Kevin Coward

Human sperm devoid of PLC, zeta 1 fail to induce Ca(2+) release and are unable to initiate the first step of embryo development.

Egg activation, which is the first step in the initiation of embryo development, involves both completion of meiosis and progression into mitotic cycles. In mammals, the fertilizing sperm delivers the activating signal, which consists of oscillations in free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Intracytoplasmic sperm injection (ICSI) is a technique that in vitro fertilization clinics use to treat a myriad of male factor infertility cases. Importantly, some patients who repeatedly fail ICSI also fail to induce egg activation and are, therefore, sterile. Here, we have found that sperm from patients who repeatedly failed ICSI were unable to induce [Ca(2+)](i) oscillations in mouse eggs. We have also shown that PLC, zeta 1 (PLCZ1), the sperm protein thought to induce [Ca(2+)](i) oscillations, was localized to the equatorial region of wild-type sperm heads but was undetectable in sperm from patients who had failed ICSI. The absence of PLCZ1 in these patients was further confirmed by Western blot, although genomic sequencing failed to reveal conclusive PLCZ1 mutations. Using mouse eggs, we reproduced the failure of sperm from these patients to induce egg activation and rescued it by injection of mouse Plcz1 mRNA. Together, our results indicate that the inability of human sperm to initiate [Ca(2+)](i) oscillations leads to failure of egg activation and sterility and that abnormal PLCZ1 expression underlies this functional defect.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

Oocyte activation, phospholipase C zeta and human infertility

BACKGROUND Mammalian oocytes are activated by intracellular calcium (Ca(2+)) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C zeta, PLCζ, which is introduced into the oocyte following membrane fusion, as the responsible factor. This review summarizes the current understanding of human oocyte activation failure and describes recent discoveries linking certain cases of male infertility with defects in PLCζ expression and activity. How these latest findings may influence future diagnosis and treatment options are also discussed. METHODS Systematic literature searches were performed using PubMed, ISI-Web of Knowledge and The Cochrane Library. We also scrutinized material from the United Nations and World Health Organization databases (UNWHO) and the Human Fertilization and Embryology Authority (HFEA). RESULTS AND CONCLUSIONS Although ICSI results in average fertilization rates of 70%, complete or virtually complete fertilization failure still occurs in 1-5% of ICSI cycles. While oocyte activation failure can, in some cases, be overcome by artificial oocyte activators such as calcium ionophores, a more physiological oocyte activation agent might release Ca(2+) within the oocyte in a more efficient and controlled manner. As PLCζ is now widely considered to be the physiological agent responsible for activating mammalian oocytes, it represents both a novel diagnostic biomarker of oocyte activation capability and a possible mode of treatment for certain types of male infertility.

Gamete physiology, fertilization and egg activation in teleost fish

The fertilization and activation of fishoocytes are vital, but unfortunatelyoverlooked, processes in fisheries research.This paper sets out to review our presentunderstanding of these important events inteleost fish and, drawing comparisons withmammalian research, to highlight areas in whichresearch effort is urgently required. Presently, the commercial culture of manyimportant freshwater, but especially marine,teleosts is beset by problems associated withfertilization, hatching and early embryonicdevelopment. These problems have beenparticularly acute in certain species leadingto the application of spawning inductiontechnologies in an effort to optimizeproduction. Increased knowledge of theprocesses of egg activation and fertilizationin these groups of fish is likely to makesignificant contribution to commercialaquaculture. Studies of a wide variety ofanimal and plant species has demonstrated thatdevelopment at fertilization is triggered by anincrease in intracellular Ca2+concentration within the egg that occurs aseither a single transient or a series ofdistinctive oscillations depending upon thespecies under investigation. This increase inintracellular Ca2+ activates the egg andalso appears to play an important role in laterembryonic development. Teleost reproductivestrategies and more importantly, teleostoocytes and spermatozoa, exhibit a remarkablevariety of adaptations. Currently, studies ofegg activation in teleosts are confined tolaboratory species such as medaka Oryziaslatipes and zebrafish Brachydanio rerio.Nevertheless, even between these two species,although an increase in intracellular Ca2+appears to be the trigger in both cases, themechanism of Ca2+ release may be quitedifferent. Activation in medaka is initiatedonly through direct contact with conspecificsperm, suggesting the involvement of asperm-specific factor, while zebrafish eggsappear to require only contact with theexternal spawning medium. In view of the highlyvariable fertility rates evident in manycommercially cultured teleosts, it could bevery rewarding to investigate the mechanism ofegg activation in representative teleost groupsusing the findings and theories emerging fromother animal groups as a starting point. Inorder to successfully conduct such aninvestigation, it will be necessary to employ acombination of physiological, molecular andrecombinant approaches.

