Post-fertilization Development Research Articles

Aspartame Causes Developmental Defects and Teratogenicity in Zebra Fish Embryo: Role of Impaired SIRT1/FOXO3a Axis in Neuron Cells.

Aspartame, a widely used artificial sweetener, is present in many food products and beverages worldwide. It has been linked to potential neurotoxicity and developmental defects. However, its teratogenic effect on embryonic development and the underlying potential mechanisms need to be elucidated. We investigated the concentration- and time-dependent effects of aspartame on zebrafish development and teratogenicity. We focused on the role of sirtuin 1 (SIRT1) and Forkhead-box transcription factor (FOXO), two proteins that play key roles in neurodevelopment. It was found that aspartame exposure reduced the formation of larvae and the development of cartilage in zebrafish. It also delayed post-fertilization development by altering the head length and locomotor behavior of zebrafish. RNA-sequencing-based DEG analysis showed that SIRT1 and FOXO3a are involved in neurodevelopment. In silico and in vitro analyses showed that aspartame could target and reduce the expression of SIRT1 and FOXO3a proteins in neuron cells. Additionally, aspartame triggered the reduction of autophagy flux by inhibiting the nuclear translocation of SIRT1 in neuronal cells. The findings suggest that aspartame can cause developmental defects and teratogenicity in zebrafish embryos and reduce autophagy by impairing the SIRT1/FOXO3a axis in neuron cells.

Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Mitochondrial UQCC3 controls embryonic and tumor angiogenesis by regulating VEGF expression

Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5-10.5days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

P-248 The meiotic spindle is preserved during freezing but post-thawing rehydration causes its destabilization in human eggs

Abstract Study question Is the human egg spindle integrity impaired by vitrification and thawing procedures? Summary answer Cryoprotective-caused dehydration stabilizes the MII spindle, but the re-entry of water during thawing induces depolymerization of microtubules, thus promoting instability of the division apparatus. What is known already Freezing sperm and preimplantation embryos have become routine IVF procedures with excellent clinical results, whereas cryopreservation of oocytes remains problematic. The major factor underlying the human oocytés notorious propensity to cryoinjury is the temperature sensitivity of the meiotic spindle, the fidelity of which is critical for faithful post-fertilization development. Most studies focus on evaluating vitrified-thawed oocytes' post-fertilization outcome, but behavior of the meiotic spindle during the freezing process has received only a little attention. Study design, size, duration The experimental study involved a total of 165 human oocytes donated for research. The presence and morphology of the meiotic spindle were examined by polarized light microscopy (PLM) and fluorescent confocal imaging to map out the MII spindle dynamics during the vitrification-thawing procedure. Participants/materials, setting, methods A total of 114 women gave informed consent for their surplus immature oocytes to be used in this project. Oocytes that completed maturation in vitro were subjected to spindle imaging (Oosight) and vitrified using a Kitazato/Cryotop open system. The PLM-positive/negative oocytes were fixed at 6 steps of the vitrification-thawing protocol and 2 hours after thawing (15 oocytes in each subgroup). Microtubules and DNA were fluorescently labeled to inspect chromosome-spindle configuration. Main results and the role of chance Time-course experiments showed that PLM-detectable bipolar spindle remained intact in all but one oocyte (44/45) fixed during pre-vitrification equilibration. Notably, the PLM signal became more prominent after adding a cryoprotectant that displaced intracellular water. In contrast, the MII spindle signal progressively disappeared during thawing. Specific tubulin labeling revealed that the microtubule mass was still present in most (41/45) oocytes from the vitrified-thawed sample group. However, the division apparatus tended to lose its bipolarity and disintegrate (8/45) during the washing steps. This trend was accented in the group of oocytes that lacked PLM-detectable spindles before freezing. Here, only a minority of cells (11/45) undergoing rehydration displayed characteristic chromosome-spindle configuration. The difference in eggś capability to ensure spindle bipolarity was apparent even 2 hours after thawing when 13/15 of initially PLM-positive but only 7/15 PLM-negative oocytes exhibited normal-shaped spindles. Based on these data, we hypothesize that the transient disappearance of the PLM spindle signal during warming may be attributed to the restoration of microtubule dynamics and spindle destabilization. The speed and efficiency of spindle reconstruction seem to depend on the overall egǵs fitness. Limitations, reasons for caution This research study involved a limited number of hormonally-primed oocytes that extruded a polar body in vitro shortly after retrieval. The obtained imaging data represent snapshots of the process. A technically challenging live imaging study is needed to complement the description of the oocyte spindle dynamics during post-vitrification recovery. Wider implications of the findings Our results demonstrate that meiotic spindles in frozen-thawed eggs are not products of de novo spindle assembly but are built on preexisting microtubule mass. Suboptimal handling and reducing the interval between thawing and ICSI may enhance unavoidable spindle instability and thus compromise the developmental potential of vitrified eggs. Trial registration number not-applicable

