Post-translational modifications generally involve the addition or removal of various functional groups to or from the protein residues. However, citrullination, which is catalyzed by the peptidylarginine deiminases (PADs), involves conversion of one kind of amino acid residue into another. One of five isoforms, PAD4 is a nuclear enzyme known to play a role in development, differentiation and apoptosis through gene regulation. To investigate the possible role of PAD4 in mammalian preimplantation embryonic development, we first studied localization and expression of PAD4 and citrullinated proteins in pig and mouse oocytes, and parthenogenetic or in vitro fertilized (IVF) embryos. Immunofluorescence study revealed that PAD4 primarily localizes in the cytoplasm in pig oocytes and parthenogenetic embryos. However, the nuclear translocation of PAD4 was observed in late germinal vesicle (GV) stage oocytes prior to GV breakdown and was localized around the metaphase (M)I and II spindle. Nucleus localized PAD4 was noticed partially again in blastocysts. In mouse IVF embryos, nuclear translocation started from the 2-cell stage and gradually increased up to blastocyst. Western blot studies confirmed that PAD4 was expressed in oocytes, and parthenogenetic embryos of pig. Citrullinated proteins were detected in granular form on the chromatin in GV, MI and MII oocytes and nuclei in all the stages of the embryos studied. It was found that the target of citrullination was histone protein (H3), not B23. Therefore the presence of PAD4 and citrullinated histone H3 in oocytes and embryos suggested a possible role for PAD4 in preimplantation embryonic development.

The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen–thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

The integrity of sperm chromatin structure has proven to be of great importance for human fertility. In this study, we investigated whether sperm cryopreservation has an effect on nuclear DNA tertiary structure, (i.e. condensation), measured by aniline blue staining, in 103 male patients who required consultation for hypo-fertility. Sperm DNA damage was significantly higher in patients showing oligospermia and severe morphological abnormalities than in native sperm populations. Furthermore we observed that chromatin decondensation was related to the cryostorage technique and to the duration of storage. This increase in decondensation was highly significant (P < 0.01) immediately after cryopreservation and from 90 days of cryostorage onwards. The possible mechanisms involved in sperm chromatin cryoinjury and the need to incorporate new methods for testing sperm nuclear structure alteration into the routine spermiogram are discussed.

The objective of the present study was to investigate the effect of Gram-negative bacteria infection on ovarian steroid receptors, i.e. progesterone receptor (PR) and estradiol receptor (ER) during preimplantation days of pregnancy. A well established mouse model of Gram-negative bacteria infection was used to test this objective. Mice were treated with normal saline or lipopolysaccharide (LPS) on day 0.5 of pregnancy and used to collect embryos and uterine horns on day 1.5 to day 4.42 preimplantation day of pregnancy. Total RNA was extracted and reverse-transcription polymerase chain reaction (PCR) was performed to check the expression of PR and ER genes. The mRNA expression of PR and ER was altered in embryos and uterus of LPS-treated animals during preimplantation days of pregnancy studied. These results suggest that PR and ER play an important role in Gram-negative bacteria infection and induced implantation failure in mouse.

The present study investigates the effect of different slow chilling curves on the storage of pacu (Piaractus mesopotamicus) embryos submitted to chilling at −8°C. Embryos at the blastopore closure stage were divided into two groups: G1 – embryos exposed to cryoprotectant solution containing methanol (10%) and sucrose (0.5 M), treated as follows: (T1) taken directly from room temperature to the refrigerator without being submitted to the curve; (T2) chilling curve of 0.5°C/min; and (T3) chilling curve of 1°C/min; and G2 – the cryoprotectant solution alone was submitted to these same temperatures, receiving the embryos only after temperature had decreased, corresponding to treatments T4, T5 and T6, respectively. Treatments were kept at −8°C for a period of 6 h. Embryo development was evaluated for each treatment, with six replicates in an entirely randomized design. Survival among embryos not submitted to refrigeration was 94.3 ± 8.05%. Percentage of total larvae (TL) and addled eggs (AE) did not differ statistically between the groups, although percentage of swimming larvae (SL) exhibited higher values in G1 for the 1°C/min curve. Furthermore, when comparing the three chilling curves, a decrease of 1°C/min resulted in the highest TL percentage (90.85%), followed by the 0.5°C/min curve (78.52%). Thus, the use of 1°C/min chilling curves is recommended for P. mesopotamicus embryos stored for 6 h at −8°C.

