Phospholipase C-zeta Research Articles

Novel PLCZ1 compound heterozygous mutations indicate gene dosage effect involved in total fertilisation failure after ICSI.

PLCZ1 mutations are related to total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), characterised by abnormal oocyte oscillations. The novel PLCZ1 compound heterozygous mutations reported by this study were associated with TFF after ICSI, with one of the mutations indicating a gene dosage effect. Oocyte activation failure is thought to be one of the main factors for total fertilisation failure (TFF) after intracytoplasmic sperm injection (ICSI), which could be induced by abnormal calcium oscillations. Phospholipase C zeta (PLCZ), a sperm factor, is associated with Ca2+ oscillations in mammalian oocytes. To date, some mutations in PLCZ1 (the gene that encodes PLCZ) have been linked to TFF, as demonstrated by the observed reduction in protein levels or activity to induce Ca2+ oscillations. In this study, normozoospermic males whose sperms exhibited TFF after ICSI and their families were recruited. First, mutations in the PLCZ1 sequence were identified by whole exome sequencing and validated using Sanger sequencing. Then, the locations of PLCZ1/PLCZ and the transcript and protein levels in the sperm of the patients were studied. Subsequently, in vitro function analysis and in silico analysis were performed to investigate the function-structure correlation of mutations identified in PLCZ1 using western blotting, immunofluorescence, RT-qPCR, and molecular simulation. Ca2+ oscillations were detected after cRNA microinjection into MII mouse oocytes to investigate calcium oscillations induced by abnormal PLCZ. Five variants with compound heterozygosity were identified, consisting of five new mutations and three previously reported mutations distributed across the main domains of PLCZ, except the EF hands domain. The transcript and protein levels decreased to varying degrees among all detected mutations in PLCZ1 when transfected in HEK293T cells. Among these, mutations in M138V and R391* of PLCZ were unable to trigger typical Ca2+ oscillations. In case 5, aberrant localisation of PLCZ in the sperm head and an increased expression of PLCZ in the sperm were observed. In conclusion, this study enhances the potential for genetic diagnosis of TFF in clinics and elucidates the possible relationship between the function and structure of PLCZ in novel mutations.

Novel PLCZ1 mutation caused polyspermy during in vitro fertilization.

Failure of oocyte activation, including polyspermy and defects in pronuclear (PN) formation, triggers early embryonic developmental arrest. Many studies have shown that phospholipase C zeta 1 ( PLCZ1 ) mutations cause failure of PN formation following intracytoplasmic sperm injection (ICSI); however, whether PLCZ1 mutation is associated with polyspermy during in vitro fertilization (IVF) remains unknown. Whole-exome sequencing (WES) was performed to identify candidate mutations in couples with primary infertility. Sanger sequencing was used to validate the mutations. Multiple PLCZ1 -mutated sperm were injected into human and mouse oocytes to explore whether PN formation was induced. Assisted oocyte activation (AOA) after ICSI was performed to overcome the failure of oocyte activation. We identified three PLCZ1 mutations in three patients who experienced polyspermy during IVF cycles, including a novel missense mutation c.1154C>T, p.R385Q. PN formation failure was observed during the ICSI cycle. However, injection of multiple PLCZ1- mutated sperm induced PN formation, suggesting that the Ca 2+ oscillations induced by the sperm exceeded the necessary threshold for PN formation. AOA after ICSI enabled normal fertilization, and all patients achieved successful pregnancies. These findings expand the mutational spectrum of PLCZ1 and suggest an important role for PLCZ1 in terms of blocking polyspermy. Furthermore, this study may benefit genetic diagnoses in cases of abnormal fertilization and provide potential appropriate therapeutic measures for these patients with sperm-derived polyspermy.

Phospholipase C Zeta 1 (PLCZ1): The Function and Potential for Fertility Assessment and In Vitro Embryo Production in Cattle and Horses.