Reproduction, Early Development, and Larval Rearing of the Climbing Perch Anabas testudineus (Teleostei: Anabantidae) in Captivity

Induced spawning, early development, and larval rearing of the climbing perch Anabas testudineus were examined under captive conditions. Five female climbing perch [125.33 ± 3.27 mm SL; 70.67 ± 5.59 g BW] were paired with apparently mature males (108.50 ± 3.97 mm SL; 39.27 ± 4.70 g BW) and induced to spawn with 5,000 IU human chorionic gonadotropin (hCG) + 0.5 mL Ovaprim kg BW-1. All hCG + Ovaprim-injected fish spawned 9 – 11 h after hormone administration but saline-injected fish (control group) failed to spawn. Mean egg production per female, fertilization and hatching rates, and larval production per female were 7,667 ± 1,313, 98.11 ± 1.63, 61.01 ± 19.92, and 4,435 ± 1,041 %, respectively. Fertilized eggs (1.52 – 1.96 mm in dia) were almost spherical, non-adhesive, transparent, and buoyant due to a large, single oil globule (515 ± 33 μm in dia). First cleavage appeared 30 min post-fertilization (MPF) and egg development lasted for 20 – 24 h post-fertilization (HPF) (29°C). Newly hatched climbing perch larvae [1.73 ± 0.08 mm in total length (TL)] with yolk volume (YV) and oil globule volume (OGV) of 0.545 ± 0.104 and 0.072 ± 0.013 mm3, respectively, had no pigmented eyes or functional mouth parts and digestive tract, and immobile with the yolk sac pointed upward. Eyes became pigmented and both mouth and anus opened in 1 d old larvae (2.09 ± 0.04 mm TL) in preparation for exogenous feeding. Except for body depth (BD) and pre-anal length (PAL), a general increase in TL (1.73 – 3.72 mm), eye diameter (ED) (0.164 – 0.378 mm), head length (HL) (0.205 – 0.923 mm), and mouth gape (MG) size (0.308 – 0.552 mm) was observed, coinciding with yolk resorption in 7 d post-hatched larvae. Climbing perch larvae were reared at 4 stocking densities (25, 50, 75, 100 larvae L-1) and fed solely on live food (Brachionus rotundiformis from 2 – 12 d and Artemia nauplii from 13 – 40 d) or co-fed live food and microparticulate diet (MPD) (Artemia nauplii from 2 – 15 d and Artemia + MPD from 16 – 40 d). Mean survival rates at lower densities (25 – 50 larvae L-1) were significantly higher than those reared at 75 and 100 larvae L-1 (P &lt; 0.05). Furthermore, climbing perch larvae co-fed Artemia nauplii and MPD showed better survival (28.67 – 81.00%) than those fed solely on live food (24.88 – 64.00%). However, at the end of the 40 d trial period, growth parameters did not vary significantly among densities (P &gt; 0.05), except for the condition factor in larvae co-fed Artemia nauplii and MPD. Taken together, results demonstrate the effectiveness of hCG + Ovaprim for captive breeding and observations on the early development of climbing perch, and the feasibility of rearing the larvae at 25 – 50 L-1 on a combination of Artemia nauplii and MPD under laboratory conditions.

