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The present study aimed to detect and quantify the expression of buffalo PLCZ1 mRNA (buPLCZ1 mRNA) in buffalo semen and to determine its oocyte-activation and fertilizing ability through IVF. Buffalo semen samples were collected and subjected to standard subjective physical and microscopic evaluation of semen characteristics. Small amount of fresh semen sample was taken for molecular experiment. The rest of the samples were processed into frozen semen. Both fresh and frozen semen were subjected to Reverse Transcriptase quantitative PCR (RT-qPCR) technique to determine and quantify buPLCZ1 mRNA expression. Subsequently, 40 bulls with known buPLCZ1 mRNA content were evaluated for its oocyte-activation activity through IVF using frozen semen. Correlation analysis was done to determine the relationship between the expression of buPLCZ1 mRNA and percentage of IVF, and other semen variables. PLCZ1 mRNA expression in buffalo semen was observed variable among donor bulls, but was not significantly different (P > 0.05) between fresh and frozen semen. The result suggests that the cryopreservation procedure does not affect the expression of buPLCZ1 mRNA in semen. Statistical analysis showed that the expression of buPLCZ1 mRNA in fresh semen was strongly correlated with that of frozen semen (r = 0.992; P 0.05) nor with Post-Thaw Motility (r = −0.0313; P > 0.05). A strong relationship between buPLCZ1 mRNA concentration and IVF rate indicates that buPLCZ1 mRNA concentration can be used to assess oocyte-activation and fertilizing potential of the buffalo semen. The present study essentially demonstrated buPLCZ1 mRNA as biological marker for male fertility and that molecular technique serves as an objective approach of semen quality evaluation to enhance bull selection for genetic improvement in water buffaloes.

Ca2+ influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2, to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double-knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and, following sperm entry or Plcz (also known as Plcz1) cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wild-type eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting that the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also showed that they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

To become fertile, mammalian sperm must undergo a series of biochemical and physiological changes known as capacitation. These changes involve crosstalk between metabolic and signaling pathways and can be recapitulated in vitro. In this work, sperm were incubated in the absence of exogenous nutrients (starved) until they were no longer able to move. Once immotile, energy substrates were added back to the media and sperm motility was rescued. Following rescue, a significantly higher percentage of starved sperm attained hyperactivated motility and displayed increased ability to fertilize in vitro when compared with sperm persistently incubated in standard capacitation media. Remarkably, the effects of this treatment continue beyond fertilization as starved and rescued sperm promoted higher rates of embryo development, and once transferred to pseudo-pregnant females, blastocysts derived from treated sperm produced significantly more pups. In addition, the starvation and rescue protocol increased fertilization and embryo development rates in sperm from a severely subfertile mouse model, and when combined with temporal increase in Ca2+ ion levels, this methodology significantly improved fertilization and embryo development rates in sperm of sterile CatSper1 KO mice model. Intracytoplasmic sperm injection (ICSI) does not work in the agriculturally relevant bovine system. Here, we show that transient nutrient starvation of bovine sperm significantly enhanced ICSI success in this species. These data reveal that the conditions under which sperm are treated impact postfertilization development and suggest that this “starvation and rescue method” can be used to improve assisted reproductive technologies (ARTs) in other mammalian species, including humans. Fil: Navarrete, Felipe A.. University of Massachussets; Estados Unidos Fil: Aguila, Luis. University of Massachussets; Estados Unidos Fil: Martin Hidalgo, David. University of Massachussets; Estados Unidos. Universidad de Extremadura ; España Fil: Tourzani, Darya A.. University of Massachussets; Estados Unidos Fil: Luque, Guillermina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ardestani, Goli. University of Massachussets; Estados Unidos Fil: Garcia Vazquez, Francisco A.. Universidad de Murcia; España Fil: Levin, Lonny R.. Cornell University; Estados Unidos Fil: Buck, Jochen. Cornell University; Estados Unidos Fil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biología; México Fil: Buffone, Mariano Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Mager, Jesse. University of Massachussets; Estados Unidos Fil: Fissore, Rafael A.. University of Massachussets; Estados Unidos Fil: Salicioni, Ana M.. University of Massachussets; Estados Unidos Fil: Gervasi, María G.. University of Massachussets; Estados Unidos Fil: Visconti, Pablo E.. University of Massachussets; Estados Unidos

