Sperm–zona pellucida (ZP) binding is a necessary event for successful fertilization. The aim of this study was to determine the effect of trace minerals such as copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) on bovine spermatozoa binding to ZP. Sperm viability, functional membrane integrity, acrosomal status (AS), total antioxidant capacity (TAC) and sperm lipid peroxidation (LPO) were also evaluated. For the present study, in vitro fertilization (IVF) medium was supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), all minerals (Cu+Mn+Se+Zn), or tested without supplement (Control). Considerably more sperm bound to ZP when Cu, Se or Zn were added to the IVF medium, but there were no difference compared with the Control, Mn and Cu+Mn+Se+Zn groups. After 1 h of incubation, viability was increased by the addition of Cu, Mn and Se with respect to the Control but, after 2 h, viability was higher only with the addition of Mn to IVF medium. Functional membrane integrity improved in sperm treated with Cu. Acrosome integrity was higher in sperm treated with Zn after 1 h of incubation. LPO was significantly higher in sperm treated with Cu or Cu+Mn+Se+Zn. The mean TACs of sperm treated with Cu, Mn, Zn or Cu+Mn+Se+Zn were lower than in the Control. In conclusion, the results obtained in the present study determined that the presence of Cu, Se and Zn in the IVF medium increased the number of spermatozoa bound to the ZP, highlighting the importance of these minerals in the fertilization process.

Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

Male germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves the incorporation of testis-specific histone variants, mitotic and meiotic divisions, haploid gene expression, histone–protamine transitions and massive epigenetic reprogramming. Understanding the protein players and the epigenetic mark network involved in the setting of the epigenetic programme in spermatogenesis is an exciting new clue in the field of reproductive biology with translational outcomes. As information in the existing literature regarding cross-talk between DNA methylation and histone hyperacetylation in the advanced stages of murine spermatogenesis is still scarce and controversial we have investigated the effect of a DNA-methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological and molecular level (by transmission electron microscopy, immunocytochemistry and immunoprecipitation methods). Our results revealed an important role for regulation of DNA methylation in controlling histone hyperacetylation and chromatin remodelling during spermatogenesis.

The cortical actin cytoskeleton, consisting of actin filaments and actin binding proteins, immediately underlies the inner surface of the plasma membrane and is important both structurally and in relaying signals from the surface to the interior of the cell. Signal transduction processes, initiated in the cortex, modulate numerous cellular changes ranging from modifications of the local cytoskeleton structure, the position in the cell cycle, to cell behaviour. To examine the molecular mechanisms and events associated with cortical changes. We have investigated targets of the protein tyrosine kinase, Src, which is associated with the cortical cytoskeleton, in Xenopus laevis oocytes. When a mRNA encoding an activated form of Src tyrosine kinase (d-Src) is injected into oocytes several changes are observed: proteins are phosphorylated, the rate at which progesterone matures an oocyte to an egg is accelerated, and the cortex at the site of injection appears to contract. Previous studies have implicated actin filaments in the Src-stimulated cortical rearrangements. In this study we identify two actin binding proteins – cortactin and moesin – as Src substrates in Xenopus oocytes that are Src substrates. We cloned and characterised the cDNA encoding one of those, Xenopus moesin, a member of the ezrin/radixin/moesin (ERM) family of actin binding proteins. In addition, we have determined that moesin is recruited to the cortex at the site of Src mRNA injection.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.

A monoclonal antibody (6964M) was generated against the envelope component gp69/64 of Xenopus laevis eggs. On indirect immunofluorescence using this antibody, the positive reaction was seen on the surface of both vitelline envelope (VE) and coelomic envelope (CE). On immunoelectron microscopy, gp69/64 was preferentially distributed on the thick bundles forming the edge of the tunnel openings on CE, and this distribution pattern was fundamentally inherited by VE. Counting the number of immunogold particles indicated that VE has about twice as many particles as CE, with a 3-4 times higher density at the animal pole than vegetal pole. The number of sperm bound to CE was small, being approximately one-twentieth of the number of sperm bound to VE. An extremely small number of sperm (< 2 per animal hemisphere) was found to bind to VE* of activated eggs as a background. The sperm binding to CE was inhibited by pretreatment of the envelopes with 6964M or in the presence of purified gp69/64 from VE on insemination, confirming that sperm binding is mediated by gp69/64 exposed on the CE surface. In spite of at most a 2-fold increase in the amount of exposed gp69/64, the sperm binding increased about 20-fold upon CE-to-VE conversion, suggesting that the increase in the amount of exposed gp69/64 is itself insufficient to explain the increase in the number of bound sperm.

