential

AbstractSignificant evidence now supports the assertion that cytosolic calcium oscillations during fertilization in mammalian eggs are mediated by a testis-specific phospholipase C (PLC), termed PLC-zeta (PLCζ) that is released into the egg following gamete fusion. Herein, we describe the current paradigm of PLCζ in this fundamental biological process, summarizing recent important advances in our knowledge of the biochemical and physiological properties of this enzyme. We describe the data suggesting that PLCζ has distinct features amongst PLCs enabling the hydrolysis of its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2) at low Ca2+ levels. PLCζ appears to be unique in its ability to target PIP2 that is present on intracellular vesicles. We also discuss evidence that PLCζ may be a significant factor in human fertility with potential therapeutic capacity

Diffusion MR Characteristics Following Concurrent Radiochemotherapy Predicts Progression-Free and Overall Survival in Newly Diagnosed Glioblastoma.

The standard of care for newly diagnosed glioblastoma (GBM) is surgery, then radiotherapy (RT) with concurrent temozolomide (TMZ), followed by adjuvant TMZ. We hypothesized patients with low diffusivity measured using apparent diffusion coefficient (ADC) histogram analysis evaluated after RT+TMZ, prior to adjuvant TMZ, would have a significantly shorter progression-free (PFS) and overall survival (OS). To test this hypothesis we evaluated 120 patients with newly diagnosed GBM receiving RT+TMZ followed by adjuvant TMZ. MRI was performed after completion of RT+TMZ, prior to initiation of adjuvant TMZ. A double Gaussian mixed model was used to describe the ADC histograms within the enhancing tumor, where ADCL and ADCH were defined as the mean ADC value of the lower and higher Gaussian distribution, respectively. An ADCL value of 1.0 um2/ms and ADCH value of 1.6 um2/ms were used to stratify patients into high and low risk categories. Results suggest patients with low ADCL had significantly shorter PFS (Cox Hazard Ratio = 0.12, P = 0.0006). OS was significantly shorter with low ADCL tumors, showing a median OS of 407 vs. 644 days (Cox Hazard Ratio = 0.31, P = 0.047). ADCH was not predictive of PFS or OS when accounting for age and ADCL. In summary, newly diagnosed glioblastoma patients with low ADCL after completion of RT+TMZ are likely to progress and die earlier than patients with higher ADCL. Results suggest ADC histogram analysis may be useful for patient risk stratification following completion of RT+TMZ

Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca release via the inositol 1,4,5-trisphosphate (InsP) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca oscillations within mammalian eggs. Moreover, studies describing PLCζ "knockout" mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca oscillations and egg activation in mammals, while also discussing the nature of a potential "alternative" sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.Qatar University student grant QUST-1-CMED-2020-3. Healthcare Research Fellowship Award (HF-14-16) made by Health and Care Research Wales (HCRW). National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST)

Editorial: The role of calcium signaling in gametogenesis and early embryogenesis

Calcium homeostasis exerts a profound role in determining the quality of gametogenesis, the resultant efficacy of fertilisation and ultimately the competency of embryogenesis. Perhaps no other single ion plays such a profound and determinative role in reproductive success as calcium, since it is critical for germinal vesicle breakdown, sperm capacitation and hyperactivation, oocyte activation, maintenance of zygote calcium homeostasis and embryogenic developmental gradients. This Research Topic aimed to collect, collate, and summarize the most recent and cutting-edge advances and propositions studying the molecular determinants and effectors of calcium signalling and homeostasis throughout the early events of fertilisation and preimplantation embryogenesis, with a specific view to examine how calcium can affect the efficacy of such important phenomena.JK was supported by a Healthcare Research Fellowship Award (HF-14–16) made by Health and Care Research Wales (HCRW), alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST)

Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Cζ

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca2+ ionophores to produce a single cytosolic Ca2+ increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca2+ oscillations indistinguishable from those occurring during fertilization, but remains untested for its efficacy in a scenario of ICSI fertilization failure. Here, we compare PLCζ with other activation stimuli in a mouse model of failed oocyte activation after ICSI, in which heat-treated sperm are injected into mouse oocytes. We show that increasing periods of 56°C exposure of sperm produces a progressive loss of Ca2+ oscillations after ICSI. The decrease in Ca2+ oscillations produces a reduction in oocyte activation and embryo development to the blastocyst stage. We treated such oocytes that failed to activate after ICSI either with Ca2+ ionophore, or with Sr2+ media which causes Ca2+ oscillations, or we injected them with recombinant human PLCζ. All these treatments rescued oocyte activation, although Sr2+ and PLCζ gave the highest rates of development to blastocyst. When recombinant PLCζ was given to oocytes previously injected with control sperm, they developed normally to the blastocyst stage at rates similar to that after control ICSI. The data suggest that recombinant human PLCζ protein is an efficient means of rescuing oocyte activation after ICSI failure and that it can be effectively used even if the sperm already contains endogenous Ca2+ releasing activity

Identification of an amino-terminus determinant critical for ryanodine receptor/Ca2+ release channel function

Aims The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer. Methods and Results We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively-charged side chains of two aspartate residues (D179 and D180) within the β8-β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8-β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8-β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8-β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8-β9 domain in channel closing. Conclusions These results suggest that efficient N-terminus inter-subunit communication mediated by the β8-β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression. Translational Potential Our findings that the RyR2 β8-β9 loop is involved in both Ca2+ release channel opening and closing have important clinical implications. This RyR2 domain is a known “hot-spot” for mutations associated with arrhythmogenic cardiac disease, which could produce hypersensitive as well as hyposensitive channels. Therapeutic strategies currently focus on gain-of-function RyR2 channels to suppress sarcoplasmic reticulum Ca2+ release either indirectly with class I/II anti-arrhythmic drugs, or by directly targeting RyR2 to inhibit channel activity. These strategies may not only be ineffective, but they may exacerbate the malignant phenotype in the case of loss-of-function RyR2 mutations