In mammals, fertilization initiates Ca(2+) oscillations in metaphase II oocytes, which are required for the activation of embryo development. Germinal vesicle (GV) oocytes also display Ca(2+) oscillations, although these unfold spontaneously in the absence of any known agonist(s) and their function remains unclear. We found that the main intracellular store of Ca(2+) in GV oocytes, the endoplasmic reticulum ([Ca(2+)](ER)), constitutively ‘leaks’ Ca(2+) through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca(2+)](ER) leak ceases around the resumption of meiosis, the GV breakdown (GVBD) stage, which coincides with the first noticeable accumulation of Ca(2+) in the stores. It also concurs with downregulation of the Ca(2+) influx and termination of the oscillations, which seemed underpinned by the inactivation of the putative plasma membrane Ca(2+) channels. Lastly, we demonstrate that mitochondria take up Ca(2+) during the Ca(2+) oscillations, mounting their own oscillations that stimulate the mitochondrial redox state and increase the ATP levels of GV oocytes. These distinct features of Ca(2+) homeostasis in GV oocytes are likely to underpin the acquisition of both maturation and developmental competence, as well as fulfill stage-specific cellular functions during oocyte maturation.

More than 15 years have elapsed since the identification of phospholipase C ζ1 (PLCζ) from a genomic search for mouse testis/sperm-specific PLCs. This molecule was proposed to represent the sperm factor responsible for the initiation of calcium (Ca2+ ) oscillations required for egg activation and embryo development in mammals. Supporting evidence for this role emerged from studies documenting its expression in all mammals and other vertebrate species, the physiological Ca2+ rises induced by injection of its messenger RNA into mammalian and nonmammalian eggs, and the lack of expression in infertile males that fail intracytoplasmic sperm injection. In the last year, genetic animal models have added support to its role as the long sought-after sperm factor. In this review, we highlight the findings that demonstrated the role of Ca2+ as the universal signal of egg activation and the experimental buildup that culminated with the identification of PLCζ as the soluble sperm factor. We also discuss the structural-functional properties that make PLCζ especially suited to evoke oscillations in eggs. Lastly, we examine unresolved aspects of the function and regulation of PLCζ and whether or not it is the only sperm factor in mammalian sperm.

After receiving the LH surge, a mature oocyte (egg) is collected in the oviduct of mammalian females. Following fusion of the gametes, the sperm elicits in the egg repeated calcium (Ca 2 + ) transients known as oscillations, which trigger egg activation. Completion of egg activation renders the meiotic and transcriptionally silent egg into a mitotically competent, transcriptionally active zygote that eventually undergoes cell differentiation and embryo development. Here, we discuss the major events of egg activation and the molecular players that underlie them. We describe the universal role of Ca 2 + on egg activation as well as the egg's Ca 2 + toolkit. We explain how failure of egg activation results in infertility, and the value of ICSI and genetic models in identifying the molecular causes of this infertility. Finally, we point to areas where additional research is needed and how this knowledge will allow better control of fertility.

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whetherin vitromaturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI,in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, althoughin vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation byin vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu ofin vitro-matured oocytes and to replicate the molecular changes associated within vivocapacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

Phospholipase C zeta, a novel sperm-specific protein which is widely known to induce oocyte activation following fertilization, had already been characterized in various mammalian species, but not in water buffaloes thus far. The present study was conducted to initially characterize and compare the sequences of PLCZ1 gene of swamp and riverine buffaloes. Semen samples were collected; total RNA was extracted and reverse-transcribed. PLCZ1 cDNA was then amplified, and submitted for sequencing. Buffalo PLCZ1 gene yielded a sequence of 1905 base pair nucleotides translated into 634 bp amino acids. In general, the buffalo PLCZ1 gene was found to have high sequence identity with cattle and other domestic species. Similarly, significant residues and motifs in PLCZ1 gene sequence are found conserved in water buffaloes. However, there are variations in sequences identified between types of water buffaloes that may play a role in species-specific differences in terms of gene and protein expression, physiological mechanisms, and biological functions. The molecular information on buffalo PLCZ1 gene is highly valuable in subsequent works such as correlation studies on the identified gene variations with semen quality and fertility, and the development of biomarkers for bull fertility.

We recently identified the DPY19L2 gene as the main genetic cause of human globozoospermia (70%) and described that Dpy19l2 knockout (KO) mice faithfully reproduce the human phenotype of globozoospermia making it an excellent model to characterize the molecular physiopathology of globozoospermia. Recent case studies on non-genetically characterized men with globozoospermia showed that phospho- lipase C, zeta (PLCz), the sperm factor thought to induce the Ca 2+ oscillations at fertilization, was absent from their sperm, explaining the poor fertilization potential of these spermatozoa. Since 30% of globozoospermic men remain genetically uncharacterized, the absence of PLCz in DPY19L2 globozoospermic men remains to be formally established. Moreover, the precise localization of PLCz and the reasons underlying its loss during spermatogenesis in globozoospermic patients are still not understood. Herein, we show that PLCz is absent, or its presence highly reduced, in human and mouse sperm with DPY19L2-associated globozoospermia. As a consequence, fertilization with sperm from Dpy19l2 KO mice failed to initiate Ca 2+ oscillations and injected oocytes remained arrested at the metaphase II stage, although a few human oocytes injected with DPY19L2-defective sperm showed formation of 2-pronuclei embryos. We report for the first time the subcellular localization of PLCz in control human sperm, which is along the inner acrosomal membrane and in the perinuclear theca, in the area corresponding to the equa- torial region. Because these cellular components are absent in globozoospermic sperm, the loss of PLCz in globozoospermic sperm is thus con- sistent and reinforces the role of PLCz as an oocyte activation factor necessary for oocyte activation. In our companion article, we showed that chromatin compaction during spermiogenesis in Dpy19l2 KO mouse is defective and leads to sperm DNA damage. Together, these defects explain the poor fertilization potential of DPY19L2-globozoospermic sperm and the compromised developmental potential of embryos obtained using sperm from patients with a deletion of the DPY19L2 gene.

