We isolated and characterized Xenopus tropicalis hb4 flanking DNA and showed that the −3076/+29 sequence was able to drive stage-specific transcription in the developmental process. Transgenic reporter analysis indicated that green fluorescent protein was expressed in the ovaries of female frogs at 3 months of age and in both the ovaries and testis of frogs at 6 months of age. A series of experiments with deletion of the flanking sequence and a subsequent luciferase reporter assay revealed that there were two positive regulatory regions and that the most proximal sequence of the promoter region had a certain level of transcriptional activity in oocytes. Subsequently, we showed that a conserved sequence containing Nobox-binding element (NBE) was essential for transcriptional activation and that Nobox expressed in the ovary had a crucial role in hb4 transcription through the NBE sequence.

The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus–oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 μM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.

In assisted reproductive technology (ART) programmes, approximately 10% of infertile patients have at least two or three repeated implantation failures (RIFs) after an in vitro fertilization (IVF) protocol. Successful implantation mainly depends on local immune tolerance mechanisms involving a spectrum of cytokines, interleukins and growth factors. The latter have played pivotal roles in the recruitment of immune cells (and notably T-lymphocyte cells). In total, 250 couples participating in frozen–thawed embryo transfer programme were incorporated in a randomized clinical trial (peripheral blood mononuclear cells (PBMC) subgroup: n=122; control subgroup: n=128). In the PBMC group, a blood sample was collected 5 days before the scheduled frozen–thawed embryo transfer; PBMCs were isolated using Ficoll separation and then cultured for 72 h. Two days prior to embryo transfer, 0.4 ml of cultured PBMCs were transferred into the patient’s uterus. Although the clinical pregnancy rate was higher in the PBMC group (34.4%) than in the control group (23.4%), this difference was not statistically significant (P=0.05 in a chi-squared test). Nevertheless, when we limited the analysis to patients with ≥3 RIFs (n=138), there was a significant difference in the clinical pregnancy rate between the PBMC group (38.6%) and the control group (19.7%; P=0.01). Our results imply that PBMC transfer can be part of effective fertility treatment for patients with RIF.

This is a retrospective study over a 5-year period. In total, 3139 embryos were individually cryopreserved (Cryotop®) and warmed using the Kitazato vitrification/warming kit. They were classified into three categories based on their expansion degree. Transfer, implantation and pregnancy rates were assessed for each embryo category and compared using SPSS (Statistical Package for the Social Sciences) software. In total, 1139 couples enrolled in infertility treatment programme benefitted from embryo vitrification at day 5. After warming, embryos belonging to the three categories showed similar success rates. Although there was a trend towards better outcomes when grade 3 embryos were transferred, the differences did not reach statistical significance: implantation rates (n fetal sac/n embryo transferred) grade 1: 21.9%, grade 2: 22.7% and grade 3: 30.3% (=0.19). Pregnancy rate (n clinical pregnancy/n transfer) (21.9%, 22.7%, 30.3%, respectively; P=0.11). Miscarriage rate was not statistically different in the three categories (14.5%, 20.4%, 20%, respectively, P=0.51). Our overall results show that it is worth vitrifying slow kinetics embryos as they provide a non-negligible chance to give rise to a pregnancy.

Male gamete chemotaxis towards the female gamete is a general strategy to facilitate the sexual reproduction in many marine eukaryotes. Biochemical studies of chemoattractants for male gametes of brown algae have advanced in the 1970s and 1980s, but the molecular mechanism of male gamete responses to the attractants remains elusive. In sea urchin, a K+ channel called the tetraKCNG channel plays a fundamental role in sperm chemotaxis and inhibition of K+ efflux through this channel by high K+ seawater blocks almost all cell responses to the chemoattractant. This signalling mechanism could be conserved in marine invertebrates as tetraKCNG channels are conserved in the marine invertebrates that exhibit sperm chemotaxis. We confirmed that high K+ seawater also inhibited sperm chemotaxis in ascidian, Ciona intestinalis (robusta), in this study. Conversely, the male gamete chemotaxis towards the female gamete of a brown alga, Mutimo cylindricus, was preserved even in high K+ seawater. This result indicates that none of the K+ channels is essential for male gamete chemotaxis in the brown alga, suggesting that the signalling mechanism for chemotaxis in this brown alga is quite different from that of marine invertebrates. Correlated to this result, we revealed that the channels previously proposed as homologues of tetraKCNG in brown algae have a distinct domain composition from that of the tetraKCNG. Namely, one of them possesses two repeats of the six transmembrane segments (diKCNG) instead of four. The structural analysis suggests that diKCNG is a cyclic nucleotide-modulated and/or voltage-gated K+ channel.

