This study aimed to establish a hormonal induction protocol for spermiation of Brycon cephalus males, using Ala6, Pro9Net-mGnRH + metoclopramide (Ovopel®). Thus, 20 males were used divided into three inductor treatments [⅓ pellet/kg (T1), ⅔ pellet/kg (T2) and 1⅓ pellet/kg (T3)] and one control group (CO), which only received physiological solution applications (0.9% NaCl). All treatments were applied in a single dose. For evaluation of the availability of the treatment, the following seminal parameters were analyzed: seminal volume, subjective spermatic motility, duration of motility, pH, osmolality and spermatic concentration. T3 showed the highest seminal volume (4.66 ± 1.52 ml), and was significantly different in comparison with T1 (2.0 ± 0.9 ml), T2 (3.5 ± 1.3 ml) and CO (2.3 ± 1.2 ml). In relation to spermatic motility, T2 and T3 showed significantly higher levels [5, (81–100%)]. However, T3 showed significantly lower average sperm motility duration than T1, T2 and CO (30 ± 7 s; 28 ± 6 s; 32 ± 8 s, respectively). With regard to the seminal parameters of spermatic concentration, pH and osmolality, no significant variation was verified among treatments. In conclusion, mGnRH + metoclopramide used for hormonal induction of B. cephalus reproduction does not induce changes related to spermatic concentration, pH and osmolality parameters of the seminal fluid and the most adequate doses among tested treatments were ⅔ pellet/kg live fish.

Aurora-A kinase (AURKA), a member of the serine/threonine protein kinase family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little information is known about AURKA in the early cleavage mouse embryo with respect to acentrosomal spindle assembly. In vitro experiments in which AURKA was inactivated with specific inhibitor MLN8237 during the early stages of embryogenesis documented gradual arrest in the cleavage ability of the mouse embryo. In the AURKA-inhibited 1-cell embryos, spindle formation and anaphase onset were delayed and chromosome segregation was defective. AURKA inhibition increased apoptosis during early embryonic development. In conclusion these data suggest that AURKA is essential for the correct chromosome segregation in the first mitosis as a prerequisite for normal later development after first cleavage.

Polo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes.

The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal–Wallis test and Mann–Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation.

Chinchilla lanigera, native to the Andean Mountains of Perú, Chile, Bolivia and Argentina, is a specimen of great economic importance because of its fur. In mammals, spermatozoa originate in testes and are transported to the epididymis, where they undergo morphological and biochemical modifications known as sperm maturation, a basic step in the acquisition of their fertilizing ability. The aim of this work is the macroscopic and microscopic analysis of the epididymis of Chinchilla lanigera Grey and its sectorization based on a histomorphological study. The epididymis presents a clear segmentation into four regions: initial segment, caput, corpus and cauda. The epithelium lining the seminiferous tubules is pseudostratified, with principal cells with stereocilia and basal, clear, apical, narrow and halo cells. The histological analysis showed that principal and basal cells are the prevailing populations in all regions, also revealing narrow cells and the absence of clear cells in the initial segment. Each segment presents its different histological and morphometric characteristics, which supports the idea of the specific behaviour of each region, giving a segment-specific character to the process of sperm maturation in this species. No significant differences were found in the morphometric measurements or in the histological evaluation of the epididymis of samples collected in April and October. The fact that no differences were found between the samples collected during the two periods when the reproductive ability in nature is different suggests the importance of external factors in the control of the reproductive cycle of Chinchilla lanigera.

Many studies of the main gap junction protein, Cx43, have been conducted in porcine oocyte research, but they have been limited to investigations of cumulus–oocyte complexes (COCs). In this study, we verified Cx43 not in COCs, but in porcine oocytes during maturation, and conducted a quantitative time course analysis. The location and dynamics of Cx43 were examined by immunocytochemistry and western blotting, respectively. COCs were cultured in NCSU23 medium and processed for immunocytochemistry and western blotting at 0, 14, 28, and 42 h after denuding. A Cx43 signal was detected on oolemmas, transzonal projections and the surface of zona pellucidae. Western blotting showed that Cx43 band density increased from 0 to 14 h, and gradually decreased thereafter. Our results clarified that Cx43 is localized in the ooplasmic membrane through zona pellucidae and its level changes over time during culture in porcine oocytes.