A long-acting recombinant FSH supports high-quality mouse follicle development and oocyte maturation in vitro by coordinating somatic and germ cell transcriptomes.

Strategies to maximize individual fertility chances are constant requirements of ART. In vitro folliculogenesis may represent a valid option to create a large source of immature ovarian follicles in ART. Efforts are being made to set up mammalian follicle culture protocols with suitable FSH stimuli. In this study, a new type of recombinant FSH (KN015) with a prolonged half-life is proposed as an alternative to canonical FSH. KN015 supports the in vitro development of mouse follicles from primary to preovulatory stage with higher efficiency than canonical FSH and enhanced post-fertilization development rates of the ovulated oocytes. The use of KN015 also allows us to compare the dynamic transcriptome changes in oocytes and granulosa cells at different stages, in vivo and in vitro. In particular, KN015 facilitates mRNA accumulation in growing mouse oocytes and prevents spontaneous luteinization of granulosa cells in vitro. Novel analyses of transcriptome changes in this study reveal that the in vivo oocytes were more efficient than in vitro oocytes in terms of maternal mRNA clearing during meiotic maturation. KN015 promotes the degradation of maternal mRNA during in vitro oocyte maturation, improves cytoplasmic maturation and, therefore, enhances embryonic developmental potential. These findings establish new transcriptome data for oocyte and granulosa cells at the key stages of follicle development, and should help to widen the use of KN015 as a valid and commercially available hormonal support enabling optimized in vitro development of follicles and oocytes.

Effects of salinity on pre- and post-fertilization developmental events in the clam Anomalocardia flexuosa (Linnaeus, 1767)

The knowledge about the effect of salinity on the physiological mechanism of bivalve reproduction is fundamental to improve production strategies in hatcheries. The present work evaluated the influence of different salinity concentrations (15, 20, 25, 30, 35 and 40 g⋅L-1) on pre- and post-fertilization development processes in the clam, Anomalocardia flexuosa, oocytes obtained by stripping. Salinity directly interfered with the germinal vesicle breakdown (GVBD) rate and in the cellular stability of unfertilized oocytes. Salinity concentrations between 30 and 35 g⋅L-1 provided better percentages of stable GVBD within 120 min, and incubation of oocytes in the salinity range of 30-35 g⋅L-1 for a time interval of 80-120 min provided > 80% GVBD. In the post-fertilization analysis, salinity affected the rate of the extrusion of the first and second polar bodies (PB1 and PB2). The release of 50% of the PBs was faster at a salinity of 35 g⋅L-1, with an estimated time of 10 min for PB1 and 30 min for PB2. Thus, chromosome manipulation methodologies aiming triploids should be applied at 35 g⋅L-1 salinity, with application of post-fertilization shock before 10 min for PB1 retention or before 30 min for PB2 retention.

304 The Paternal Programming of Periconception Development

Abstract There is now a significant body of human and animal model data which identify common associations between perturbed development during gestation, disproportionate fetal growth and poor adult cardiovascular and metabolic health. Typically, these studies focus on the impact of poor maternal health on offspring development and well-being. However, a growing evidence base now indicates that sub-optimal paternal factors (such as diet) can also program adult offspring ill-health. While the link between paternal health and offspring well-being is becoming established, attention is being focused on defining the underlying mechanism(s). Here, the father may influence post-fertilization development through two main pathways, the integrity and status of the sperm genome (including epigenome) and through components of the seminal plasma. We have used mouse models of poor-quality diet to study the impact sub-optimal paternal nutrition has on the quality and epigenetic status of the sperm, embryonic development and fetal growth. We have also used our models to study the relative seminal plasma and sperm contributions to adult offspring cardiovascular and metabolic health. Our studies identify a continuum of changes ranging from testicular morphology, sperm epigenetic status, preimplantation embryo development, fetal growth and adult offspring cardio-metabolic ill-health in response to either over or undernutrition. Interestingly, we also observed that these long-term offspring effects appear mediated through specific sperm and seminal plasma specific mechanisms. Our data highlight the significance of a father’s diet at the time of conception for the development and well-being of his offspring.

