Rapid thawing of frozen bull spermatozoa by transient exposure to 70 °C improves the viability, motility and mitochondrial health.

Up to now, the definitive conclusion of the positive effects of rapid transient thawing at higher temperatures for shorter durations has not been obtained yet and is still under discussion due to some contradictory findings and limited assessment of post-thawed parameters. The purpose of the current study was to evaluate the effectiveness of rapid thawing in water at 70 °C by using various post-thawed parameters of frozen bull spermatozoa. Experiment 1, monitoring the change of temperature inside frozen bull straw thawed in water at different temperatures. Experiment 2, evaluation of various post-thawed characteristics of frozen bull spermatozoa thawed in water at different temperatures by using a computer-assisted sperm analysis, flow cytometry and immunocytochemistry. The time it took for the temperature inside the straw to warm up to 15 °C was nearly twice as faster when the straw was thawed in 70 °C water compared with 39 °C. Although there were differences among bulls, viability, motility, and mitochondrial membrane potential of spermatozoa thawed at 70 °C for 8 seconds and stabilized at 39 °C for 52 seconds were significantly higher than those of controls (thawed at 39 °C for 60 seconds) at 0 and 3 h after thawing. Just after thawing, however, there were no differences in acrosome integrity and distribution of phospholipase C zeta1, whereas mitochondrial reactive oxygen species production was significantly lower in spermatozoa thawed at 70 °C. From these results, we conclude that rapid thawing at 70 °C and then stabilization at 39 °C significantly improves viability, motility and mitochondrial health of bull spermatozoa rather than conventional thawing at 39 °C. The beneficial effect of rapid transient thawing could be due to shorter exposure to temperatures outside the physiological range, consequently maintaining mitochondrial health.

Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo.

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

Negative correlations of mitochondrial DNA copy number in commercial frozen bull spermatozoa with the motility parameters after thawing.

The purpose of the current study was to investigate the relationship between mitochondrial content of commercial frozen-thawed bull spermatozoa and motility. Firstly, mitochondrial DNA copy number per spermatozoon (MDCN), mitochondrial content (MC), the percentage of spermatozoa with high mitochondrial membrane potential (HMMP), intracellular reactive oxygen species (ROS) and motility parameters of frozen-thawed spermatozoa derived from five bulls were determined by using qPCR, flow cytometry and CASA, respectively, and analyzed the relationships. Results showed that all parameters examined, including MDCN, MC, HMMP, ROS and motility indicators, significantly differed among frozen spermatozoa from different bulls. Both MDCN and MC were negatively correlated with HMMP and motility indicators, but positively with ROS, of course, whereas there was a highly positive relationship between MDCN and MC. Secondly, when MDCN and MC were examined in frozen spermatozoa prepared at different points in the lives of four bulls, those did not correlate overall throughout their lives (1.3-14.3 years old), but did correlate significantly in two sires. From these results, we conclude that MDCN and MC of frozen spermatozoa differ among sires, and are negatively correlated with HMMP and sperm motility parameters, probably due to mitochondrial oxidative stress resulted in the presence of ROS, demonstrating that these appear to be useful markers to assess sires' spermatozoa. It should be noted that the MDCN and MC of bull spermatozoa may not vary overall with the age of the sire, whereas those changes with age in some individuals and may affect sperm motility.

Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos.

GATA factors are essential transcription factors for embryonic development that broadly control the transcription of other genes. This study aimed to examine GATA2 protein localization in mouse embryos at the 2-cell stage, when drastic transformation in gene expression occurs for subsequent development in early embryos. We first analyzed GATA2 localization in 2-cell embryos at the interphase and mitotic phases by immunofluorescence analysis. In the interphase, GATA2 protein was localized in the nucleus, as a common transcription factor. In the mitotic phase, GATA2 protein was observed as a focally-aggregated spot around the nucleus of each blastomere. To explore the relationship between GATA2 protein localization and cell cycle progression in mouse 2-cell stage embryos, GFP-labeled GATA2 protein was overexpressed in the blastomere of 2-cell embryos. Overexpression of GFP-labeled GATA2 protein arrested cellular mitosis, focally aggregated GATA2 protein expression was not observed. This mitotic arrest by GATA2 overexpression was not accompanied with the upregulation of a 2-cell stage specific gene, murine endogenous retrovirus-L. These results suggest that GATA2 protein localization changes dynamically depending on cell cycle progression in mouse 2-cell embryos; in particular, focally aggregated localization of GATA2 in the mitotic phase requires appropriate cell cycle progression.