Apoptosis in most cell types is accompanied by altered Ca(2+) homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP3 R1) generates a 95-kDa C-terminal fragment (C-IP3 R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca(2+) homeostasis and express C-IP3 R1, although whether or not C-IP3 R1 expression contributes to Ca(2+) misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding C-IP3 R1. We found that: (1) expression of C-IP3 R1 in eggs lowered the Ca(2+) content of the endoplasmic reticulum (ER), although, as C-IP3 R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; (2) expression of C-IP3 R1 in eggs enhanced fragmentation associated with aging; (3) endogenous IP3 R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP3 R1 in eggs with downregulated IP3 R1 partly restored fragmentation; (4) C-IP3 R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca(2+) pool that occurs during maturation, undermined the Ca(2+) oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3 R1 and C-IP3 R1 in regulating Ca(2+) homeostasis and the ER Ca(2+) content during oocyte maturation. Nevertheless, the role of C-IP3 R1 on Ca(2+) homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3 R1 remains intact and C-IP3 R1 undergoes rapid turnover.

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.

Changes in the intracellular concentration of calcium ([Ca2+]i) represent a vital signaling mechanism enabling communication between and among cells as well as with the environment. Cells have developed a sophisticated set of molecules, "the Ca2+ toolkit," to adapt [Ca2+]i changes to specific cellular functions. Mammalian oocytes and eggs, the subject of this review, are not an exception, and in fact the initiation of embryo devolvement in all species is entirely dependent on distinct [Ca2+]i responses. Here, we review the components of the Ca2+ toolkit present in mammalian oocytes and eggs, the regulatory mechanisms that allow these cells to accumulate Ca2+ in the endoplasmic reticulum, release it, and maintain basal and stable cytoplasmic concentrations. We also discuss electrophysiological and genetic studies that have uncovered Ca2+ influx channels in oocytes and eggs, and we analyze evidence supporting the role of a sperm-specific phospholipase C isoform as the trigger of Ca2+ oscillations during mammalian fertilization including its implication in fertility.

SummaryIn mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3−/− eggs failed to conduct Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.

A sperm-specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca(2+) ]i ) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT-PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long-lasting [Ca(2+) ]i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca(2+) ]i oscillation-inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.

Voltage-gated calcium (CaV) channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1–1.4; CaV2.1–2.3) and Low-voltage activated (LVA, CaV3.1–3.3). HVA channels are highly expressed in brain (neurons), heart, and adrenal medulla (chromaffin cells), among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, “conopeptides”, which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

The activation of the developmental program in mammalian eggs relies on the initiation at the time of fertilization of repeated rises in the intracellular concentration of free calcium ([Ca(2+)](i)), also known as [Ca(2+)](i) oscillations. The ability to mount the full complement of oscillations is only achieved at the end of oocyte maturation, at the metaphase stage of meiosis II (MII). Over the last decades research has focused on addressing the mechanisms by which the sperm initiates the oscillations and identification of the channels that mediate intracellular Ca(2+) release. This review will describe the up-to-date knowledge of other aspects of Ca(2+) homeostasis in mouse oocytes, such as the mechanisms that transport Ca(2+) out of the cytosol into the endoplasmic reticulum (ER), the Ca(2+) store of the oocyte/egg, into other organelles and also those that extrude Ca(2+). Evidence pointing to channels in the plasma membrane that mediate Ca(2+) entry from the extracellular milieu, which is required for the persistence of the oscillations, is also discussed, along with the modifications that these mechanisms undergo during maturation. Lastly, we highlight areas where additional research is needed to obtain a better understating of the molecules and mechanisms that regulate Ca(2+) homeostasis in this unique Ca(2+) signaling system.

