Dimitra Nikiforaki

Loss of activity mutations in phospholipase C zeta (PLCζ) abolishes calcium oscillatory ability of human recombinant protein in mouse oocytes

BACKGROUNDnMammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ(H398P)), leading to abnormal Ca(2+) release profiles and reduced oocyte activation efficiency.nnnMETHODS AND RESULTSnIn the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLCζ(H398P) was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ(H398P) and an artificially induced frameshift mutation (PLCζ(ΔYC2)) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLCζ(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ(WT) > PLCζ(H398P) > PLCζ(ΔYC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ(H398P) compared with fertile controls.nnnCONCLUSIONSnWe demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency.

Diagnostic and prognostic value of calcium oscillatory pattern analysis for patients with ICSI fertilization failure

STUDY QUESTIONnDoes calcium oscillatory pattern analysis following heterologous intra-cytoplasmic sperm injection (ICSI) of human sperm into mouse oocytes lead to diagnostic and prognostic information for patients suffering from ICSI fertilization failure?nnnSUMMARY ANSWERnWe found that calcium oscillatory pattern analysis following heterologous ICSI has the strength to reveal, for the individual patient, the most probable underlying reason for low or failed fertilization after conventional ICSI.nnnWHAT IS KNOWN ALREADYnFertilization failure occurs in 1-3% of the couples undergoing conventional ICSI, for whom the mouse oocyte activation test (MOAT) or a similar heterologous ICSI model is the only diagnostic test available to evaluate the oocyte-activating capacity of human sperm cells. The MOAT classifies the patients into three groups: a low (group 1), an intermediate (group 2) and a high (group 3) activating group. In MOAT group 1 patients, a sperm-related deficiency is likely to be the cause of previous fertilization failures, while in MOAT group 3 patients a sperm-related deficiency can most probably be refuted. For MOAT group 2 patients, the result is called inconclusive; hence, both sperm and oocyte deficiencies may still contribute to the previous ICSI fertilization failure.nnnSTUDY DESIGN, SIZE, DURATIONnThe calcium-releasing ability of sperm from 26 MOAT patients with a history of zero or low fertilization following conventional ICSI was compared with the calcium-releasing ability of sperm from 4 control patients, with proven oocyte activation potential. Per case an average of 19 mouse oocytes were injected. Calcium imaging started within 5-10 min after ICSI and continued for 2 h.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnHuman sperm were demembranated with 0.02% lysolecithin for 1 min immediately before heterologous piezo-driven ICSI. For calcium imaging, metaphase II oocytes from B6D2/F1 mice were loaded with fura-2 acetoxymethyl ester. The calcium oscillatory patterns following heterologous ICSI were scored per oocyte and per patient individually based on the presence of calcium spikes and their frequency and amplitude.nnnMAIN RESULTS AND THE ROLE OF CHANCEnFor patients with low or high MOAT activating capacity (MOAT group 1 or 3, respectively), calcium analysis confirmed the MOAT result. For patients with a former inconclusive intermediate MOAT activating capacity result (MOAT group 2), no or strongly dissimilar calcium oscillatory patterns were seen, with significantly lower amplitude and frequency compared with control sperm. When the product of the amplitude and the frequency of the calcium traces was compared between the groups, MOAT group 1 and 2 cases differed significantly from MOAT group 3 cases and the control sperm (P < 0.01).nnnLIMITATIONS, REASONS FOR CAUTIONnThe results of the calcium analysis in mouse oocytes should not be directly extrapolated to human oocytes, since it is well known that human spermatozoa exhibit a greater activating potency in mouse oocytes compared with mouse spermatozoa. Furthermore, not much is known yet about the influence of aberrant calcium oscillatory patterns, such as found in MOAT group 2 patients, on pre- and post-implantation embryo development in the human.nnnWIDER IMPLICATIONS OF THE FINDINGSnBased on the current calcium oscillatory pattern analysis, we found that the product of calcium spike amplitude with its frequency allowed us to create a new threshold value, which can assist in confirming or refuting, on a single patient base, a sperm-borne activation deficiency. The latter is especially interesting for patients with a former intermediate inconclusive MOAT result (MOAT group 2 patients), for whom calcium oscillatory pattern analysis should be considered.nnnSTUDY FUNDING/COMPETING INTEREST(S)nF.V.M. is holder of an aspirant clinical research mandate by the Flemish foundation of Scientific Research (FWO-Vlaanderen). B.H. is supported by a Ghent University grant (KAN-BOF E/01321/01). P.D.S. is holder of a fundamental clinical research mandate by the same Flemish foundation of Scientific Research (FWO-Vlaanderen).