Brilliant cresyl blue (BCB) vital labelling is a powerful method for analyzing the quality of porcine cumulus–oocyte complexes. Our aim was to investigate the correlation between the selection of porcine oocytes using BCB labelling and selected intranuclear characteristics of porcine oocytes and parthenotes. Moreover, BCB labelling was correlated with the diameter of the oocyte and the developmental potential of the parthenotes. The following methods were used: BCB labelling, measurement of the diameter of the oocyte, parthenogenetic activation, immunocytochemistry, transmission electron microscopy, enucleation and relative protein concentration (RPC) analysis. We determined that the diameter of the oocytes in the BCB-positive (BCB+) group was significantly larger than in the BCB-negative (BCB−) group. Immediately after oocyte selection according to BCB labelling, we found significant difference in chromatin configuration between the analyzed groups. BCB+ oocytes were significantly better at maturation than BCB− oocytes. BCB+ embryos were significantly more competent at cleaving and in their ability to reach the blastocyst stage than BCB− embryos. Ultrastructural analyses showed that the formation of active nucleoli in the BCB+ group started at the 8-cell stage. Conversely, most BCB− embryos at the 8-cell and 16-cell stages were fragmented. No statistically significant difference in RPC in nucleolus precursor bodies (NPBs) between BCB+ and BCB− oocytes was found. We can conclude that BCB labelling could be suitable for assessing the quality of porcine oocytes. Moreover, the evaluation of RPC indicates that the quantitative content of proteins in NPB is already established in growing oocytes.

During sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy.

Oocytes of B6D2F1 (BDF1) mice are often used as recipients for intracytoplasmic sperm injection because of their cell membrane resistance against capillary penetration. It is assumed that oocytes of BDF1 mice have superior traits because of their hybrid vigour. However, the mechanisms of hybrid vigour are unclear. In this study, we focused on the membrane resistance of MII stage oocytes against changes in extracellular osmotic pressure. As a result, MII stage oocytes of inbred C57BL/6 and DBA/2 mice showed high tolerance in either a hypertonic or a hypotonic environment. Conversely, MII stage oocytes of hybrid BDF1 and D2B6F1 mice showed high tolerance in both hypertonic and hypotonic environments. Therefore, it is considered that MII stage oocytes of hybrid mice have superior traits than those of inbred mice. Our findings demonstrated that the hybrid vigour exists in the form of resistance to extracellular osmotic environment in hybrid MII stage oocytes.

This study aimed to optimize the derivation of trophectoderm from in vitro-produced camel embryos under feeder-free culture conditions using the basement membrane matrix Matrigel. Trophoblastic vesicles were obtained through mechanical microdissection of in vitro-produced camel (Camelus dromedarius) embryos. Supplementing the culture medium with 10 ng/ml of epidermal growth factor and 10 ng/ml fibroblast growth factor improved the attachment and subsequent outgrowths of cultured trophoblastic vesicles when compared with the control group and the groups supplemented individually with each growth factor. The expression levels of pluripotency genes octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), myelocytomatosis proto-oncogene (c-Myc) and anti-apoptotic gene B-cell lymphoma 2 (Bcl2) were increased in trophoblastic vesicles supplemented with both growth factors when compared with the control group. Conversely, both growth factors decreased the expression of apoptotic genes tumour protein p53 (p53) and Bcl-2-associated X protein (Bax). To the best of our knowledge, this may be the first report describing the derivation of trophoblast stem cells from in vitro-produced camel embryos.

This study aimed to describe the morphology and sperm quality of free-living adult males of cururu stingray Potamotrygon wallacei, endemic from the Rio Negro basin, Brazilian Amazon. The sperm was collected in loco from the seminal vesicle region and fixed in buffered saline formaldehyde solution for further evaluation of morphometry, sperm plasma membrane integrity and sperm concentration. The spermatozoa presented a total length of 138.25 ± 1.82 μm with a helical shape and a long head. A high percentage of cells with intact membrane (98 ± 2%) and normal spermatozoa (92 ± 1%) were observed. The cell concentration was 0.34 ± 0.05 × 1010 spermatozoa/ml of semen. These observations are unprecedented for potamotrygonid species and will serve as a basis for future management and conservation strategies.