Changes in Ca2+ homeostasis render oocytes competent to undergo [Ca2+]i oscillations and activation. During mouse oocyte maturation Ca2+ influx and SOCE are down-regulated, whereas [Ca2+]ER content increases. Bypassing the down-regulation of Ca2+ influx disturbs oocyte maturation., In preparation for fertilization, mammalian oocytes undergo optimization of the mechanisms that regulate calcium homeostasis. Among these changes is the increase in the content of the Ca2+ stores ([Ca2+]ER), a process that requires Ca2+ influx. Nevertheless, the mechanism(s) that mediates this influx remains obscure, although is known that [Ca2+]ER can regulate Ca2+ influx via store-operated Ca2+ entry (SOCE). We find that during maturation, as [Ca2+]ER increases, Ca2+ influx decreases. We demonstrate that mouse oocytes/eggs express the two molecular components of SOCE—stromal interaction molecule 1 (Stim1) and Orai1—and expression of human (h) Stim1 increases Ca2+ influx in a manner that recapitulates endogenous SOCE. We observe that the cellular distribution of hStim1 and hOrai1 during maturation undergoes sweeping changes that curtail their colocalization during the later stages of maturation. Coexpression of hStim1 and hOrai1 enhances influx throughout maturation but increases basal Ca2+ levels only in GV oocytes. Further, expression of a constitutive active form of hStim1 plus Orai1, which increases basal Ca2+ throughout maturation, disturbs resumption of meiosis. Taken together, our results demonstrate that Ca2+ influx and SOCE are regulated during maturation and that alteration of Ca2+ homeostasis undermines maturation in mouse oocytes.

The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development.

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca 2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca 2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca 2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility. Fil: Navarrete, Felipe A.. University of Massachusetts; Estados Unidos Fil: Alvau, Antonio. University of Massachusetts; Estados Unidos Fil: Lee, Hoi Chang. University of Massachusetts; Estados Unidos Fil: Levin, Lonny R.. Weill Cornell Medical College; Estados Unidos Fil: Buck, Jochen. Weill Cornell Medical College; Estados Unidos Fil: Leon, Patricia Martin De. University of Delaware; Estados Unidos Fil: Santi, Celia M.. University of Washington; Estados Unidos Fil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Mager, Jesse. University of Massachusetts; Estados Unidos Fil: Fissore, Rafael A.. University of Massachusetts; Estados Unidos Fil: Salicioni, Ana M.. University of Massachusetts; Estados Unidos Fil: Darszon, Alberto. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Visconti, Pablo E.. University of Massachusetts; Estados Unidos

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.

International audience; In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).

An intracellular calcium ([Ca(2+)]i) rise has been described in cumulus-oocyte complexes (COCs) following luteinizing hormone (LH) exposure. Together with cAMP, Ca(2+) is a candidate signal for resumption of meiosis. Here, we analyzed if the most common hormones involved in oocyte maturation can induce the same Ca(2+) signal. In addition, we characterized the source of this signal. Immature, in vitro-matured, and roscovitine-meiotically arrested COCs were loaded with Fluo-4 AM, stimulated with hormones/growth factors, and tested for [Ca(2+)](i) variations in cumulus cells. Reagents known to inhibit or stimulate [Ca(2+)](i) rises were used to characterize these [Ca(2+)](i) dynamics. Finally, expression of LH receptors (LHRs) in COCs was analyzed by immunofluorescence. In immature COCs, follicle-stimulating hormone (FSH) elicited a single [Ca(2+)](i) rise that was higher than those induced by LH and growth hormone (GH), whereas epithelial growth factor failed to induce any changes in [Ca(2+)](i). The [Ca(2+)](i) rise induced by FSH was higher in immature COCs; was reduced in roscovitine-arrested, immature COCs; and was negligible in gonadotropin-induced, in vitro-matured COCs. In the case of spontaneous- and GH-matured COCs, however, FSH stimulation caused a lower [Ca(2+)](i) rise. The hormone-induced [Ca(2+)](i) rise was due to: (i) external Ca(2+) entry; (ii) intercellular communication; and (iii) intracellular Ca(2+) stores. Immunofluorescence revealed that LHRs were expressed throughout the cumulus cells. The above results show that: (i) gonadotropins and GH cause a [Ca(2+)](i) rise in cumulus cells; (ii) this [Ca(2+)](i) rise results from extra-, inter-, and intra-cellular cumulative Ca(2+) fluxes; and (iii) LHRs are distributed on either outer or inner cumulus cells.

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.