Phospholipase C Zeta 1 (PLCZ1) is considered a major sperm-borne oocyte activation factor. After gamete fusion, PLCZ1 triggers calcium oscillations in the oocyte, resulting in oocyte activation. In assisted fertilization, oocyte activation failure is a major cause of low fertility. Most cases of oocyte activation failures in humans related to male infertility are associated with gene mutations and/or altered PLCZ1. Consequently, PLCZ1 evaluation could be an effective diagnostic marker and predictor of sperm fertilizing potential for in vivo and in vitro embryo production. The characterization of PLCZ1 has been principally investigated in men and mice, with less known about the PLCZ1 impact on assisted reproduction in other species, such as cattle and horses. In horses, sperm PLCZ1 varies among stallions, and sperm populations with high PLCZ1 are associated with cleavage after intracytoplasmic sperm injection (ICSI). In contrast, bull sperm is less able to initiate calcium oscillations and undergo nuclear remodeling, resulting in poor cleavage after ICSI. Advantageously, injections of PLCZ1 are able to rescue oocyte failure in mouse oocytes after ICSI, promoting full development and birth. However, further research is needed to optimize PLCZ1 diagnostic tests for consistent association with fertility and to determine whether PLCZ1 as an oocyte-activating treatment is a physiological, efficient, and safe method for improving assisted fertilization in cattle and horses.

Sperm-Induced Ca2+ Release in Mammalian Eggs: The Roles of PLCζ, InsP3, and ATP.

Mammalian egg activation at fertilization is triggered by a long-lasting series of increases in cytosolic Ca2+ concentration. These Ca2+ oscillations are due to the production of InsP3 within the egg and the subsequent release of Ca2+ from the endoplasmic reticulum into the cytosol. The generation of InsP3 is initiated by the diffusion of sperm-specific phospholipase Czeta1 (PLCζ) into the egg after gamete fusion. PLCζ enables a positive feedback loop of InsP3 production and Ca2+ release which then stimulates further InsP3 production. Most cytosolic Ca2+ increases in eggs at fertilization involve a fast Ca2+ wave; however, due to the limited diffusion of InsP3, this means that InsP3 must be generated from an intracellular source rather than at the plasma membrane. All mammalian eggs studied generated Ca2+ oscillations in response to PLCζ, but the sensitivity of eggs to PLCζ and to some other stimuli varies between species. This is illustrated by the finding that incubation in Sr2+ medium stimulates Ca2+ oscillations in mouse and rat eggs but not eggs from other mammalian species. This difference appears to be due to the sensitivity of the type 1 InsP3 receptor (IP3R1). I suggest that ATP production from mitochondria modulates the sensitivity of the IP3R1 in a manner that could account for the differential sensitivity of eggs to stimuli that generate Ca2+ oscillations.

MACS-DGC sperm preparation method resulted in high-quality sperm, top-quality embryo, and higher blastocyst rate in male factor infertile couples with high DNA fragmented sperm

Conventional sperm selection based on motility and morphology fails to provide detailed information on sperm functional and molecular status. Magnetic-activated cell sorting (MACS) protocol aims to optimize this process by selecting apoptotic sperm cells. Phospholipase C zeta-1 (PLCz1) is a physiological stimulus for oocyte activation and early embryonic development. The purpose of this study was to examine seminal parameters, DNA fragmentation index (DFI), and PLCz1 expression levels in MACS-DGC sorted specimens (DFI > 30%) and assess early development in resulting embryos. Semen specimens from 60 patients diagnosed with male factor infertility were collected and processed by either density gradient centrifugation (DGC) or MACS-DGC protocols. Pre and post-preparation analysis was performed. PLCz1 expression was assessed using the RT-PCR method. Retrieved eggs from their partners were divided into two groups in which they were injected with different sorted sperm. The fertilization rate and embryonic development were evaluated. While sperm’s progressive motility and morphology significantly improved, there was a substantial decline in DFI following MACS-DGC. Fertilization rates were almost the same between the groups, and the latter resulted in remarkably more top-quality embryos and more blastocysts. PLCz1 expression was considerably higher in the MACS-DGC group. By eliminating apoptotic cells, the MACS-DGC technique could sort highly PLCz1-expressed sperm, optimize sperm selection in individuals with elevated DFI, development of resulting embryos.