Qualitative and quantitative assessment of sperm miRNAs identifies hsa-miR-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p as potential biomarkers of male infertility and sperm quality

BackgroundIn contrast with the preceding stages of the germ cells, spermatozoa are unusually rich in small non-coding RNAs in comparison to the coding RNAs. These small RNAs may have had an essential role in the process of spermatogenesis or may have critical roles in the post-fertilization development. Sporadic efforts have identified a few differentially expressed miRNAs in infertile individuals, which do not replicate in other studies.MethodsIn order to identify miRNAs signatures of infertility or poor sperm quality, we compared miRNA differential expression data across nine datasets, followed by their analysis by real-time PCR in a case–control study. This was followed by the validation of potential biomarkers in yet another set of cases and controls. For this, total RNA was isolated from 161 sperm samples. miRNA expression levels in infertile cases and fertile controls were measured using TaqMan real-time PCR. Meta-analyses of two miRNAs (hsa-miR-9-3p and hsa-miR-122-5p) were performed using Comprehensive Meta‐Analysis Software (version 2). All statistical analyses were performed with the help of GraphPad Prism Software (version 8).ResultsLiterature search identified seven miRNAs (hsa-let-7a-5p, hsa-miR-9-3p, hsa-miR-22-5p, has-miR-30b-5p, hsa-miR-103-3p, hsa-miR-122-5p and hsa-miR-335-5p) showing consistent dysregulation in infertility across a minimum of four studies. In the discovery phase, six miRNAs showed strong association with infertility with four (hsa-miR-9-3p, hsa-miR-30b-5p, hsa-miR-103-3p and hsa-miR-122-5p) showing consistent differential regulation across all sub-groups. Receiver operating characteristic (ROC) curve analysis showed that the area under curve of > 0.75 was achieved by three (hsa-mir-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p) miRNAs. In the validation phase, these three miRNAs showed consistent association with infertility (hsa-mir-9-3p, hsa-miR-30b-5p, and hsa-miR-122-5p). Meta-analysis on hsa-miR-122-5p showed its significant quantitative association with infertility [Hedge’s g = -2.428, p = 0.001 (Random effects)].ConclusionsThree miRNAs (hsa-miR-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p) have strong linkage with infertility and a high potential as sperm quality biomarkers.

Control of Maternal-to-Zygotic Transition in Human Embryos and Other Animal Species (Especially Mouse): Similarities and Differences.

Maternal-to-zygotic transition (MZT) of the control of early post-fertilization development is a key-event conditioning the fate of the future embryo, fetus and newborn. Because of the relative paucity of data concerning human embryos, due to ethical concerns and the poor availability of human embryos donated for research, most data have to be derived from animal models, among which those obtained using mouse embryos are most prevalent. However, data obtained by studies performed in non-mammalian specie can also provide useful information. For this reason, this review focuses on similarities and differences of MZT control mechanisms in humans and other species, with particular attention to the mouse. A number of molecular pathways controlling MZT in mice and humans are compared, pointing out those that could be at the origin of further focused experimental studies and the development of new diagnostic tools based on the translational medicine principles. Data concerning possible candidate molecules to be included in these studies are identified.

Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes.

Although it is not a well-established technology, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies in response to the growing demands of patients’ sociological and pathological conditions. Oocyte cryopreservation can adversely affect the developmental potential of oocytes by causing an increase in intracellular oxidative stresses and damage to the mitochondrial structure. In this study, we studied whether autologous adipose stem cell (ASC) mitochondria supplementation with vitrified and warmed oocytes could restore post-fertilization development that decreased due to mitochondrial damage following cryopreservation. ASC mitochondria showed similar morphology to oocytes’ mitochondria and had a higher ATP production capacity. The vitrified-warmed oocytes from juvenile mice were supplemented with ASC mitochondria at the same time as intracellular sperm injection (ICSI), after which we compared their developmental capacity and the mitochondria quality of 2-cell embryos. We found that, compared to their counterpart, mitochondria supplementation significantly improved development from 2-cell embryos to blastocysts (56.8% vs. 38.2%) and ATP production in 2-cell embryos (905.6 & 561.1 pmol), while reactive oxygen species levels were comparable. With these results, we propose that ASC mitochondria supplementation could restore the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach for mitochondrial transplantation therapy.