THE PATTERN OF LOCALIZATION OF THE PUTATIVE OOCYTE ACTIVATION FACTOR, PHOSPHOLIPASE CZETA, IN UNCAPACITATED, CAPACITATED, AND IONOPHORE-TREATED HUMAN SPERMATOZOA

BACKGROUND Recent studies suggest that in mammals, oocyte activation at fertilization is triggered by a sperm-specific phospholipase C, PLCzeta. We investigated PLCzeta localization in human spermatozoa. METHODS A polyclonal antibody was generated against human PLCzeta and used in immunoblotting and immunofluorescence studies of ejaculated human sperm in uncapacitated and capacitated states. An ionophore was also used to induce the acrosome reaction in vitro. RESULTS After verifying specificity of the anti-PLCzeta antibody by immunoblotting, immunofluorescence studies showed that the predominant localization of PLCzeta in uncapacitated sperm was in the equatorial region, a pattern maintained following capacitation and ionophore treatment. The analysis of pooled samples showed approximately 88% of uncapacitated sperm expressed PLCzeta in the equatorial region, whereas approximately 35% and approximately 21% of sperm expressed additional populations of PLCzeta in the acrosomal or post-acrosomal region, respectively. One population of PLCzeta was observed in the post-acrosomal region of approximately 12% of sperm. The proportion of cells with post-acrosomal PLCzeta increased following capacitation and ionophore treatment (P < 0.05). The same tendency was found in individual samples. There was a strong correlation (r = 0.716, P < 0.0001) between presence of an intact acrosome and proportion of sperm immunoreactive to PLCzeta in the acrosomal region. CONCLUSIONS PLCzeta was variably detectable in three localities within the sperm head: the equatorial segment and acrosomal/post-acrosomal region. Variability in PLCzeta localization in sperm from fertile males may reflect differences in oocyte activation capabilities between individuals or within an ejaculate. This approach may help in investigating the possible links between PLCzeta and certain types of male infertility.

Phospholipase C zeta undergoes dynamic changes in its pattern of localization in sperm during capacitation and the acrosome reaction.

OBJECTIVE To evaluate the localization of phospholipase C zeta (PLC zeta) in non-capacitated, capacitated, and ionophore-treated sperm. DESIGN Phospholipase C zeta was cloned from the hamster, an important model organism for studying fertilization. Next, we used hamster and mouse models to investigate the localization of PLC zeta in non-capacitated and capacitated sperm and in sperm treated with ionophore to induce the acrosome reaction. SETTING University laboratory. ANIMAL(S) Male mice and hamsters, 4-6 weeks old. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Phospholipase C zeta localization in non-capacitated, capacitated, and ionophore-treated sperm. RESULT(S) Full-length hamster PLC zeta complementary DNA is 1953 base pairs in size, encoding an open reading frame of 651 amino acids, sharing 85% amino acid similarity with the mouse. Phospholipase C zeta was localized in acrosomal and post-acrosomal regions of sperm. The post-acrosomal localization, which became more evident after capacitation and was maintained after ionophore treatment, is in line with PLC zeta being the endogenous agent of egg activation. However, the acrosomal PLC zeta population, which was lost after ionophore treatment, suggests that PLC zeta could have other functions besides egg activation. CONCLUSION(S) Phospholipase C zeta is localized to acrosomal and post-acrosomal regions and undergoes dynamic changes during capacitation and the acrosome reaction, indicating a potential role regulating not only egg activation but other sperm functions.