P-260 A more physiological approach to support oocyte activation in cases with fertilization failure

Abstract Study question Can we use a physiological compound to trigger oocyte activation in couples undergoing ICSI with history of fertilization failure due to sperm activating factor deficiency? Summary answer In a pilot sibling oocyte study, oocyte activation using recombinant human phospholipase C-zeta (rhPLCζ) yielded a comparable fertilization compared to conventional method using ionomycin. What is known already ICSI overcomes most forms of male gamete defects, however partial or total fertilization failure still occurs regardless of adequate semen parameters and optimal oocyte maturity. This has been attributed to the lack of a cytosolic factor identified as PLCζ. To address this, various approaches of assisted oocyte activation have been proposed, including gamete exposure to chemical or electrical stimuli. The most popular approach remains to be ionomycin. Despite its apparent safety, this stimulus is unnatural and may be aggressive on the oocytes. Therefore, we propose a more physiological approach by utilizing a recombinant form of this specific enzyme. Study design, size, duration In the past 2 months, couples with poor fertilization at their initial ICSI cycles were identified and counseled for assisted oocyte activation (AOA) in the subsequent cycle after confirmation of a PLCζ deficiency. Oocytes were separated into 2 equal cohorts activated either by ionomycin or co-injection with recombinant human rhPLCζ during ICSI. Fertilization rates between ionomycin-activated and rhPLCζ-activated cohorts were compared, as well as with the couples’ history cycles. Participants/materials, setting, methods Six couples with suboptimal fertilization outcomes consented for the study (IRB 0712009553) and were offered AOA with their subsequent ICSI cycles. Conventional AOA was carried out by exposing post-ICSI oocytes to 50 mM ionomycin. The rhPLCζ had a confirmed >95% purity. Our physiological rhPLCζ-activation method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5mg/mL in glycerol) during ICSI. Main results and the role of chance Couples (n = 6, female age: 36.2 ± 6yrs, male age: 38.0 ± 7yrs) had negative infertility workups with normal semen parameters (3.3 ± 3 mL volume, 42.1 ± 35 x106/mL concentration, 30.8 ± 23% motility, 2.0 ± 1% normal morphology). They underwent 7 unsuccessful ICSI cycles with 20.0% (12/60) fertilization rate and yielded a 91.7% cleavage rate, of those, 3 were transferred at cleavage stage but did not yield clinical pregnancy. A total of 2 blastocysts were obtained but were all aneuploid. In their subsequent treatment cycles, overall AGT achieved a significantly improved fertilization rate of 51.6% (32/62) (P<0.001). We then compared the efficiency between the two AGT methods in equally distributed sibling oocyte cohort. Although mathematically insignificant, we observed a trend that rhPLCζ-activation outperforms ionomycin-activation in total activation (67.9% vs 50.0%) as well as fertilization (57.1% vs 47.0%). The new rhPLCζ-activation method yielded a comparable embryo cleavage rate at 87.5% compared to the conventional method at 81.3%. Although fresh embryo transfer at cleavage stage did not yield pregnancy rate yet, rhPLCζ-activated cohort had one euploid blastocyst while the ionomycin-activated cohort did not yield euploid blastocyst. Limitations, reasons for caution While we observed that a more physiological rhPLCζ-activation yields a comparable fertilization compared to the conventional ionomycin method, the study is preliminary, and a confirmatory clinical outcome is not yet available. Wider implications of the findings This is the first attempt to use recombinant sperm-specific protein to mimic physiological oocyte activation for patient treatment. If this data is confirmed in a larger series, this may represent a way to normalize fertilization in cases of sperm activating factor deficiency even in different sources of spermatozoa. Trial registration number N/A

THE LEVEL OF EXPRESSION OF MESSENGER PLCZ IN SPERM MIGHT BE CORRELATED TO EMBRYONIC DEVELOPMENTAL COMPETENCE IN ART

Sperm are special cells with minimal cytoplasm to ensure motility and protect DNA during sperm formation and migration to the egg. The sperm-specific phospholipase C zeta (PLCZ) is identified as candidate sperm-borne oocyte-activating factor that triggers a cytoplasmic Ca2+ oscillations during fertilization. Such oscillations are known to be important for optimal embryonic development. Mutations in PLCZ1 cause male infertility due to fertilization failure or poor fertilization. The RNA profile through microarrays include approximately 3000∼7000 types of coding transcripts.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca2+ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] substrate to produce the Ca2+-mobilising messenger, inositol 1,4,5-trisphosphate (IP3), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.