The autophagic inducer and inhibitor display different activities on the meiotic and developmental competencies of porcine oocytes derived from small and medium follicles.

This study aimed to examine the effect of rapamycin (autophagy inducer) and 3-methyladenine (3-MA, autophagy inhibitor) on the meiotic and developmental competencies of porcine oocytes derived from medium follicles (MF, 3-6 mm in diameter) and small follicles (SF, 1-2 mm in diameter) during in vitro maturation (IVM) process. The presence of 1 nM but not 10 nM rapamycin significantly increased the maturation rate of MF-derived oocytes (P < 0.05). However, the maturation rate of SF-derived oocytes was not affected by rapamycin at both concentrations (1 nM and 10 nM). The maturation rate of MF-derived oocytes decreased significantly (P < 0.05) in the presence of 0.2 mM but not 2 mM 3-MA than non-supplemented control. In contrast, in SF-derived oocytes, 3-MA at both 0.2 and 2 mM concentrations did not affect the maturation rates. The presence of 1 nM rapamycin significantly increased the blastocyst formation rate of MF-derived mature oocytes following parthenogenetic activation (P < 0.05). However, the blastocyst formation rate of SF-derived mature oocytes was not affected by the presence of rapamycin. The presence of 3-MA significantly reduced the blastocyst formation rate of MF-derived mature oocytes but did not change that of SF-derived oocytes. In conclusion, our study results show differences in activity of the autophagy inducer and inhibitor on the meiotic and developmental competencies of MF- and SF-derived porcine oocytes.

Ca2+ Signaling and Homeostasis in Mammalian Oocytes and Eggs.

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.

Removal of cumulus cells around 20 h after the start of in vitro maturation improves the meiotic competence of porcine oocytes via reduction in cAMP and cGMP levels.

We examined the effect of the timing of removing cumulus cells surrounding porcine oocytes from small follicles (SFs, < 3 mm in diameter) and medium follicles (MFs; 3-6 mm in diameter) on the meiotic and developmental competence of the oocytes. Cumulus-oocyte complexes (COCs) were collected from SFs and MFs, and the oocytes were denuded at 0, 20, and 44 h after the start of in vitro maturation (IVM), and the meiotic progression of the oocytes was assessed at the end of the IVM period. The incidence of mature oocytes was significantly affected by both the origin of the COCs and the time when the oocytes were denuded. Although the percentage of mature oocytes was always higher when the COCs were collected from MFs than that when the COCs were collected from SFs, the maturation rate was significantly higher when the oocytes were denuded at 20 h than when they were denuded at 44 h after the start of IVM. When the mature oocytes were activated electrically, the developmental competence of the oocytes denuded at 20 and 44 h to reach the blastocyst stage did not differ, whereas the competence of the MF-derived oocytes was significantly higher than that of SF-derived oocytes. When the intracellular cAMP and cGMP levels in SF-derived oocytes were examined at 24 h of IVM, the levels of both were significantly decreased only in the oocytes denuded at 20 h. In conclusion, denuding oocytes at 20 h of IVM caused a significant reduction in ooplasmic cAMP and cGMP levels and increased the meiotic competence of the oocytes without any reduction in blastocyst formation, even in the case of SF-derived oocytes.

Constitutive IP3R1-mediated Ca2+ release reduces Ca2+ store content and stimulates mitochondrial metabolism in mouse GV oocytes.