The developmental competence of mammalian eggs is compromised by postovulatory aging. We and others have found that in these eggs, the intracellular calcium ([Ca(2+)](i)) responses required for egg activation and initiation of development are altered. Nevertheless, the mechanism(s) underlying this defective Ca(2+) release is not well known. Here, we investigated if the function of IP(3)R1, the major Ca(2+) release channel at fertilization, was undermined in in vitro-aged mouse eggs. We found that in aged eggs, IP(3)R1 displayed reduced function as many of the changes acquired during maturation that enhance IP(3)R1 Ca(2+) conductivity, such as phosphorylation, receptor reorganization and increased Ca(2+) store content ([Ca(2+)](ER)), were lost with increasing postovulatory time. IP(3)R1 fragmentation, possibly associated with the activation of caspase-3, was also observed in these eggs. Many of these changes were prevented when the postovulatory aging of eggs was carried out in the presence of caffeine, which minimized the decline in IP(3)R(1) function and maintained [Ca(2+)](ER) content. Caffeine also maintained mitochondrial membrane potential, as measured by JC-1 fluorescence. We therefore conclude that [Ca(2+)](i) responses in aged eggs are undermined by reduced IP(3)R1 sensitivity, decreased [Ca(2+)](ER) , and compromised mitochondrial function, and that addition of caffeine ameliorates most of these aging-associated changes. Understanding the molecular basis of the protective effects of caffeine will be useful in elucidating, and possibly reversing, the signaling pathway(s) compromised by in vitro culture of eggs.

A major unresolved issue in developmental biology is the precise mechanism whereby the sperm activates the oocyte. With the discovery that calcium signals are the primary trigger for oocyte activation, a key remaining question became the identification of the signaling protein that mediates such calcium signals at fertilization. A major step forward came in 2002 with the discovery of a sperm-specific mammalian phospholipase C called phospholipase C zeta (PLCζ), which had the expected properties of the mammalian oocyte activation factor and was subsequently identified in other vertebrate groups. Most recently, defects in PLCζ have been shown to be linked to certain types of male infertility in humans. Despite these advances, many questions remain about the precise mechanism of action of PLCζ and the extent of its role during oocyte activation in the vertebrate kingdom. In this review, we will look at the current state of understanding of PLCζ's mechanism of action and physiological role in mammals and other vertebrates, and identify areas of uncertainty that still remain to be resolved.

Under the influence of gonadotropins or growth factors, a close cooperation develops between cumulus cells and the oocyte that is implicated in transmitting signals involved in maintaining or releasing the meiotic arrest in the oocyte. While cyclic adenosine 5'-monophosphate (cAMP) is a key molecule in maintaining the meiotic arrest, calcium (Ca(2+)) may play a role in controlling either spontaneous or gonadotropin-induced oocyte maturation, possibly by modulating intracytoplasmic cAMP concentrations via Ca(2+)-sensitive adenylate cyclases. This review focuses on the mechanisms related to the origin of the Ca(2+) wave that travels from the cumulus cells to the oocyte, and discusses the source of variations affecting the dynamics of this wave.

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+]i) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1 with eggs after the vitrification performed using ethylene glycol (EG) alone or EG + dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG + DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG + DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+]i oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+]i oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.

Recent studies suggest that egg activation in mammals is triggered by a sperm-specific phospholipase C, PLCzeta. In other vertebrate species such as medaka fish, chickens, and quail, PLCzeta is also expressed as a testis-specific mRNA. Functional studies suggest that PLCzeta plays a similar role as a trigger of egg activation in these species. Here, we report the identification of PLCzeta orthologues in pufferfish species Takifugu rubripes (Fugu) and Tetraodon nigroviridis (Tetraodon). Unexpectedly in these species PLCzeta is expressed not in the testis, but in ovary and brain. Injection of pufferfish PLCzeta copy ribonucleic acid (cRNA) into mouse eggs failed to trigger calcium oscillations, unlike medaka PLCzeta cRNA. Our findings provide the first evidence that PLCzeta may be expressed in the egg, rather than the sperm, in some vertebrate species, and that its mechanism of action and physiologic role at fertilization may differ in different vertebrate species.

One of the most important processes in fertilization is the fusion of egg and sperm; however, the molecular mechanisms involved in this process are not well understood. So far, using genetic approaches, only two proteins have been demonstrated to be necessary for this process: Izumo in sperm and CD9 in the egg. Here we demonstrate that sperm produced by Tssk6 (Sstk)-null mice present defects that prevent the successful fertilization of eggs in vitro and the fusion to zona-pellucida-free eggs. Tssk6 is a member of the testis-specific serine kinase family of proteins and is expressed postmeiotically in male germ cells. In order for fusion to occur, during the process known as acrosome reaction Izumo needs to relocate from the anterior head to other regions, including the postacrosomal compartment. Tssk6-null sperm fails to relocate Izumo during the acrosome reaction. Agents that interfere with actin dynamics blocked the acrosome-reaction-associated translocation of Izumo that is required for fusion in wild-type sperm. Additionally, actin polymerization was compromised in Tssk6-null sperm. Taken together, our results indicate that Tssk6 is involved in sperm-egg fusion through the regulation of actin polymerization and changes in Izumo localization.