Chinchilla lanigera is an endangered species therefore the development of cryopreservation protocols for its gametes is a useful tool in the application of assisted reproduction techniques. A study of the functionality of the spermatozoa punctured from the cauda epididymis was performed on fresh or frozen–thawed samples with three cryoprotective media (test-yolk buffer, sucrose and glycerol). The effect that these media had on sperm physiology during the freezing, storage and later thawing process was analysed. A decrease in the percentages of viability, motility, membrane integrity and capacity to undergo the induced acrosome reaction was found with all the media assayed, an increase in the percentages of DNA fragmentation was also observed. The comparative analysis of the effect of the different cryoprotectants assayed showed that the best medium to use to cryopreserve epididymal sperm in this species is test-yolk buffer. This medium had the least effect on the abovementioned physiological parameters, especially at the level of genetic material.

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus–cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey–rabbit embryo). The monkey–rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey–rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey–rabbit ISCNT and rabbit–rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey–rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit–rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey–rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey–rabbit ISCNT embryos is similar to that of rabbit–rabbit SCNT embryos; and (4) the mitochondrial fate of monkey–rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

To ascertain whether the Kunming (KM) mouse is an available model for age-related decline in female fertility in human or not, oocytes from young (6–8 weeks), middle-aged (9 months) and aged (12 months) female mice were compared with respect to number of oocytes, frequency of in-vitro maturation (IVM) and in-vitro fertilization (IVF), and meiotic chromosome segregation and alignment. The mean number of pups born per mouse decreased significantly from the young to the middle-aged and the aged mice. The mean number of ovarian follicles, ovarian germinal vesicle oocytes and ovulated MII oocytes decreased significantly with maternal age. The rate of IVM in oocytes from young mice (73.9%) was less significantly than that in oocytes from middle-aged and aged mice (86.1% and 84.4%, respectively). Immunocytochemical analysis showed that ageing caused a significantly higher rate (49.3%) of chromosome misalignment than that (15.7%) of the young mice. The presence of premature chromatids was also significantly higher in MII oocytes of aged mice as compared with young mice (37.8 versus 8.3%). Pronuclear formation was delayed in oocytes of middle-aged and aged females (35.5 and 42.3% respectively in 5 h of IVF) as compared with young mice (88.1%). The study suggests that KM mouse exhibits an age-related decline in female fertility. Significant reduction of germinal vesicle (GV) and MII oocytes and significant increase of metaphase chromosome misalignment and premature chromatid segregation after meiotic maturation of oocytes, similar to human, presumably contribute to the decline in aged KM mice.

Heat stress (HS) is especially harmful for bovine ovarian follicle development and oocyte competence. Furthermore, HS causes premature aging in oocytes due to high levels of reactive oxygen species (ROS), involved in the harmful effects over the oocyte maturation and the steroidogenic activity of follicular cells. In this study, the presumptive protective effects of antioxidant agents on heat-stressed oocytes were evaluated. Heifer oocytes were matured for 22 h under control (38°C) and HS conditions (41.5°C at 18–21 h of maturation). For each oocyte, nuclear stage and cortical granule (CG) distribution were evaluated. Steroidogenic activity of cumulus cells was also recorded. The antioxidant agents used in the study were: retinol (1.43 μg/ml), retinyl (0.28 μg/ml) and oleic acid (0.05 mg/ml). Based on a chi-squared test (P < 0.05), HS affected negatively the metaphase II (MII) progression and produced a premature CG exocytosis. Retinol improved the oocyte MII progression. However, retinyl and oleic acid, at the concentrations used in this study, could not counteract adverse effects of HS. A decrease in progesterone and increase in estradiol availability were observed when retinyl and oleic acid were supplemented to the maturation medium, respectively. In conclusion, retinol proved to be valuable in heat-stressed oocytes protecting nuclear maturation.