Comparison of pre- and post-implantation development following the application of three artificial activating stimuli in a mouse model with round-headed sperm cells deficient for oocyte activation

STUDY QUESTIONnDoes the application of three different artificial activating stimuli lead to a difference in pre- and post-implantation embryo development in the wobbler mouse, a mouse model with oocyte activation deficient round-headed sperm cells similar to human globozoospermia?nnnSUMMARY ANSWERnNo gross differences were found between strontium chloride, electrical pulses or ionomycin with respect to the pre- and post-implantation development in the wobbler mouse.nnnWHAT IS KNOWN ALREADYnFertilization failure following intra-cytoplasmic sperm injection (ICSI) occurs in 1-3% of the ICSI cycles in human assisted reproduction technology (ART) and has been successfully overcome by different artificial activating stimuli. No comparison has been made yet in terms of their efficiency and safety.nnnSTUDY DESIGN, SIZE, DURATIONnCalcium release and embryo development were compared between oocytes fertilized by wobbler and wild-type (WT) sperm following ICSI with or without three different artificial activating agents. Preimplantation development was assessed on 70 injected oocytes on average per group. On average, 10 foster mothers were used per activating group to compare post-implantation development.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnWe used the wobbler mouse model that possesses oocyte activation deficient round-headed sperm cells. First, the calcium release following ICSI using wobbler sperm was compared with that of WT sperm. Outcome measures were the percentage of oocytes that showed calcium release and their mean amount of calcium rises. Secondly, the pre- and post-implantation development was assessed following ICSI with wobbler sperm plus artificial oocyte activation using either: (i) strontium chloride (Wob-Sr), (ii) electrical pulses (Wob-E) or (iii) ionomycin (Wob-I). Outcome measures were the activation, cleavage and blastocyst rates and the assessment of blastocyst quality by differential staining. Following mouse embryo transfer, pregnancy and birth rates as well as mean litter sizes were examined. Finally, pups were followed up until 8 weeks of age and then mated with fertile controls to assess their fertility.nnnMAIN RESULTS AND THE ROLE OF CHANCEnThe percentage of oocytes showing calcium rises as well as the number of calcium rises per oscillating oocyte were significantly lower in the wobbler group when compared with the WT group (9.3 versus 96% and 2.1 calcium rises versus 31 calcium rises) (P < 0.001). The fertilization rate was significantly lower in the wobbler group (11.4%) when compared with the WT group (92.1%) and the artificial activation groups (strontium chloride: 99%, electrical pulses: 99% and ionomycin: 81%, respectively) (P < 0.001). Post-implantation development did not differ significantly between the WT and artificial activation groups, with pregnancy rates in favor of strontium chloride and electrical pulses. The weight of the male pups did not differ between the study groups, whereas the weight of the female pups originating from Wob-Sr embryos was significantly lower at weeks 2, 3 and 4 when compared with female pups originating from WT embryos. However, the latter difference was not observed at later time points, nor in the other artificial activating groups. All offspring mated successfully with fertile controls.nnnLIMITATIONS, REASONS FOR CAUTIONnResults in animal models should be extrapolated with caution to a subfertile human population. Also, ionomycin is currently the most widely used artificial oocyte activating agent in human ART.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe low frequency of observed calcium rises and the low activation rate make the wobbler mouse a highly suitable model to study oocyte activation deficiency. Strontium chloride and electrical pulses were more efficient means to restore fertilization rates and to support pre- and post-implantation embryonic development than ionomycin.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis work was supported by the Flemish foundation of Scientific Research (FWO-Vlaanderen) (aspirant clinical research mandate to F.V.M., fundamental clinical research mandate to P.D.S.); and Ghent University grant (KAN-BOF E/01321/01 to B.H.). The authors have no competing interests to declare.