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Oligoasthenoteratozoospermia (OAT) is demonstrated to be one of the most common causes of male subfertility. Phospholipase C ζ (PLCζ), a sperm-specific protein, is considered to be one of the sperm-borne oocyte activating factors (SOAFs), which play a vital role in fertilization. The post-acrosomal sheath WW domain-binding protein (PAWP) is another candidate for SOAF. The aim of this study was to compare the PLCζ localization patterns and percentage of PLCζ- and PAWP-positive sperm cells in patients with OAT and fertile men with normozoospermia. A total of 40 men included in this study were classified into two groups: OAT (n = 25) and control group (n = 15). Semen samples were collected and analyzed using conventional semen analysis according to the World Health Organization guidelines. The percentage of PLCζ- and PAWP-positive sperm cells and localization patterns of PLCζ were evaluated using immunofluorescence staining. The mean percentage of sperm cells expressing PAWP and PLCζ was significantly lower in OAT compared to control group (52.8 ± 4.2 vs. 76.8 ± 5 and 63.4 ± 3.5 vs. 86.7 ± 2.1, respectively). In addition, statistically significant differences were found with regard to the PLCζ localization patterns, including equatorial, acrosomal + equatorial, and equatorial + post-acrosomal pattern, between the two groups (p < 0.01). The present study showed a lower percentage of sperm cells expressing PLCζ and PAWP, as well as altered localization patterns of PLCζ in men with OAT. Given the role of PLCζ and PAWP in fertilization, as two major candidates for SOAFs, our findings indicate that PLCζ and PAWP impairments may be one of the possible etiologies of decreased fertility in OAT.

Mammalian reproduction is one of the most complex and fascinating biological phenomenon, which aims to transfer maternal and paternal genetic material to the next generation. At the end of oogenesis and spermatogenesis, both haploid gametes contain a single set of chromosomes ready to form the zygote, the first cell of the newly developing individual. The mature oocyte and spermatozoa remain in a quiescent state, during which the oocyte is characterized by nuclear and cytoplasmic arrest, while the spermatozoa necessitates further maturation within the epididymis and female reproductive track prior to egg fertilization. Either in vivo or in vitro, the sperm initiates a series of irreversible biochemical and physiological modifications in the oocyte. The earliest detected signal after fertilization is cytosolic CaLa reproduction des mammifères est l’un des phénomènes biologiques les plus complexes et fascinant; son objectif est de transférer le matériel génétique maternel et paternel à la génération suivante. A la fin de l’ovogenèse et de la spermatogenèse, les deux gamètes haploïdes contiennent un ensemble unique de chromosomes prêts à former un zygote, la première cellule du nouvel individu en développement. L’ovocyte mature et les spermatozoïdes restent dans un état quiescent au cours duquel l’ovocyte est caractérisé par un arrêt cytoplasmique et nucléaire, alors que les spermatozoïdes ont besoin d’une maturation ultérieure dans l’épididyme et le tractus génital femelle avant de pouvoir féconder l’ovocyte. Que ce soit in vivo ou in vitro, le spermatozoïde amorce une série de modifications biochimiques et physiologiques dans l’ovocyte.Le premier signal détecté après la fécondation est constitué des oscillations Ca2+ cytosoliques, une condition préalable au développement embryonnaire. Ces oscillations libèrent l’ovocyte de son arrêt en seconde méiose vers l’embryogenèse, phénomène connu sous le nom ‘d’activation de l’ovocyte’. L’isoforme phospholipase C zeta (PLCζ) est la seule protéine soluble du spermatozoïde capable d’activer dans l’ovocyte la voie de signalisation inositol triphosphate/Ca2+ qui mène aux oscillations Ca2+ et par conséquent au développement de l’embryon.Par rapport aux autres PLC, la structure spécifique de PLCζ lui confère une activité spécialisée via les domaines catalytiques préservés de X et Y, ainsi que des caractéristiques propres tels un déclenchement rapide, une grande sensibilité au Ca2+ et un arrêt des oscillations à la formation du zygote. Les découvertes récentes de PLCζ ont induit des études centrées sur les possibles applications cliniques de cette protéine dans l’évaluation et la prise en charge de l’infertilité masculine lors de FIV/ICSI. L’échec de fécondation est attribué à l’absence de reprise de la seconde méiose ovocytaire, suggérant la possibilité que l’échec de l’ICSI puisse être lié à une activité défectueuse de la PLCζ dans le spermatozoïde. La micro injection de PLCζ humaine recombinante dans des ovocytes humains après échec de fécondation en ICSI pourrait déclencher des oscillations Ca2+ et permettre une fécondation réussie, offrant de nouveaux espoirs aux couples traditionnellement orientés vers le don de spermatozoïdes. Il est toutefois nécessaire de disposer d’un plus grand nombre d’études avant toute mise en œuvre de cette approche en clinique. Des orientations pour de futures études sont discutées.

International audience; Purpose Sperm-borne PLCζ protein induces Ca 2+ oscillations in the oocyte and is believed to play a major role during oocyte activation. However, its implication in fertilization failure following ICSI is still debated. We analyzed PLCζ gene sequence, protein expression level, and localization in both patients with previous failed fertilization by ICSI and sperm donors with proven fertility in order to assess the association of PLCζ with both sperm characteristics and ability to fertilize. Methods Semen from 15 patients and 13 sperm donors with proven fertility was included in the study. Analysis of the PLCζ gene sequence, protein expression through Western blot, and protein localization by immunofluorescence were performed. Results Two patients with total fertilization failure presented mutations in heterozygosis in the PLCζ gene. Comparison with donor sample sequences displayed comparable SNP al-lele frequency. Distribution pattern of PLCζ did not vary significantly between donor and patient samples. Levels of PLCζ protein in sperm cells showed an interindividual variability both in patient and donor samples. Several SNPs previously reported in infertile patients were also present in fertile men. Conclusion Failed fertilization occurs even when levels and distribution of PLCζ protein are within normal range. PLCζ seems to be a necessary but not sufficient factor in determining the molecular pathway involved in oocyte activation.

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca(2+) ionophores to produce a single cytosolic Ca(2+) increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca(2+) 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 Ca(2+) oscillations after ICSI. The decrease in Ca(2+) 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 Ca(2+) ionophore, or with Sr(2+) media which causes Ca(2+) oscillations, or we injected them with recombinant human PLCζ. All these treatments rescued oocyte activation, although Sr(2+) 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 Ca(2+) releasing activity.

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) ζ-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:51Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:30:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-10-15 In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca2+]i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca2+]i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ~70 kDa in whole sperm lysates of domestic cat as well as in both soluble and insoluble fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males. © 2013 Elsevier Inc.. Department of Animal Reproduction and Veterinary Radiology College of Veterinary Medicine and Animal Science São Paulo State University, UNESP, Botucatu, São Paulo Department of Veterinary and Animal Sciences University of Massachusetts, Amherst, MA Universidade de Franca UNIFRAN, Franca, São Paulo Department of Animal Reproduction and Veterinary Radiology College of Veterinary Medicine and Animal Science São Paulo State University, UNESP, Botucatu, São Paulo