P-218 Mitochondria transfer from adipose stem cell ameliorates the development potential of cryopreserved oocytes

Abstract Study question Could autologous adipose stem cell (ASC) mitochondria supplementation restore the post-fertilization development potentialin vitrified-thawed oocytes? Summary answer Autologous ASC mitochondria supplementation could rejuvenate the quality of vitrified-thawed oocytes and enhance the embryo’s developmental capacity. What is known already Pre- and post-implantation developmental potential of embryos derived from vitrified oocytes are remarkedly lower than those of fresh oocytes. Despite its’ low efficacy, oocyte cryopreservation is becoming prevalent in assisted reproductive technologies to cater the growing demands due to patients' sociological and pathological conditions. Unfavorable effects to mitochondrial membrane potential, mitochondrial structure, ATP production, reactive oxygen species (ROS), meiotic spindle and microfilaments, and Ca2+ ion regulation are reported as detrimental damages in oocytes following the cryopreservation, that adversely affect the development potential. Furthermore, autologous stem cell mitochondria supplementation can rescue the aging-related oocyte mitochondrial damages. Study design, size, duration The mature oocytes, autologous ASC, and mitochondria were collected from young mice and analyzed at Osaka City University, Japan. In total, 600 young mouse mature oocytes were occupied in this prospective study. Participants/materials, setting, methods Young C57BL/6JJmsmouse (8weeks) mature oocytes that have been pre-vitrified were thawed before the start of the ICSI procedure.ASC specificity and ASC mitochondria function and ultrastructure were pre-analyzed. ASC mitochondria were isolated on the same day and supplemented with intracellular sperm injection (ICSI) and as the control, the mitochondrial buffer was injected. The survival rate, fertilization rate, blastulation, mitochondria function, ROS level in 2 cell embryos, and live birth rates were compared between the 2 groups. Main results and the role of chance The ASC mitochondriashowed higher membrane potential compared to the somatic cells and were spherical in shape with low cristae numbers. The survival rate and the fertilization ratewere comparable in both mitochondria supplemented and control groups. However, theASC mitochondria supplementation seemed to havesignificantly improvedthe blastocyst development capacity from 2cell embryos compared to the control group (P &amp;lt; 0.05;56.8% &amp; 38.2%, respectively).And interestingly, a significantly higher ATP level was found in the mitochondria supplemented group’s 2 cell embryos thanin the control group (P &amp;lt; 0.05;905.6pmol &amp; 561.1pmol respectively). And though it was not statistically significant, a higher potential ofgetting live birth was found in the mitochondria supplemented group thanthe control group after 2 cell embryo transfer. Limitations, reasons for caution We acknowledge that the absence of compared data with fresh oocytes’ ICSI, the detailed cellular mechanism behind theimprovement of embryo development, and transgenerational safety in offspring developed fromthe mitochondria supplementation werethe limitations of this study. Wider implications of the findings With these results, we propose that ASC mitochondria supplementation could rejuvenate the quality of cryopreserved oocytes and enhance the embryo developmental capacity, signifying another possible approach of mitochondrial transplantation therapy. Trial registration number not applicable

Developmental Competence of equine MI oocytes following in vitro maturation culture