Arginine modification to citrulline (citrullination) is catalyzed by peptidylarginine deiminases (PADs) and one of the isomers PAD4 is shown to be involved in the gene regulation. In our previous paper we studied the localization and expression of PAD4 and the target of PAD4 in mammalian gametes and preimplantation embryos. In this study the role of PAD4 was examined in the pig diploid parthenogenetic preimplantation embryonic development. Knockdown of PAD4 by RNAi resulted in delayed development. Inhibition of PAD4 by a potent PAD4 inhibitor Cl-amidine from the time of activation for 24 h resulted in developmental arrest at the first cleavage. Inhibition at the later stages of development resulted in delayed or arrested development. A shorter exposure to Cl-amidine for 6 h at any stage of growth resulted in slow development. Thus, this study suggests that PAD4 activity is essential for the normal development of the embryos.

Tissue transplantation and in vitro ovarian follicle culture have been investigated as alternative techniques to restore fertility in young women who are facing fertility-threatening diseases or treatments following ovarian tissue cryopreservation. Although transplants of fresh or frozen ovarian tissue have successfully yielded healthy live births in different species including humans, the risks of reintroducing cancer cells back into the patient, post treatment, have limited its clinical purpose. The in vitro ovarian follicle culture minimizes these risks and provides a way to harvest more mature oocytes, however its clinical translation has yet to be determined. Not only is it possible for tissue cryopreservation to safeguard fertility in cancer patients, this technique also allows the maintenance of germplasm banks for animals of high commercial value or for those animals that are at risk of extinction. Given the importance of managing female genetic material, this paper reviews the progress of the methods used to preserve and restore female fertility in different species to demonstrate the results obtained in the past 50 years of research, the current achievements and the future directions on this field.

The cell-cycle regulators that control meiotic divisions also regulate the events that accompany the oocyte-to-zygote transition. Thus, the meiotic machinery functions as an internal pacemaker that propels the oocyte toward embryogenesis. The preimplantation embryo expresses a number of receptors that are important for initial activity of the phosphatidylinositol 3-kinase–protein kinase B (PI3K-Akt/PKB) pathway. The complete PI3K-Akt/PKB-CDK1 cascade is implicated as a key regulator of a number of cellular functions. Selective inhibition of protein kinase B (Akt/PKB) with inhibitor SH6 and cyclin-dependent kinase 1 (CDK1) with inhibitor roscovitine arrest development of the 1-cell preimplantation mouse embryo before entry into the first mitosis. The pronuclei of these inhibited embryos migrate to one another, but do not progress to pronuclei envelope breakdown and pronuclear fusion running immediately before the onset of mitosis. SH6-treated 1-cell mouse embryos showed a high occurrence of apoptosis features (nuclear fragmentation, positive terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), active caspase-3 in both cytoplasm and nucleoplasm). In the Akt/PKB-inhibited embryos, the active phosphorylated form Ser473Akt/PKB was not detected in pronuclear areas when compared with inhibitor-free controls. Although CDK1-inhibited 1-cell embryos also failed to enter into the first mitosis, the presence of apoptotic cell death features was not observed. In the roscovitine-treated embryos, Ser473Akt/PKB was detected in the pronuclei independently of CDK1 activity. We conclude that Akt/PKB plays an important role during entry of the 1-cell mouse embryo into the first mitosis, and probably functions as a relay in the cell-cycle stage. We assume that Akt/PKB is the primary target responsible for mediating anti-apoptotic signals in the 1-cell mouse embryo.