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).

The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

Although much information about such processes as cell cycle control, second messenger systems, protein kinases and steroid hormone action has been collected from studies of Xenopus oocyte maturation, we still have very little idea about how the steroid hormone, progesterone, signals the resumption of meiosis from the oocyte plasma membrane. In this review we re-examine the data on second messenger systems in Xenopus oocytes and discuss some of the unresolved questions about hormone signal transduction during maturation. We outline some reasons for the contradictions in the literature and offer some suggestions for avenues of future research.

For decades, scientists have considered that female mammals are born with a lifetime reserve of oocytes in the ovary, irrevocably fated to decline after birth. However, controversy in the matter of the possible presence of oocytes and granulosa cells that originate from stem cells in the adult mammalian ovaries has been expanded. The restricted supply of oocytes in adult female mammals has been disputed in recent years by supporters of neo-oogenesis, who claim that germline stem cells (GSCs) exist in the ovarian surface epithelium (OSE) or the bone marrow (BM). Differentiation of ovarian stem cells (OSCs) into oocytes, fibroblast-like cells, granulosa phenotype, neural and mesenchymal type cells and generation of germ cells from OSCs under the contribution of an OSC niche that consists of immune system-related cells and hormonal signalling has been claimed. Although these arguments have met with intense suspicion, their confirmation would necessitate the revision of the current classic knowledge of female reproductive biology.

Viable sperm cells of Nicotiana tabacum were isolated by the semi-vivo technique. After pollination, excised styles were floated, cut end immersed, in a solution of 15% sucrose with 0.01% boric and 0.03% Ca(NO3)2 at 27°C in a growth chamber until pollen tubes emerged. After sperm cells were formed (at least 8 h after pollination) tubes were immersed in a 9% mannitol solution. In this solution, sperm cells are nearly ellipsoidal and retain viability for over 6 h.

M5 ganglioside (NeuGcα2–6Glcβl-' Cer) is the predominant glycosphingolipid in sea urchin eggs. Distribution of M5 ganglioside was studied in unfertilised and fertilised eggs of the sea urchin Hemicentrotus pulcherrimus by indirect immunofluorescence microscopy. In the cortices of unfertilised eggs, anti-M5 antibody strongly stained the submembranous, polygonal and tubular network of endoplasmic reticulum that was revealed by a membrane-staining dye, DiIC18(3). In addition to the cortical network of endoplasmic reticulum, at least two morphologically distinct vesicles were positive to the antibody. In the cortices isolated from fertilised eggs 30 min after insemination, the antibody stained only a similar network of endoplasmic reticulum, presumably the one reconstructed 5–10 min after fertilisation. During mitosis the endoplasmic reticulum is known to aggregate within the asters of the mitotic apparatus. Indeed, the antibody stained the asters and (more strongly) the vesicular components attaching to the periphery of the mitotic apparatus.

The ovulatory follicle has the dual role of releasing a viable oocyte capable of fertilisation and the production of key endocrine signals that result in mating behaviour, the induction of the preovulatory LH surge and ovulation. Further, following ovulation the ovulatory follicle must be capable of forming a viable corpus luteum if pregnancy is to be maintained. Follicle growth is therefore a developmental process during which the follicle progressively acquires a number of properties, each of which is an essential prerequisite for further development. Failure to acquire these properties at the correct time and in an exact sequence will lead to failure of the developmental process and to the deterioration of the follicle through atresia and degeneration of the oocyte.