With an increasing demand for the in vitro production (IVP) of equine embryos over the past 10 years, the value of each oocyte recovered has also increased. Intracytoplasmic sperm injection (ICSI) is performed to fertilize the oocytes reaching MII stage of nuclear maturation, which is determined by microscopic evidence of an extruded polar body (PB). Cytoplasmic maturation, during which replication of organelles, proteins and RNA's necessary for postfertilization development occurs, is more difficult to evaluate. However, equine oocytes contain a large amount of cytoplasmic lipids and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. Furthermore, the distribution of lipids may reflect a level of cytoplasmic capacitation and developmental competence, although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization (Ambruosi et al. Therio 2009; 71:1093-1104). The present study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can occur in the absence of PB extrusion. Cumulus-oocyte-complexes (COC; n=411) obtained from ovarian follicles by transvaginal aspiration were stored in a holding medium overnight at 20°C before undergoing incubation at 38.5°C in 50ul drops of equilibrated maturation media as described by Foss. et al (EVJ Supp 2013;45:39-43). After 28.5 hours, oocytes were denuded followingexposure to 80IU hyaluronidase and evaluated for the presence of an extruded PB as well as lipid reorganization. The cytoplasm of MI oocytes that did not exhibit PB extrusion was graded according to the percentage with a dark granular appearance. All oocytes were fertilized by conventional ICSI with thawed spermatozoa from ICSI-fertile stallions. Fertilized oocytes were cultured as described by Foss et al (EVJ Suppl 2013;45:39-43). When examined under an inverted 1000X light microscope, 66% (N=273/411) of the denuded oocytes exhibited an intact membrane with an extruded polar body and were considered mature (MII), while 26% (N=106/411) of the oocytes without a visible polar body were classified as immature (MI). Of the MI oocytes injected, 23% (N=24/106) were deemed to have attained some level of cytoplasmic capacitation (Cc=40-70% granularity). A cleavage rate of 25.5% was recorded for all injected MI oocytes regardless of Cc status. Of the 24 MI oocytes with some Cc, nine (37%) cleaved and continued to develop, and seven of those developed to the blastocyst stage. Of the 82 MI oocytes that did not demonstrate Cc, 18 (22%) cleaved but no blastocysts (0%) were obtained. This study found a blastocyst developmental potential of 6.6% per total MI's injected and 29% per MI oocytes with at least a 40-70% Cc. Therefore, injecting oocytes that do not show nuclear maturation, yet have reached a level of cytoplasmic capacitation, may still result in blastocyst development.

The role of growth hormone for fertility in women with hypopituitarism

Growth hormone (GH) is an important regulator of the female reproductive system. In vitro and non-human in vivo studies demonstrate a role of GH in steroidogenesis, folliculogenesis, and post-fertilization development. Given its ability to modulate the reproductive system and potentiate the effects of gonadotropins, a beneficial role of GH replacement therapy to optimize fertility has been suggested. Women with hypopituitarism have lower pregnancy and live birth rates. Limited data suggest a role of GH in enhancing fertility management in women with hypopituitarism. GH replacement therapy may be especially relevant in women with hypopituitarism as well as in women considered poor ovarian responders and require assisted reproductive techniques.

Hydrogen peroxide signalling mediates fertilization and post-fertilization development in the red alga Bostrychia moritziana.

Reactive oxygen species (ROS) signalling has a multitude of roles in cellular processes throughout biology. We hypothesized that red algal fertilization may offer an interesting model to study ROS-mediated signalling, as the stages of fertilization are complex and unique. We detected the localization of ROS production microscopically and monitored the expression of three homologues of NADPH oxidase in reproductive cells during fertilization. ROS were instantaneously produced by spermatia (sperm) when they attached to female trichogynes, diffused across the cell membrane in the form of H2O2, and triggered ROS generation in the carpogonium (egg) as well as carpogonial branch cells which are not in direct contact with spermatia. The expression of NADPH oxidase homologues, RESPIRATORY BURST OXIDASE HOMOLOGUES (BmRBOHs), began to be up-regulated in the female plant upon gamete binding, peaking during the fertilization process and descending back to their original level after fertilization. Pre-treatment with diphenylene iodonium or caffeine blocked gene expression as well as H2O2 production. Post-fertilization development was also inhibited when the redox state of the plants was perturbed with H2O2 at any time before or after the fertilization. Our results suggest that H2O2 acts as an auto-propagating signalling molecule, possibly through Ca2+ channel activation, and regulates gene expression in fertilization as well as post-fertilization development in red algae.

