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.

Reference gene selection in mouse oocytes is an important task required to perform further adequate analysis of target gene expression levels. In the current work we have analyzed expression stability of the seven most commonly used reference genes (Actb, Eef1e1, Gapdh, H2afz, Ppia, Rpl4 and Ubc) in mouse oocytes at the germinal vesicle (GV) stage. We have performed analysis of expression stability of the above-mentioned reference genes with the three most commonly used software tools: geNorm, BestKeeper and NormFinder. Taking into account the results obtained from all of these programmes Gapdh, Rpl4 and H2afz seem to be suitable candidate reference genes in GV oocytes of mouse.

Around 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

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.

Prolyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles.

Quality of in vitro-produced embryos is influenced by changes in gene expression in response to adverse conditions. Gene markers for predicting ‘good embryos’ do not exist at present. We propose that the expression of pluripotency markers OCT4–SOX2–NANOG in D9 (day 9) bovine demi-embryos correlated with development at D13 (day 13). Day 8 in vitro-produced blastocysts were split in two cloned halves, one half (D9) was subjected to analysis of pluripotency markers and the other was kept in culture until D13 of development. Embryo development was scored and correlated with its own status at D9 and assigned to one of two categories: G1, arrested/dead; or G2, development up to D13. SOX2 and NANOG expression levels were significantly higher in embryos from G1 and there was also negative correlation between SOX2 and embryo survival to D13 (G3; r = −0.37; P = 0.03). We observed a significant reduction in the expression of the three studied genes from D9 to D13. Furthermore, there was a correlation between the expression of pluripotency markers at D9 and embryo diameter and the expression of trophoblastic markers at D13 (TP1–EOMES–FGF4–CDX2–TKDP1). Finally, the quotient between the relative expression of SOX2 and OCT4 in the D9 blastocysts from G1 and G2 showed that embryos that were considered as competent (G2) had a quotient close to one, while the other group had a quotient of 2.3 due to a higher expression of SOX2. These results might indicate that overexpression of SOX2 at the blastocyst stage had a negative effect on the control of embryonic developmental potential.

Cryptorchidism, characterized by the presence of one (unilateral) or both (bilateral) undescended testes, is a common male urogenital defect. Cryptorchidism can lead to male infertility, testicular cancer being the most extreme clinical symptom, as well as psychological issues of the inflicted individual. Despite this, both knowledge about the aetiology of cryptorchidism and the mechanism for cryptorchidism-induced male infertility remain limited. In this present study, by using an artificial cryptorchid mouse model, we investigated the effects of surgery-induced cryptorchidism on spermatogenic cells and seminiferous epithelial cycles. We found that surgery-induced cryptorchidism led to a reduced testicular weight, aberrant seminiferous epithelial cycles and impaired spermatogenesis characterized by degenerating spermatogenic cells. We also observed multinucleated giant cells after surgery-induced cryptorchidism. Transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and western blot assays demonstrated cryptorchidism-induced apoptosis of spermatogenic cells. Moreover, we identified the occurrence of autophagy in germ cells after surgery-induced cryptorchidism. Interestingly, apoptosis and autophagy were synchronous, suggestive of their synergetic roles in promoting germ cell death. Our results provide novel insights into the cryptorchidism-induced male infertility, thereby contributing to the development of male contraceptive strategies as well as treatment options for male infertility caused by cryptorchidism.

Rabbits play an important role in people’s lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

Mammalian Pou5f1 encodes the POU family class V (POU-V) transcription factor which is essential for the pluripotency of embryonic cells and germ cells. In vertebrates, various POU-V family genes have been identified and classified into the POU5F1 family or its paralogous POU5F3 family. In this study, we cloned two cDNAs named CpPou5f1 and CpPou5f3, which encode POU-V family proteins of the Japanese red bellied newt Cynops pyrrhogaster. In the predicted amino acid sequence encoded by CpPou5f1, the typical MAGH sequence at the N-terminus and deletion of arginine at the fifth position of POU-homeodomain were recognized, but not in the sequence encoded by CpPou5f3. Phylogenetic analysis using Clustal Omega software indicated that CpPou5f1 and CpPou5f3 are classified into the clade of the POU5F1 and POU5F3 families, respectively. In a real-time polymerase chain reaction (RT-PCR) analysis, the marked gene expression of CpPou5f1 was observed during oogenesis and early development up to the tail-bud stage, whereas weak gene expression of CpPou5f3 was detected only in the early stages of oogenesis and gastrula. In adult organs, CpPou5f1 was expressed only in the ovary, while gene expression of CpPou5f3 was recognized in various organs. A regeneration experiment using larval forelimb revealed that transient gene expression of CpPou5f1 occurred at the time of wound healing, followed by gene activation of CpPou5f3 during the period of blastema formation. These results suggest that CpPou5f1 and CpPou5f3 might play different roles in embryogenesis and limb regeneration.