A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility

BACKGROUND Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζ(H398P)), leading to abnormal PLCζ function and infertility. METHODS AND RESULTS Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζ(H233L)), which is predicted to disrupt local protein interactions in a manner similar to PLCζ(H398P) and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζ(H233L) and PLCζ(H398P) exist on distinct parental chromosomes, the former inherited from the patients mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. CONCLUSIONS Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms.

Protamine 1 to protamine 2 ratio correlates with dynamic aspects of DNA fragmentation in human sperm

OBJECTIVE To investigate the relationship between the protamine 1 to protamine 2 (P1/P2) ratio and the rate of sperm DNA fragmentation in sperm samples from human males with proven fertility and three different cohorts of male patients. DESIGN P1/P2 ratio was analyzed using acid-urea polyacrylamide acid-urea gels electrophoresis (PAGE). Sperm DNA fragmentation using sperm chromatin dispersion methodology was analyzed after 0, 4, 8, and 24 hours of incubation at 37°C. SETTING University medical school and hospital. PATIENT(S) A total of 32 human males: six with proven fertility, seven carriers of chromosome reorganizations, nine clinical varicocele patients, and ten subclinical varicocele patients. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) P1/P2 ratio, sperm DNA fragmentation (SDF) and the rate of sperm DNA fragmentation (rSDF). RESULT(S) P1/P2 ratio correlated with SDF and rSDF. Statistical differences were detected between fertile controls and patients for the three pathologies studied. rSDF yielded information that differed from baseline SDF. No differences were detected for P1/P2 ratio among patient groups, in reference to the three pathologies studied. CONCLUSION(S) SDF and rSDF correlates with P1/P2 ratio in human sperm, and statistical differences were detected when fertile controls were compared with three different cohorts of patients.

Nanotechnology in reproductive medicine: Emerging applications of nanomaterials

UNLABELLED In the last decade, nanotechnology has been extensively introduced for biomedical applications, including bio-detection, drug delivery and diagnostic imaging, particularly in the field of cancer diagnostics and treatment. However, there is a growing trend towards the expansion of nanobiotechnological tools in a number of non-cancer applications. In this review, we discuss the emerging uses of nanotechnology in reproductive medicine and reproductive biology. For the first time, we summarise the available evidence regarding the use of nanomaterials as experimental tools for the detection and treatment of malignant and benign reproductive conditions. We also present an overview of potential applications for nanomaterials in reproductive biology, discuss the benefits and concerns associated with their use in a highly delicate system of reproductive tissues and gametes, and address the feasibility of this innovative and potentially controversial approach in the clinical setting and for investigative research into the mechanisms underlying reproductive diseases. FROM THE CLINICAL EDITOR This unique review paper focuses on the emerging use of nanotechnology in reproductive medicine and reproductive biology, highlighting the role of nanomaterials in the detection and treatment of various reproductive conditions, keeping in mind the benefits and potential concerns associated with nanomaterial use in the delicate system of reproductive tissue and gametes.

Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca2+) in all animals that have been examined, and such Ca2+ elevations, in turn, provide key activating signals that are required for non‐parthenogenetic development. Several lines of evidence indicate that the Ca2+ transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad‐based analysis of Ca2+ dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non‐mammalian species, and subsequently reviews various topics related to a sperm‐specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca2+ signals in fertilized oocytes or eggs are compared in a taxon‐by‐taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization‐induced Ca2+ signals are typically propagated as global waves that depend on Ca2+ release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5‐trisphosphate (IP3). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less‐common modes of producing Ca2+ transients are also examined. In addition, the importance of fertilization‐induced Ca2+ signals for activating development is underscored by noting some major downstream effects of these signals in various animals. Mol. Reprod. Dev. 80: 787–815, 2013.