Microfluidic chips as a method for sperm selection improve fertilization rate in couples with fertilization failure.

Sperm quality plays a vital role in successful fertilization and pregnancy. Patients with fertilization failure (total failure or low-fertilization rate) despite having normal semen parameters are a challenging group whose sperm cannot fertilize the oocyte via the intracytoplasmic sperm injection (ICSI) technique. Microfluidics is offered as a new method for proper sperm sorting. This study aimed to evaluate sperm parameters, DNA fragmentation index (DFI), expression of phospholipase C zeta 1 (PLCZ1), and transition nuclear proteins 1 (TNP1) mRNAs in sperm selected by microfluidic sperm sorting (MSS) chip compared with conventional density gradient centrifugation technique in patients with fertilization failure following ICSI. Subsequence fertilization rate and embryo quality were assayed. Normal morphology and total motility were significantly higher, and DFI was significantly lower in sperm selected by the MSS chip in fertilization failure and control groups. The RT-PCR results demonstrated a significant increase in the expression of PLCZ1 and TNP1 genes in sperm of both groups selected by MSS chips compared to the DGC method. In addition, with the selected sperm by MSS chip, an increase in fertilization rate and improvement of embryo quality was obtained. The present study findings show that sperm sorting by the microfluidic method improves fertilization rate in patients with poor fertilization outcomes following ICSI.

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca2+ ([Ca2+]i): transient elevations in [Ca2+]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca2+ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca2+]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca2+ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca2+]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca2+ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca2+]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca2+ wave and spiral-like Ca2+ oscillations, respectively.

Phospholipase C-zeta levels are not correlated with fertilisation rates in infertile couples.

In mammals, 'oocyte activation' is triggered by certain proteins, one of which is phospholipase C-zeta. Recent evidence suggests that low expression of phospholipase C-zeta might be associated with male infertility, while a limited number of studies claimed the opposite. This study was designed to test whether quantity of phospholipase C-zeta and in vitro fertilisation rates are correlated or not, assessed by flow cytometry. Semen samples from 43 infertile couples were analysed for the percentage and mean fluorescent intensity (MFI) of phospholipase C-zeta protein. Results were confirmed by immunofluorescent labelling. Patients with a fertilisation rate of 40% or lower were involved in the low fertilisation group, while the high fertilization group consisted of patients with a fertilisation rate of 60% and higher. Quantitative analyses by flow cytometry showed no significant difference among the low fertilisation and high fertilisation groups when phospholipase C-zeta ratio or MFI was considered. No correlation was found between pregnancy rates and phospholipase C-zeta quantity. None of the total fertilisation failure cases were lack of phospholipase C-zeta. In fact, fertilisation was possible even when phospholipase C-zeta levels were very low. Thus, we concluded that phospholipase C-zeta quantity cannot be considered as a diagnostic tool for male infertility.