We evaluated the effect of different activation methods on blastocyst development after equine nuclear transfer. All activation treatments were followed by incubation in 2 mM 6-dimethylaminopurine for 4 h. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 sec using a FemtoJet injection device, then treated with ionomycin. The blastocyst rate (9.8%) for 0.1-sec injection was significantly higher than that for 0.2 sec (0%) or 0.8 sec (1.4%). In Experiment 2, injection of murine PLCzeta cRNA before or after ionomycin treatment did not increase blastocyst development (0 and 4.5%) over a control treatment (injection of sperm extract after ionomycin exposure; 5.6%). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in five pregnancies, all lost before 70 days of gestation. In Experiment 3, cells from a second biopsy sample from the same horse produced significantly more blastocysts than did the original sample (4/44 vs. 0/58; p0.05). Transfer of these four blastocysts produced two viable foals. In Experiment 4, blastocyst development rates did not differ between oocytes in metaphase I or II at the time of nuclear transfer (16.7 and 3.0%, respectively). A healthy foal was produced from a blastocyst originating from a metaphase I oocyte.

Xenopus oocyte death is partly controlled by the apoptotic initiator, caspase-2. We reported previously that oocyte nutrient depletion activates caspase-2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. We now show that caspase-2 phosphorylated at S135 binds 14-3-3ζ, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3ζ from caspase-2 is controlled by metabolism and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3ζ is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death.

This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4–23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8–54.5%) was also achieved with oocytes collected at 10–12 hpi than from those collected 14–16 hpi (8.3–14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2–42.8 %) were significantly higher than from those collected at 16 hpi (6.8–8.4 %) (p

Reviews in Developmental Biology have covered the pathways that generate the all-important intracellular calcium (Ca(2+)) signal at fertilization [Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., 1993a. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel in Ca(2+) waves and Ca(2+) oscillations at fertilization of mammalian eggs. Dev. Biol. 158, 62-78; Runft, L., Jaffe, L., Mehlmann, L., 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237-254] and the different temporal responses of Ca(2+) in many organisms [Stricker, S., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157-176]. Those reviews raise the importance of identifying how Ca(2+) causes the events of egg activation (EEA) and to what extent these temporal Ca(2+) responses encode developmental information. This review covers recent studies that have analyzed how these Ca(2+) signals are interpreted by specific proteins, and how these proteins regulate various EEA responsible for the onset of development. Many of these proteins are protein kinases (CaMKII, PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly regulated by Ca(2+), and whose amount increases during late oocyte maturation. We cover biochemical progress in defining the signaling pathways between Ca(2+) and the EEA, as well as discuss how oscillatory or multiple Ca(2+) signals are likely to have specific advantages biochemically and/or developmentally. These emerging concepts are put into historical context, emphasizing that key contributions have come from many organisms. The intricate interdependence of Ca(2+), Ca(2+)-dependent proteins, and the EEA raise many new questions for future investigations that will provide insight into the extent to which fertilization-associated signaling has long-range implications for development. In addition, answers to these questions should be beneficial to establishing parameters of egg quality for human and animal IVF, as well as improving egg activation protocols for somatic cell nuclear transfer to generate stem cells and save endangered species.

The World Health Organization declaimed that infertility is a major global public health issue of the last few decades. Infertility is commonly defined as the failure to conceive after 1 year of unprotected intercourse and is estimated to concern 72.4 million people worldwide with 40.5 million currently seeking medical care. The overall burden of subfertility/infertility is significant, is likely underestimated, and has not displayed any decrease over the last 20 years. Male factors are estimated to be involved, at least partially, in half of the cases. While the diagnosis, medical treatment, and psychosocial management of infertility have rapidly evolved over the past 4 decades, some difficulties still persist. Little is known about the physiopathology of altered sperm production, its genetic causes, or the genetic and epigenetic consequences for the gamete and the forthcoming conceptus. The information generated by conventional semen analysis has historically classified patients into categories lacking knowledge of causality and leaving conventional therapy as somewhat empirical. One of the reasons for this lack of fundamental understanding is the heterogeneity of causal factors as male infertility is a typical multifactorial disorder with a strong genetic basis and additional factors such as urogenital infections, immunological or endocrine diseases, attack from reactive oxygen species (ROS), or perturbations from endocrine disruptors. Since assisted reproduction technology (ART) is widely used to achieve conception with gametes produced by compromised spermatogenesis, there is a clear need to detail the molecular pathogenesis of male infertility to improve long-term risk assessment on a case-by-case basis. In this context, research on the male partner will shed a much-needed light on the physiopathology of male reproduction, will enhance patient management, and constitutes a prerequisite for the development of new therapeutic solutions.