The sea urchin embryo is recognized as a model system to reveal developmental mechanisms involved in human health and disease. In Part I of this series, six carbohydrates were tested for their effects on gastrulation in embryos of the sea urchin Lytechinus pictus. Only l-rhamnose caused dramatic increases in the numbers of unattached archenterons and exogastrulated archenterons in living, swimming embryos. It was found that at 30 h post-fertilization the l-rhamnose had an unusual inverse dose-dependent effect, with low concentrations (1–3 mM) interfering with development and higher concentrations (30 mM) having little to no effect on normal development. In this study, embryos were examined for inhibition of archenteron development after treatment with α-l-rhamnosidase, an endoglycosidase that removes terminal l-rhamnose sugars from glycans. It was observed that the enzyme had profound effects on gastrulation, an effect that could be suppressed by addition of l-rhamnose as a competitive inhibitor. The involvement of l-rhamnose-containing glycans in sea urchin gastrulation was unexpected, since there are no characterized biosynthetic pathways for rhamnose utilization in animals. It is possible there exists a novel l-rhamnose-containing glycan in sea urchins, or that the enzyme and sugar interfere with the function of rhamnose-binding lectins, which are components of the innate immune system in many vertebrate and invertebrate species.

This study examined the impact of cow body condition on the quality of bovine preimplantation embryos. The embryos (n = 107) were flushed from dairy cows and classified according to a five-point scale body condition score (BCS2 n = 17; BCS3 n = 31; BCS4 n = 11) on the 7th day after insemination and then analyzed for development, dead cell index (DCI), cell number and actin cytoskeleton quality. The highest embryo recovery rate (P < 0.05) was recorded in the BCS3 group and the lowest in the BCS4 group. More transferable (morula, blastocyst) embryos were obtained from the BCS4 cows (79%), compared with the BCS2 (64%) or BCS3 (63%) animals. However, cell numbers were higher in the BCS2 and BCS3 groups (P < 0.05) compared with the BCS4 embryos. Conversely, the DCI was lowest in the BCS2 (3.88%; P < 0.05) and highest in the BCS4 (6.56%) embryos. The proportion of embryos with the best actin quality (grade I) was higher in the BCS2 and BCS3 cows compared with the BCS4 group. Almost 25% of all embryos showed fragmented morphology and a higher DCI (5.65%) than normal morulas (1.76%). More fragmented embryos were revealed in the BCS2 (28.6%) and BCS4 (31.25%) groups, and less (19.15%) in the BCS3 group. The cell numbers in such embryos were lower in the BCS4 (22.57) than in the BCS2 (46.25) or BCS3 (42.4) groups. In conclusion, the body condition of dairy cows affects the quality of preimplantation embryos. A BCS over 3.0 resulted in a higher incidence of poor (fragmented) embryos.

Preovulatory binding of viable spermatozoa in the oviduct isthmus is widely accepted as a preliminary to fertilization, but details of physiological events associated with epithelial binding and release from binding are themselves little understood. Important questions include the potential number, distribution and stability of such sites in the caudal isthmus, whether multiple molecular forms of binding exist within a single-mated individual, and whether some sites are more favourable than others for the maintenance of preovulatory sperm viability. Also to be resolved is whether spermatozoa interact with the first available binding sites in the isthmus, whether spermatozoa from second or subsequent matings bind closer to the site of fertilization, and whether the first spermatozoa entering the oviduct are those that will be released first with impending ovulation. Ideally, future research needs to focus on a fertilizing spermatozoon monitored in vivo and not on spermatozoa destined to remain on or in the zona pellucida or in the lower reaches of the oviduct.

Vertebrate pigmentation provides an ideal system for studying the intersections between evolution, genetics, and developmental biology. Teleost fish, with their accessible developmental stages and intense and diverse colours produced by chromatophores, are an ideal group for study. We set out to test whether Betta splendens is a good model organism for studying the evolution and development of diverse pigmentation. Our results demonstrate that B. splendens can be bred to produce large numbers of offspring with easily visualized pigment cells. Depending on the colour of the parents, there was variation in larval pigmentation patterns both within and between breeding events. In juveniles the developing adult pigmentation patterns showed even greater variation. These results suggest that B. splendens has great potential as a model organism for pigmentation studies.

The aim of this study was to explore whether the morphology of polar bodies (PBs) estimated at 16–18 h after insemination can be used as an additional marker for predicting human embryo quality or pregnancy outcome. The data from 355 patients who received standard in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment after controlled ovarian hyperstimulation were recruited. Normal fertilized 3048 zygotes from 382 cycles were divided into two groups, PBs intact or fragmented, according to the morphology of PBs assessed at 16–18 h after insemination. Embryo quality and pregnancy outcome were compared between the two groups. It was shown that the day 3 (D3) good embryo rate, good quality blastocyst rate and available embryo rate of the PBs intact group were all significantly higher than that of the corresponding fragmented groups. However, no significant differences in pregnancy rate (PR) or implantation rate (IR) were observed between the intact and fragmented groups. Although PBs morphology estimated at 16–18 h after insemination had little effect on PR or IR in fresh embryo transfer cycles, a better embryo quality can be achieved in the PB-intact group, which is valuable for embryo selection.