The postovulatory aging of oocytes eventually affects the development of oocytes and embryos. Oxidative stress is known to accelerate the onset of apoptosis in oocytes and influence their capacity for fertilisation. This study aimed to reveal the roles of temperature and the antioxidant N-acetyl-l-cysteine in preventing the aging of postovulatory mouse oocytes. First, newly ovulated mouse oocytes were cultured at various temperature and time combinations in HCZB medium with varying concentrations of N-acetyl-l-cysteine to assess signs of aging and developmental potential. When cultured in HCZB with 300 μM N-acetyl-l-cysteine at different temperature and incubation time combinations (namely 25°C for 12 h, 15°C for 24 h and 5°C for 12 h), the increase in the susceptibility of oocytes to activating stimuli was efficiently prevented, and the developmental potential was maintained following Sr2+ activation or in vitro fertilisation. After incubation at either 15°C for 36 h or 5°C for 24 h, oocytes that had decreased blastocyst rates displayed unrecoverable abnormal cortical granule distribution together with decreased BCL2 levels, total glutathione concentrations and glutathione/glutathione disulphide (GSH/GSSG) ratios. In conclusion, postovulatory oocyte aging could be effectively inhibited by appropriate N-acetyl-l-cysteine addition at low temperatures. In addition, a simple method for the temporary culture of mature oocytes was established.

Ovarian cortical fragments from five adult ewes were in vitro cultured for 1, 3 or 5 days in the presence of minimum essential medium either supplemented or not by follicle-stimulating hormone (FSH) (100 ng/ml) or indole-3-acetic acid (IAA) (10, 20, 40 or 100 ng/ml), alone or in combination. After in vitro culture, ovarian fragments were submitted to follicular isolation and viability test was performed using trypan blue. Addition of IAA (10 ng/ml) to a free-FSH medium resulted in the highest percentages of viable follicles, but was progressively deleterious in higher concentrations (20, 40 and 100 ng/ml) if in absence of FSH. Follicular development was observed only when FSH was added to an IAA-free medium. In conclusion, IAA at a concentration of 10 ng/ml increases follicular survival in vitro. However, at high concentrations (20, 40 or 100 ng/ml), this auxin may be deleterious to preantral follicles, the addition of FSH to the medium being necessary.

The ZP3 gene encodes for a zona glycoprotein that serves as both a cell-specific binding site for capacitated spermatozoa and an inducer of acrosomal exocytosis during fertilisation. In this study we have determined the nucleotide sequence of rat ZP3 (accession no. Y10823), predicted primary amino acid structure and determined the cellular origin of this molecule within the ovary. Rat ZP3 was found to have an open reading frame of 1272 nucleotides encoding a polypeptide chain of 424 amino acids that was expressed exclusively by the actively growing oocyte population. Rat ZP3 exhibited 91%, 78% and 66% identity with the mouse, hamster and human homologues, respectively. Key features of mouse ZP3, including the number and location of cysteine and proline residues and N-linked glycosylation sites, were also conserved in the rat homologue. The putative O-linked glycosylation sites, a series of serine residues at ZP3329–334, were also conserved in rat and mouse ZP3, although immediately downstream of this site the amino acid sequences deviated over a short stretch of amino acids. The hydropathicity profile revealed two hydrophobic domains. The first was associated with a putative N-terminal signal sequence which is unusual in the rat in possessing a proline residue at the -1 position relative to the signal cleavage site, a feature it shares with human and marmoset ZP3 but not mouse. The second hydrophobic domain was observed at the C-terminus downstream of a TGF-β type III receptor domain that appears to be common to all ZP3 sequences examined to date.