One important result of egg activation is the establishment of blocks to prevent polyspermic fertilization; these blocks are established on the zona pellucida and the egg plasma membrane. This study examines what the sperm brings to the egg to induce the establishment of the membrane block to polyspermy, building on past evidence that membrane block establishment does not occur in response to parthenogenetic stimuli that induce a single transient increase in cytosolic Ca2+ or intracytoplasmic sperm injection (ICSI). We test the hypotheses that (i) sperm-associated Ca2+ release activity triggers membrane block establishment; (ii) introduction of sperm contents via variations on ICSI protocols (resulting in improved Ca2+ transients, egg activation and embryo development over traditional ICSI protocols) triggers membrane block establishment and (iii) sperm adhesion [binding of an extracellular sperm ligand(s) to an egg receptor(s)] combined with sperm-associated Ca2+ release activity triggers membrane block establishment. Interestingly, none of these stimuli induced establishment of the membrane block to polyspermy in mouse eggs. However, the sperm-associated remodeling of the egg cortical cytoskeleton differs between conventionally fertilized and ICSI-fertilized eggs; taken with our previous data implicating actin microfilaments in membrane block establishment, this raises the possibility that cortical reorganization may be a contributing factor. In sum, fertilization-like Ca2+ transients, either alone or combined with sperm-egg binding, are not sufficient for membrane block establishment, but that an event(s) associated with gamete interaction plays a role in this membrane function change.

The oocytes of B6.YTIR sex-reversed female mice can be fertilized but the resultant embryos die at early cleavage stages. In the present study, we examined chromosome segregation at meiotic divisions in the oocytes of XY female mice, compared to those of XX littermates. The timing and frequency of oocyte maturation in culture were comparable between the oocytes from both types of females. At the first meiotic division, the X- and Y-chromosomes segregated independently and were retained in oocytes at equal frequencies. However, more oocytes retained the correct number of chromosomes than anticipated from random segregation. The oocytes that had reached MII-stage were activated by fertilization or incubation with SrCl2. As expected, the majority of oocytes from XX females completed the second meiotic division and reached the 2-cell stage in 24 h. By contrast, more than half of oocytes from XY females initially remained at the MII-stage while the rest precociously entered interphase after SrCl2 activation; very few oocytes were seen at the second anaphase or telophase and they often showed impairment of sister–chromatid separation. Eventually the majority of oocytes entered interphase and formed pronuclei, but very few reached the 2-cell stage. Similar results were obtained after fertilization. We conclude that the XY chromosomal composition in oocyte leads to impairment in the progression of the second meiotic division.

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. Fil: Bello, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Cappa, Andrea Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: de Paola, Maria Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Zanetti, Maria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Fukuda, Mitsunori. Tohoku University; Japón Fil: Fissore, Rafael A.. Dep. Veterinary And Animal Science-univ.massachusetts A; Estados Unidos Fil: Mayorga, Luis Segundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Michaut, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Exactas y Naturales; Argentina

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) mediates increases in the intracellular concentration of Ca(2+) ([Ca(2+)]i) during fertilization in mammalian eggs. The activity of IP3R1 is enhanced during oocyte maturation, and phosphorylations by M-phase kinases are thought to positively regulate the activity of IP3R1. Accordingly, we and others have found that IP3R1 is phosphorylated at S(421), T(799) (by Cdk1) and at S(436) (by ERK). Nevertheless, the effects of these phosphorylations on the function of the receptor and their impact on [Ca(2+)]i oscillations in eggs have not been clearly examined. To address this, we expressed in mouse oocytes an IP3R1 variant with the three indicated phosphorylation sites replaced by acidic residues, IIIE-IP3R1, such that it would act like a constitutively phosphorylated IP3R1, and examined [Ca(2+)]i parameters in response to stimuli. We found that overexpression of wild type (wt-IP3R1) or IIIE-IP3R1 in oocytes containing endogenous receptors caused dominant negative-like effects on Ca(2+) release and oscillations. Therefore, we first selectively removed the endogenous IP3R1, and subsequently expressed the exogenous receptors. We found that in response to injection of PLCζ cRNA, eggs without endogenous IP3R1 failed to mount persistent Ca(2+) oscillations, although expression of wt-IP3R1 restored their [Ca(2+)]i oscillatory activity. We also observed that the Ca(2+) oscillatory ability and the sensitivity to IP3 in eggs expressing IIIE-IP3R1 were greater than in those expressing wt-IP3R1. Lastly, we found that exogenous IP3R1s are resistant to downregulation and support longer oscillations and of higher amplitude. Altogether, our results show that phosphorylations by Cdk1 and MAPK enhance the activity of IP3R1, which is consistent with its maximal activity observed at the time of fertilization and the role of Ca(2+) release in egg activation.