The Prochilodontidae prepare for reproduction in captivity, but neither ovulation nor spawning occurs, thus requiring induced reproduction for breeding and restocking. This study analyzed for the first time the embryogenesis and larval ontogeny of P. hartii submitted to induced reproduction by hypophysation with crude common carp pituitary extract. The extrusion of oocytes and sperm was performed manually and fertilization was done using the dry method. After fertilization, the eggs were kept in incubators at 23°C. A stereomicroscope was used to measure egg diameter and to monitor embryo development. Samples of larvae were collected daily for 7 days for histological and biometric analyses. The recently extruded oocytes, non-hydrated, are spherical, grey, and non-adhesive with a diameter of 1480 ± 39 μm and after hydration, have a diameter of 2860 ± 120 μm. The positive response to hypophysation was 100% for females and 80% for males. Spawning occurred 7 h after the third hormonal dosage. The fertilization rate was 77% at 23°C. Blastopore closure occurred at 6 h 45 min and embryonic development was completed 36 h 10 min after fertilization. After 204 h post fertilization (hpf) the larvae reached a standard length of 6.56 ± 0.14 mm with the yolk sac completely resorbed. In P. hartii, the oral cavity opening occurred 132 hpf. The results of this study provide knowledge to better understand induced reproduction, breeding, and management of P. hartii, a species with a high potential for pisciculture, and which is commercially important in the Jequitinhonha River basin.

We evaluated the usefulness of lissamine green B (LB) staining of cumulus–oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

The objective of this study was to characterize the early development and allometric growth of the grumatã (Prochilodus vimboides). We describe a sample of 266 eggs and larvae obtained through induced spawning. The eggs were spherical (mean 3.7 mm diameter), exhibited a yellow yolk and were non-adhesive and pelagic after fertilization and hydration. The time elapsed between the early cleavage and post-flexion stages was considered short (328 hours, 8054 hour-degrees) in regard to the development times of other Neotropical rheophilic species, but time to hatching was considerably longer than in other Prochilodus species. The most notable anatomical changes were observed between the end of the yolk larval stage and the beginning of the pre-flexion stage, when the larvae displayed directed swimming and the digestive system became functional, enabling the transition from endogenous to exogenous feeding. After hatching, the larvae grew from 6.04 to 15.15 mm in total length average. Two growth phases were observed at this stage: a non-linear asymptotic curve in yolk-sac larvae, and a linear constant-rate growth phase after exogenous feeding started. Allometric growth related to standard length was positive for head length, negative for eye diameter, and switched between phases from negative to positive in body depth and head height. Morphological development and allometric growth in different larval phases impose drastic anatomical and physiological changes that are synchronic with habitat changes and the flood cycles during the reproductive period.

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

Gap junctional intercellular communication is assumed to play an important role during pre- and peri-implantation development. In this study, we eliminated connexin43 (Cx43) and connexin45 (Cx45), major gap junctional proteins in the pre- and peri-implantation embryo. We generated Cx43−/−Cx45−/− embryos by Cx43+/−Cx45+/− intercrossing, because mice deficient in Cx43 (Cx43−/−) exhibit perinatal lethality and those deficient in Cx45 (Cx45−/−) exhibit early embryonic lethality. Wild-type, Cx43−/−, Cx45−/−, and Cx43−/−Cx45−/− blastocysts all showed similar outgrowths in in vitro culture. Moreover, Cx43−/−Cx45−/− embryos were obtained at the expected Mendelian ratio up to embryonic day 9.5, when the Cx45−/− mutation proved lethal. The Cx43−/−Cx45−/− embryos seemed to have no additional developmental abnormalities in comparison with the single knockout strains. Thus, pre- and peri-implantation development does not require Cx43 and Cx45. Other gap junctional proteins are expressed around these stages and these may compensate for the lack of Cx43 and Cx45.

The developmental potential of in vitro matured porcine oocytes is still lower than that of oocytes matured and fertilized in vivo. Major problems that account for the lower efficiency of in vitro production include the improper nuclear and cytoplasmic maturation of oocytes. With the aim of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus–oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10 or 50 ng/ml) and/or GDNF (0, 10 or 50 ng/ml) for 44 h, and subsequently subjected to fertilization and cultured for 7 days in vitro. The in vitro-formed blastocysts derived from selected growth factor groups (i.e. EGF = 50 ng/ml; GDNF = 50 ng/ml; EGF = 50 ng/ml + GDNF = 50 ng/ml) were also used for mRNA expression analysis, or were subjected to Hoechst staining. The results showed that the addition of EGF and/or GDNF during oocyte maturation dose dependently enhanced oocyte developmental competence. Compared with the embryos obtained from control or single growth factor-treated oocytes, treatment with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P < 0.05), and also simultaneously induced the expression of BCL-xL and TERT and suppressed the expression of caspase-3 in resulting blastocysts (P < 0.05). These results suggest that both GDNF and EGF may play an important role in the regulation of porcine in vitro oocyte maturation and the combination of these growth factors could promote oocyte competency and blastocyst quality.