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.

Androgenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.

Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8–16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

Recovery from decreased cell volume is accomplished by a regulated increase of intracellular osmolarity. The acute response is activation of inorganic ion transport into the cell, the main effector of which is the Na+/H+ exchanger NHE1. NHE1 is rapidly activated by a cell volume decrease in early embryos, but how this occurs is incompletely understood. Elucidating cell volume-regulatory mechanisms in early embryos is important, as it has been shown that their dysregulation results in preimplantation developmental arrest. The kinase JAK2 has a role in volume-mediated NHE1 activation in at least some cells, including 2-cell stage mouse embryos. However, while 2-cell embryos show partial inhibition of NHE1 when JAK2 activity is blocked, NHE1 activation in 1-cell embryos is JAK2-independent, implying a requirement for additional signalling mechanisms. As focal adhesion kinase (FAK aka PTK2) becomes phosphorylated and activated in some cell types in response to decreased cell volume, we sought to determine whether it was involved in NHE1 activation in the early mouse embryo. FAK activity requires initial autophosphorylation of a tyrosine residue, Y397. However, FAK Y397 phosphorylation levels were not increased in either 1- or 2-cell embryos after cell volume was decreased. Furthermore, the selective FAK inhibitor PF-562271 did not affect NHE1 activation at concentrations that essentially eliminated Y397 phosphorylation. Thus, autophosphorylation of FAK Y397 does not appear to be required for NHE1 activation induced by a decrease in cell volume in early mouse embryos.

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.

The aim of this study was to evaluate the effects of different timing for frozen–thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen–thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.

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.

The aim of this study was to investigate if there is an adverse effect of multiple controlled ovarian stimulation (COS) on the maturity of oocytes (MI and MII), fertilization rate, embryo developmental qualities and clinical pregnancy rates in donation cycles. In total, 65 patients undergoing oocyte donation cycles multiple times were included in this study. Patients were grouped as group A that consisted of donors with ≤2 stimulation cycles while B consisted of donors with ≥3 stimulation cycles; and group C included donors who had ≤15 oocytes, while group D had donors with ≥16 oocytes. Numbers of oocytes obtained, MI and MII oocytes, fertilization, embryo quality and clinical pregnancy outcomes were compared. Significant statistical differences were observed in total number of oocytes obtained, maturity of oocytes (MI and MII), fertilization rate, embryo qualities and clinical pregnancy outcomes of donors in groups A–D. Donors with ≤2 ovarian stimulation cycles had lower numbers of immature oocytes than donors with three or more stimulation cycles. However, donors with ≥3 stimulation cycles had higher numbers of mature oocytes, zygotes, with better day 3 embryo qualities and higher clinical pregnancy rates than donors with ≤2 stimulation cycles. Repeated COS does not seem to have any adverse effect on ovarian response to higher dose of artificial gonadotropin, as quality of oocytes collected and their embryological developmental potential were not affected by the number of successive stimulation cycles. The effect of multiple COS on the health of the oocyte donor needs to be assessed for future purpose.

Heat shock may disrupt oocyte function by increasing the generation of reactive oxygen species (ROS). We evaluated the capacity of the antioxidant melatonin to protect oocytes using two models of oxidative stress – heat shock and the pro-oxidant menadione. Bovine cumulus–oocyte complexes (COC) were exposed in the presence or absence of 1 µM melatonin to the following treatments during maturation: 38.5°C, 41°C and 38.5°C+5 µM menadione. In the first experiment, COC were matured for 3 h with 5 µM CellROX® and analyzed by epifluorescence microscopy to quantify production of ROS. The intensity of ROS was greater for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin reduced ROS intensity for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. In the second experiment, COC were matured for 22 h. After maturation, oocytes were fertilized and the embryos cultured for 7.5 days. The proportion of oocytes that cleaved after fertilization was lower for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin increased cleavage for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. Melatonin tended to increase the developmental competence of embryos from heat-shocked oocytes but not for embryos from oocytes exposed to menadione or from control oocytes. In conclusion, melatonin reduced production of ROS of maturing oocytes and protected oocytes from deleterious effects of both stresses on competence of the oocyte to cleave after coincubation with sperm. These results suggest that excessive production of ROS compromises oocyte function.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.