EFFECT OF MICROINJECTION OF MESSENGER RNA OF PLCZ INTO ZYGOTE IN MOUSE EMBRYO DEVELOPMENT IN VITRO

Sperm are special cells with minimal cytoplasm to ensure motility and protect DNA during sperm formation and migration to the egg. Therefore, it is known that a series of gene expression mechanisms such as DNA replication-RNA transcription-translation do not occur in mature sperm. And it is known that most of the cytoplasmic components required for fertilization and embryonic development are derived from the egg, and sperm have only paternal gene transfer function. However, spermatozoa RNAs are also delivered to the oocyte contributing to early embryo development. Mature spermatozoa, in spite of being transcriptionally and translationally inactive, possess several types of RNAs that accumulate in the sperm cell. The study of sperm RNAs has been challenging because of the difficulty associated with sperm RNA isolation. The sperm-specific phospholipase C zeta (PLCZ) is identified as candidate sperm-borne oocyte-activating factor that triggers a cytoplasmic Ca2+ oscillations during fertilization. Such oscillations are known to be important for optimal embryonic development. In this study, we investigated the purifying of messenger RNA of PLCZ from mouse sperm, and produced cDNA of PLCZ. Also we investigated the role of cDNA of PLCZ in embryonic development. cDNA library originated from mouse testis. cDNA was produced by in vitro transcription with or without GFP tagging. Total mRNA from mouse testes and sperm were isolated by Dynabeads™ mRNA Purification Kit. To evaluate the [Ca2+]i oscillatory activity, we performed microinjection of cDNA into mouse mature eggs and [Ca2+]i monitoring. To investigate the effect of microinjection of cDNA of PLCZ on mouse embryo development, we produces artificially activated zygote with 10 mM strontium, and examined microinjection of cDNA PLCZ into zygote stage. Further embryonic development were observed blastocyst formation for 5 days culture in KSOM. mRNA of PLCZ were purified from mouse sperm and testes, but not in mouse granulosa cells. Microinjection of cDNA of PLCZ into zygote stage induced higher blastocyst formation than control eggs significantly (P<0.02). The ability of PLCZ to increase [Ca2+]i appears to have influenced intracellular signaling and induced embryogenesis in vitro. SUPPORT: This research was supported by a grant from Republic of Korea, NRF-2018R1D1A1B07043250.

The identification of novel mutations in PLCZ1 responsible for human fertilization failure and a therapeutic intervention by artificial oocyte activation.

Fertilization involves a series of molecular events immediately following egg-sperm fusion; Ca2+ oscillations are the earliest signaling event, and they initiate the downstream reactions including pronucleus formation. Successful human reproduction requires normal fertilization. In clinical IVF or ICSI attempts, some infertile couples suffer from recurrent fertilization failure. However, the genetic reasons for fertilization failure are largely unknown. Here, we recruited several couples diagnosed with fertilization failure even though their gametes are morphologically normal. Through whole-exome sequencing and Sanger sequencing, we identified biallelic mutations in gene-encoding phospholipase C zeta 1 (PLCZ1) in four independent males in couples diagnosed with fertilization failure. Western blotting showed that missense mutations decreased the level of PLCZ1 and that nonsense or frameshift mutations resulted in undetectable or truncated proteins. Expression of these mutations in mice significantly reduced the levels of oocyte activation. Artificial oocyte activation in patient oocytes could rescue the phenotype of fertilization failure and help establish pregnancy and lead to live birth. Our findings expand the spectrum of PLCZ1 mutations that are responsible for human fertilization failure and provide a potentially feasible therapeutic treatment for these patients.

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C-zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ-related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid-binding ability, and PLCζ level along with its distribution were evaluated using computer-assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid-binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? New mutations in the PLCZ1 sequence are associated with FFs after ICSI. FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. Patients were divided into two groups based on oocyte evaluation 19h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19h after injection. In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