Mouse embryos secrete molecules homologous to platelet-activating factor (PAF), termed embryo-derived PAF (EPAF), which act in an autocrine/paracrine fashion to stimulate embryonic development in vitro. Mouse EPAF is thought to consist predominantly of hexadecyl (C16) and octadecyl (C18) PAF-like components. Mouse pre-implantation embryos cultured with exogenous PAF from the early cleavage stages exhibit increased blastocyst cell numbers and rates of mitosis around the 8-cell stage. We investigated whether exogenous PAF could specifically stimulate embryonic cell proliferation prior to the blastocyst stage in the mouse and also compared the biological activities of the C16 and C18 PAF isoforms as follows. Embryos were cultured for either 24 h or 120 h from the 2-cell stage and their total cell numbers were determined or their development assessed in terms of their incidence of successful zona-hatching respectively. In each case, embryos were cultured in unsupplemented medium or in medium supplemented with either C16 or C18 PAF (0.5 μM). Compared with controls, culture with C16 PAF produced a significant stimulation of mean total per number per embryo and a significant increase in the incidence of successful zona-hatching, whilst culture with C18 PAF had no significant effect. We then cultured 1-cell zygotes for 48 h in unsupplemented medium or medium supplemented with either an equimolar mixture of C16 and C18 PF or with either C16 or C18 PF alone (each at 0.2 μM). Embryos were also scored for cell number at 4 h and 30 h of culture. Although no significant effect on mean cell number per embryo was seen following 4 h or 30 h of culture with a mixed C16/C18 PAF preparation, culture for 48 h with a mixed C16/C18 PAF preparation or with C16 PAF alone produced a significant increase in mean cell number per embryo compared with controls - an effect that is likely to be receptor-mediated, since culture with an equivalent concentration of C18 PAF had no significant effect compared with controls. We have demonstrated that mouse zygotes/embryos can respond in a specific manner to exogenous hexadecyl PAF in terms of increased rates of cell proliferation prior to cavitation, and must be capable of doing so at some time between the first and third, and also between the second and fourth, cell cycles. Such embryos presumably express one or more classes of functional PAF-receptor molecule during this period (i.e. as early as during the 1-, 2- or 4-cell stages). We have also demonstrated that embryonic response to exogenous PAF is significantly isoform-specific, which may reflect differences between the two isoforms either in affinity for binding to putative embyronic PAF-receptor molecules or in their ability to elicit a stimulatory response following binding. This observation calls into question the use of preparations containing a mixture of hexadecyl and octadecyl PAF isoforms, particularly in dose-response studies, in the mouse.

The expression of nine serotonin (5-HT) receptor transcripts was studied using reverse transcription polymerase chain reaction (RT-PCR) in germ cells, cleavage and gastrulation stages of Japanese quail, and qPCR for 5-HT3 and 5-HT4 receptors in oocytes and embryos. We show the presence/absence of nine serotonin transcripts known in birds for receptors 5-HT1A, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A in avian germ cells and early embryos. The absence of 5-HT3 and 5-HT5A in primordial germ cells and of 5-HT3 and 5-HT7A in sperm is characteristic. All transcripts appeared in oocytes at all stages (except for 5-HT3 and 5-HT5A transcripts) and all were present in cleaving embryos and at gastrulation, except for 5-HT3, which was permanently observed as late as in stage 4. Interestingly, 5-HT3 and 5-HT5A receptors accumulated in 3-mm and F1 oocytes but were degraded at ovulation and started to be re-transcribed in cleavage stage II embryos and beyond. The selective appearance of 5-HT receptors in germ cells and early embryos supports the hypothesis that serotonin may act as a signalling molecule at early stages of germ line and embryo differentiation via individual receptors present during different stages, when specialized communication systems are not yet developed.

Minor and major zygotic genome activation (ZGA) are crucial for preimplantation development. During this process, histone variants and methylation influence chromatin accessibility and consequently regulated the expression of zygotic genes. However, the detailed exchanges of these modifications during ZGA remain to be determined. In the present study, the epigenetic modifications of histone 3 on lysine 9 (H3K9), 27 (H3K27) and 36 (H3K36), as well as four histone variants were determined during minor and major ZGA and in post-ZGA stages of mouse embryos. Firstly, microH2A1, H3K27me3 and H3K36me3 were asymmetrically stained in the female pronucleus during minor ZGA but lost staining in major ZGA. Secondly, H3K9me2 and H3K9me3 were strongly stained in the female pronucleus, but weakly stained in the male pronucleus and disappeared after ZGA. Thirdly, H2A.Z and H3.3 were symmetrically stained in male and female pronuclei during minor ZGA. Moreover, H3K27me2 was not statistically changed during mouse early development, while H3K36me2 was only detected in 2- and 4-cell embryos. In conclusion, our data revealed dynamics of histone methylation and variants during mice ZGA and provided details of their exchange in mice embryogenesis. Moreover, we further inferred that macroH2A1, H2A.Z, H3K9me2/3 and H3K27me2/3 may play crucial roles during mouse ZGA.