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP3) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca 21 ]i) seen during mammalian fertilization. IP3 promotes Ca 21 release in eggs by binding to its receptor, the type-1 IP3 receptor (IP3R-1, also known as ITPR1), a ligand-gated Ca 21 channel located in the membrane of the endoplasmic reticulum, the main Ca 21 store of the cell. While IP3R1 has been shown to mediate all Ca 21 release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca 21 release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a ;70%‐80% reduction in the number of intact IP3R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP3 analogue, adenophostin A. Functional Ca 21 release analysis revealed that IP3R-1 is the predom

BACKGROUND: Deficiencies remain in the ability of in vitro-matured human oocytes to acquire full developmental competence and give rise to a healthy pregnancy. A clear deficiency of current systems utilizing human oocytes has been the absence of cumulus cells. In the present study, a three-dimensional (3D) co-culture system exploiting an extracellular matrix was developed and compared to conventional methods for its ability to support maturation of human oocytes. METHODS AND RESULTS: Cumulus cells were embedded into a 3D collagen gel matrix with individual oocytes added to each gel. Oocytes from the same patient cultured in the gel matrix matured to metaphase II at rates similar to those of cumulus-free oocytes cultured in individual microdrops. Following maturation of oocytes and fixation of intact gels, chromatin and cytoskeletal elements were assessed in oocytes and cumulus cells. The activities of the key cell cycle kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were compared in oocytes matured under the two culture conditions. Compared with denuded oocytes, co-cultured oocytes exhibited increased MAPK activity, but no difference in MPF levels. CONCLUSIONS: This work characterizes a novel and efficacious culture system that takes advantage of the unique properties of the extracellular matrix, a 3D microenvironment, and the presence of cumulus cells for maturing human oocytes in vitro.

A sperm-specific phospholipase (PL) C, termed PLCζ, is proposed to be the soluble sperm factor that induces Ca2+ oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCζ mRNA have reduced amounts of PLCζ protein. Sperm derived from these transgenic mice trigger patterns of Ca2+ oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca2+ oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCζ is the physiological trigger of Ca2+ oscillations required for activation of development.

Nhlh2 is a member of the basic helix-loop-helix (bHLH) transcription factor family and is expressed in developing and adult neuroendocrine tissues such as the pituitary and hypothalamus. Targeted deletion of Nhlh2 (N2KO) in mice results in hypogonadism and obesity. While gonadally intact male N2KO mice are infertile and lack male sexual behavior, female N2KO mice can become pregnant and carry litters to full term. Unlike normal females in which fertility averages 8–12 months with approximately one pregnancy per month, N2KO females have a shorter reproductive span with most females supporting only three to four pregnancies in a 9-month period. In addition, N2KO females exhibit abnormal estrous cycles characterized by a truncated estrus and a prolonged proestrus. We have found that while young female N2KO mice ovulate the same number of oocytes as normal females in response to exogenous hormones, the number of oocytes released by aged N2KO females is reduced over 50%. Interestingly, oocytes from N2KO females are equally competent for in vitro fertilization assays when compared to oocytes from similarly aged normal and heterozygous mice. We have further demonstrated that both young and old N2KO females show at least a 50% reduction in hormone-stimulated sexual behavior as measured by their lordosis quotient. This suggests that N2KO females show a lifelong behavioral hyporesponsiveness to exogenous steroid hormones accompanied by a reduction in reproductive longevity via reduced ovulation with aging. Potential gene regulatory mechanisms that involve the action of the Nhlh2 transcription factor on female fertility and sexual behavior are discussed.

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]ioscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active bothin vivoandin vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]ioscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]irelease. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]ioscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]ioscillations.

In all species studied, fertilization induces intracellular Ca21 ([Ca21]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca21]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca21]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca21]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca21]i oscillations to injection of stallion sperm, and [Ca21]i monitoring was performed. Such pairs consistently displayed [Ca21]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species. assisted reproductive technology, calcium, fertilization, gamete biology

In mammalian eggs, the fertilizing sperm evokes intracellular Ca2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor (SF), is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Based on findings showing that production of inositol 1,4,5-trisphosphate (IP3) underlies the generation of [Ca2+]i oscillations, it has been suggested that SF functions either as a phospholipase C (PLC), an enzyme that catalyzes the generation of IP3, or as an activator of a PLC(s) pre-existing in the egg. This review discusses the role of SF as the molecule responsible for the production of IP3 and the initiator of [Ca2+]i oscillations in mammalian fertilization, with particular emphasis on the possible involvement of egg- and sperm-derived PLCs, including PLCzeta, a novel sperm specific PLC.