180 Stallion sperm phospholipase C zeta affects cleavage rates after intracytoplasmic injection in bovine oocytes

Phospholipase C zeta (PLCz) is a sperm protein linked to oocyte activation and zygote development in diverse species. Human intracytoplasmic sperm injection (ICSI) success is poor when sperm PLCz is reduced or mutated. We hypothesised that the expression of PLCz in stallion sperm corresponds with cleavage rates after ICSI. For this study, we selected sperm from 4 of 21 stallions for which frozen-thawed sperm were evaluated using flow cytometry to assess mean fluorescence intensity (MFI) and percentage of sperm positively labelled with PLCz. Before flow cytometric assessment, Western blotting and immunofluorescence were performed to validate antibody binding and to identify PLCz as a 71-kDa protein in stallion sperm, located in the acrosomal and postacrosomal region, and the tail (Gonzalez-Castro et al. 2017 Reprod. Fertil. Dev. 30, 228). Frozen sperm from 4 stallions were selected based on MFI and percentage of PLCz-labelled sperm per total sperm population, respectively (High, 87% with 10,384 MFI and 84% with 10,784 MFI; Low, 56% with 4,789 MFI and 59% with 5,360 MFI). The samples were selected so that other fertility indicators, such as normal morphology (&amp;gt; 70%), DNA integrity (&amp;lt;8%, flow cytometric evaluation using sperm chromatin structure assay), and viability (SYBR14+/propidium iodide-, flow cytometric assessment) were similar for High and Low. Bovine ovaries were transported at 25°C before collection of oocytes from 2- to 8-mm follicles. Oocyte maturation and embryo culture were done as previously described using a bovine system with chemically defined media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). Oocytes were matured for 22 to 24h at 38.5°C in 5% CO2 and air before removal of cumulus cells. Before ICSI, straws of frozen sperm were cut under liquid nitrogen, with a small section thawed directly in medium. Oocytes were selected based on normal morphology and an extruded polar body. Before injection, individual sperm were selected at 200× based on normal morphology and progressive motility. Oocytes were injected with sperm from High (n=62 oocytes) and Low (n=56). A third group of oocytes (n=43) were sham injected (no sperm) to determine the rate of parthenogenetic cleavage. Cleavage rates were compared using chi-squared test. Cleavage rates differed (P&amp;lt;0.0001) among groups, with 53% (33/62) for High, 34% (19/56) for Low, and 0% (0/43) for sham injections. Sperm populations from the High group had higher (P&amp;lt;0.04) cleavage rates than those from the Low group. We concluded that PLCz in stallion sperm populations is a valuable indicator of ICSI success, and this protein is important factor for oocyte activation and initiation of embryo development after assisted fertilization.

176 Localization and Quantitative Expression of Phospholipase C Zeta in Equine Sperm Using Commercial Antibodies

Fertilization failure in vivo and in vitro (intracytoplasmic sperm injection, ICSI) can be caused by the inability of sperm to elicit intracellular calcium oscillations and to induce oocyte activation. Phospholipase C zeta (PLCz) is sperm-associated protein that can induce oocyte activation. Male infertility has been associated with PLCz deficiency in various species, although this has not been studied in the stallion. We hypothesised that the location and amount of PLCz on sperm varies among stallions. The aim of this study was to validate commercial antibodies (Ab) to detect PLCz on stallion sperm, and then to use these Ab to quantify the amount of PLCz, using flow cytometry, with the long-term goal of correlating PLCz on sperm with stallion fertility. Frozen-thawed sperm were analysed (20 stallions in 3 replicates) using 2 commercial Ab (anti-mouse PLCz M163 and anti-human PLCz H50, Santa Cruz Biotechnology, TX, USA). Western blot and immunofluorescence microscopy were used to validate Ab binding. For microscopy, sperm DNA was counterstained with 1 µg mL−1 Hoechst 33258. For flow cytometry, samples were incubated with Live Dead Fixable Far Red Stain Kit (Molecular Probes, Eugene, OR, USA), fixed, permeabilized, incubated overnight with primary Ab, and labelled with conjugated secondary Ab (anti-rabbit IgG Alexa Fluor 488, Molecular Probes). Green and far red mean fluorescence intensity (MFI) were measured for 20,000 cells per sample. Results are presented as mean ± SEM. Wilcoxon test, Spearman rank correlation, and linear regression were performed for analyses. Immunoblot analyses for both commercial Ab identified an immunoreactive band of ~70 kDa in sperm heads, tails, and whole sperm; β-tubulin was used as loading control and for normalization. Microscopy revealed PLCz in the acrosomal and post-acrosomal regions, connecting piece, midpiece, and tail. Post-acrosomal localization was the pattern most frequently observed (55%), followed by acrosomal plus post-acrosomal regions (25%). The PLCz labelling was observed on &gt;85% of midpiece and tail regions, independent of Ab used. Flow cytometric evaluation revealed that percentage of live sperm was 47 ± 2%. Similar fluorescence intensity was exhibited for both Ab (M163 and H50) with a wide range of values among stallions [M163, mean 30.7 ± 1.9 × 103 (range, 8.8-82.2 × 103); H50: 25.5 ± 3.2 × 103 (7.3-55.0 × 103)]. The percentage of live sperm within a sample was not associated with Ab MFI. However, when samples were gated for live/dead cells, live sperm exhibited higher (P &lt; 0.001) MFI than dead sperm for M163 (42.6 ± 6.0 v. 30.6 ± 3.9 × 103) and H50 (38.4 ± 4.7 v. 25.6 ± 3.7 × 103). There was a strong and positive correlation between M163 and H50 MFI for total sperm and live sperm (total: r = 0.81, P &lt; 0.001; live: r = 0.71; P &lt; 0.001). In conclusion, 2 anti-PLCz commercial antibodies detected equine PLCz, and the PLCz was localised on the sperm as described. Flow cytometric evaluation showed that stallions have different quantities of PLCz on their sperm, and this may provide a mean to determine if PLCz on stallion sperm is associated with fertility.