In mammals, fertilization initiates Ca2+ oscillations in metaphase II oocytes, which are required for the activation of embryo development. Germinal vesicle (GV) oocytes also display Ca2+ 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 Ca2+ in GV oocytes, the endoplasmic reticulum ([Ca2+]ER), constitutively 'leaks' Ca2+ through the type 1 inositol 1,4,5-trisphosphate receptor. The [Ca2+]ER leak ceases around the resumption of meiosis, the GV breakdown (GVBD) stage, which coincides with the first noticeable accumulation of Ca2+ in the stores. It also concurs with downregulation of the Ca2+ influx and termination of the oscillations, which seemed underpinned by the inactivation of the putative plasma membrane Ca2+ channels. Lastly, we demonstrate that mitochondria take up Ca2+ during the Ca2+ oscillations, mounting their own oscillations that stimulate the mitochondrial redox state and increase the ATP levels of GV oocytes. These distinct features of Ca2+ 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.

XY oocytes of sex-reversed females with a Sry mutation deviate from the normal developmental process beyond the mitotic stage†.

The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry- mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry- females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry- PGCs was associated with aberrant ß-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry- oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as "Swyer syndrome," in which patients with an XY karyotype present as typical females and are infertile.

Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation.

The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.

Presence of vascular endothelial growth factor during the first half of IVM improves the meiotic and developmental competence of porcine oocytes from small follicles.

The aim of the present study was to investigate the effect of vascular endothelial growth factor (VEGF) on the meiotic and developmental competence of porcine oocytes from small follicles (SF; 0.5-3mm diameter). When cumulus-oocyte complexes (COCs) from medium-sized follicles (MF; 3-6mm diameter) and SF were cultured for IVM, the maturation rates were significantly higher for oocytes from MF than SF. Concentrations of VEGF in the medium were significantly higher for COCs cultured from MF than SF. When COCs from SF were exposed to 200ngmL-1 VEGF during the first 20h of IVM, the maturation rate improved significantly and was similar to that of oocytes derived from MF. The fertilisability of oocytes was also significantly higher than that of VEGF-free SF controls. Following parthenogenetic activation, the blastocyst formation rate improved significantly when SF COC culture was supplemented with 200ngmL-1 VEGF, with the rate similar to that of oocytes from MF. The results of the present study indicate that VEGF markedly improves the meiotic and developmental competence of oocytes derived from SF, especially at a concentration of 200ngmL-1 during the first 20h of IVM.

Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes.

The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes.

DNA Methylation Errors in Cloned Mouse Sperm by Germ Line Barrier Evasion.

The germ line reprogramming barrier resets parental epigenetic modifications according to sex, conferring totipotency to mammalian embryos upon fertilization. However, it is not known whether epigenetic errors are committed during germ line reprogramming that are then transmitted to germ cells, and consequently to offspring. We addressed this question in the present study by performing a genome-wide DNA methylation analysis using a target postbisulfite sequencing method in order to identify DNA methylation errors in cloned mouse sperm. The sperm genomes of two somatic cell-cloned mice (CL1 and CL7) contained significantly higher numbers of differentially methylated CpG sites (P = 0.0045 and P = 0.0116). As a result, they had higher numbers of differentially methylated CpG islands. However, there was no evidence that these sites were transmitted to the sperm genome of offspring. These results suggest that DNA methylation errors resulting from embryo cloning are transmitted to the sperm genome by evading the germ line reprogramming barrier.

A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 µM), higher sildenafil concentrations (25 and 250 µM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 µM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

Mitochondrial dynamics controlled by mitofusins define organelle positioning and movement during mouse oocyte maturation.

Mitochondria are abundant in fully grown mammalian oocytes with a unique spherical morphology, but the mechanisms controlling mitochondria behavior are not well understood. Here we describe for the first time the control of mitochondrial behavior in mouse oocytes by a fusion/fission mechanism. Mitofusins (Mfn1 and Mfn2) and OPA1 proteins are required for outer and inner mitochondrial membrane fusion, respectively, whereas Drp1 is the key regulator of mitochondrial fission. We show that mouse oocytes express the Mfn1, Mfn2, Opa1 and Drp1 proteins, both in immature and mature oocytes at similar levels. Overexpression of Mfn1 or Mfn2 causes marked mitochondrial aggregation, particularly in the perinuclear region during meiotic progression. Tracking of mitochondria with chromosomes or endoplasmic reticulum (ER) throughout oocyte maturation demonstrates that Mfn1 and Mfn2-promoted mitochondrial aggregation disturbs the spatiotemporal dynamic of the chromosomes and ER, respectively. Our findings suggest that organelle dynamics are co-ordinately controlled during meiotic division, and an imbalance of mitochondrial fusion/fission leads to disorganization of the organelle compartments.

Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth.

Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten-eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.

Forced expression of DNA methyltransferases during oocyte growth accelerates the establishment of methylation imprints but not functional genomic imprinting.

In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analyzed DNA methylation imprints. The results showed that 2- to 4-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing (ng) oocytes versus fully grown oocytes derived from wild-type mice, but the analyzed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in ng oocytes is insufficient for methylation imprints, and imprinted regions are resistant to DNMT3A/DNMT3L in ng oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1 and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn and Impact was observed in ng oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.

Long exposure to mature ooplasm can alter DNA methylation at imprinted loci in non-growing oocytes but not in prospermatogonia.

DNA methylation imprints that are established in spermatogenesis and oogenesis are essential for functional gametes. However, the mechanisms underlying gamete-specific imprinting remain unclear. In this study, we investigated whether male and female gametes derived from newborn mice are epigenetically plastic and whether DNA methylation imprints are influenced by the niche surrounding the nuclei of the gametes. When prospermatogonia possessing sperm-specific DNA methylation imprints were fused with enucleated fully grown oocytes and exposed to the ooplasm for 5-6 days, the DNA methylation status of the reconstituted oocytes remained identical to that of prospermatogonia for all the imprinted regions analysed. These results suggest that the imprinting status of prospermatogonia is stable and that the epigenome of prospermatogonia loses sexual plasticity. By contrast, when non-growing oocytes lacking oocyte-specific DNA methylation imprints were fused with enucleated fully grown oocytes and the reconstituted oocytes were then cultured for 5-6 days, the Igf2r, Kcnq1ot1 and, unexpectedly, H19/Igf2 differentially methylated regions (DMRs) were methylated. Methylation imprints were entirely absent in oocytes derived from 5-day-old mice, and H19/Igf2 DMR is usually methylated only in spermatogenesis. These findings indicate that in the nuclei of non-growing oocytes the chromatin conformation changes and becomes permissive to DNA methyltransferases in some DMRs and that mechanisms for maintaining non-methylated status at the H19/Igf2 DMR are lost upon long exposure to mature ooplasm.

Regulation of endoplasmic reticulum Ca(2+) oscillations in mammalian eggs.

Changes in the intracellular concentration of free calcium ([Ca(2+)]i) regulate diverse cellular processes including fertilization. In mammalian eggs, the [Ca(2+)]i changes induced by the sperm unfold in a pattern of periodical rises, also known as [Ca(2+)]i oscillations. The source of Ca(2+) during oscillations is the endoplasmic reticulum ([Ca(2+)]ER), but it is presently unknown how [Ca(2+)]ER is regulated. Here, we show using mouse eggs that [Ca(2+)]i oscillations induced by a variety of agonists, including PLCζ, SrCl2 and thimerosal, provoke simultaneous but opposite changes in [Ca(2+)]ER and cause differential effects on the refilling and overall load of [Ca(2+)]ER. We also found that Ca(2+) influx is required to refill [Ca(2+)]ER, because the loss of [Ca(2+)]ER was accelerated in medium devoid of Ca(2+). Pharmacological inactivation of the function of the mitochondria and of the Ca(2+)-ATPase pumps PMCA and SERCA altered the pattern of oscillations and abruptly reduced [Ca(2+)]ER, especially after inactivation of mitochondria and SERCA functions. We also examined the expression of SERCA2b protein and found that it was expressed throughout oocyte maturation and attained a conspicuous cortical cluster organization in mature eggs. We show that its overexpression reduces the duration of inositol-1,4,5-trisphosphate-induced [Ca(2+)]i rises, promotes initiation of oscillations and enhances refilling of [Ca(2+)]ER. Collectively, our results provide novel insights on the regulation of [Ca(2+)]ER oscillations, which underlie the unique Ca(2+)-signalling system that activates the developmental program in mammalian eggs.

Molecular characteristics of horse phospholipase C zeta (PLCζ).

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.