The effect of prolonged incubation and temperature on oocyte activator phospholipase C-zeta activity of sperm

Amac: Kapasite olmus spermin fertilizasyon kapasitesinin zamanla ustsel olarak dustugu bilinmesine ragmen bunun nedeni hala arastirilmaktadir. Calismada amac; uzamis inkubasyonun ve sicakligin sperm fertilizasyon kapasitesi uzerinde etkisini hareketlilik ve oosit aktivasyon faktoru olarak kabul edilen fosfolipaz C-zeta (PLCZ) ile degerlendirmektir. Gerec ve Yontem: On alti infertil erkek hastadan, ciddi oligoasthenospermisi olmayan 11’inin semenlerine dansite gradyan yikamasi uygulandi. Ardindan ornekler bolunerek 3 gun oda sicakliginda veya 37°C’de kulture edildi. Ornekler hareketlilik, PLCZ boyanma yuzdesi ve yogunlugu acisindan gunluk olarak degerlendirildi. Bulgular: Hem oda sicakligi, hem de vucut sicakliginda her uc parametrede de zamanla azalma gozlendi. Ozellikle gunler arasi hareketlilikteki degisim ile PLCZ boyanan sperm yuzdesindeki degisim arasinda guclu bir korelasyon bulundu, ancak bu korelasyon zamanla azalmakta idi. Sonuc: Inkubasyon suresinin uzamasiyla, PLCZ boyanma parametreleri ve hareketlilik arasinda korelasyon gorulmektedir. PLCZ boyanmasinin semen analizi ile rutinde kullanimi, ozellikle aciklanamayan infertilite ve fertilizasyon basarisizligi vakalarinin spermlerinin fertilizasyon kapasitesinin belirlenmesinde yarar saglayabilecek ve yardimci ureme teknileri (YUT) sikluslarindaki basariyi artirabilecektir.

Relationship between phospholipase C-zeta, semen parameters, and chromatin status

ABSTRACTThe need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca2+ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.

The establishment of appropriate methods for egg-activation by human PLCZ1 RNA injection into human oocyte

Phospholipase C-zeta (PLCZ1), a strong candidate of egg-activating sperm factor, can induce Ca2+ oscillations and cause egg activation. For the application of PLCZ1 to clinical use, we examined the pattern of Ca2+ responses and developmental rate by comparing PLCZ1 RNA injection methods with the other current methods, such as cytosolic aspiration, electrical stimulation and ionomycin treatment in human oocytes. We found that the pattern of Ca2+ oscillations after PLCZ1 RNA injection exhibited similar characteristics to that after ICSI treatment. We also determined the optimal concentration of human PLCZ1 RNA to activate the human oocytes. Our findings suggest that human PLCZ1 RNA is a better therapeutic agent to rescue human oocytes from failed activation, leading to normal and efficient development.