Air Pollution and Reproduction: A focus on the clinical perspective

BACKGROUND AND AIM: Air pollution is widely known to affect human cardiopulmonary health, but only recently has research begun to focus on understanding the association between ambient air pollution and reproduction. METHODS: In this 15-minute symposia presentation, I will provide a basic overview of relevant potential biological mechanisms after air pollution exposures on reproductive physiology. The physiology of human reproduction and the pathophysiology of infertility, clinical aspects in relation to outcome definitions, exposure assessment, and will be discussed using literature review and clinical experience. Key papers will be discussed within the clinical context of reproductive endocrinology and infertility with a focus on early embryo development and the uterine micro-environment. RESULTS:Human reproduction requires gametogenesis, fertilization, implantation, and post-fertilization fetal development. Ambient in-vitro and in-vivo embryo growth environments will be compared in relation to gaseous concentrations, including oxygen and carbon dioxide. Existing research on air pollution and reproduction will be discussed in relation to clinical aspects of gamete development and embryo culture environments. CONCLUSIONS:Air pollution exposures have been studied in the clinical fertility centers as well as at a population level. Air pollution exposures, broadly defined, have an impact on human reproduction, though much is still yet to be determined. Using a physiological basis, including embryo development and the uterine air pollution exposures and reproductive outcomes. KEYWORDS: air pollution, gametogenesis, fertilization, implantation, post-fertilization development

Does supplementation with mitochondria improve oocyte competence? A systematic review.

Mitochondrial supplementation was proposed as a complementary treatment to assisted reproductive technologies to improve oocyte competence and support post-fertilization development. This strategy is based on the fact that poor-quality/aged oocytes contain lower and dysfunctional mitochondria. However, the efficacy and safety of mitochondrial supplementation are still controversial. Therefore, this review summarizes the clinical/biological outcomes of mitochondrial supplementation, aiming to improve oocyte competence or explore the safety of this technique, and was based on an online search using PubMed and Web of Science, until September 2019. The studies included reported outcomes related to the efficacy and safety of mitochondrial supplementation either in human or animal models (bovine, porcine and mouse). Extracted data were organized according to study objective, the mitochondrial source and the main outcomes: fertilization/pregnancy rates, embryo development and adverse outcomes. Clinical pregnancy was not improved in the only randomized controlled trial published, although an increase was demonstrated in other non-randomized studies. Fertilization rate and embryo development were not different from control groups in the majority of studies, although performed in different contexts and using diverse sources of mitochondria. The safety of mitochondria transfer is still a concern, however, the euploid rate and the absence of reported congenital malformation from the clinical studies are reassuring. In summary, mitochondrial supplementation does not seem to cause harm although the benefit of improving oocyte competence is still unclear due to the diversity of methodological approaches and low-quality of the data available. Analyzed data support the need to investigate further, in both pre-clinical and clinical contexts.

418 Paternal Programming of Offspring Health

Abstract There is now a significant body of human and animal model data identifying common associations between perturbed development during gestation, disproportionate fetal growth and poor adult cardiovascular and metabolic health. Typically, these studies have focused on the impact of poor maternal health on offspring development and well-being. However, a growing evidence base now indicates that sub-optimal paternal factors (such as diet) can also program adult offspring ill-health. While the link between paternal health and offspring well-being is becoming established, attention is being focused on defining the underlying mechanism(s). Here, the father may influence post-fertilization development through two main pathways, the integrity and status of the sperm genome (including epigenome) and through components of the seminal plasma. We have used a mouse model of paternal low protein diet to study the impact sub-optimal nutrition has on the quality and epigenetic status of the sperm, embryonic development and fetal growth. We have also used our model to study the relative seminal plasma and sperm contributions to adult offspring cardiovascular and metabolic health. Our studied highlight the role of a father’s diet at the time of conception for the development and well-being of his offspring.

PI-Plat: a high-resolution image-based 3D reconstruction method to estimate growth dynamics of rice inflorescence traits

BackgroundRecent advances in image-based plant phenotyping have improved our capability to study vegetative stage growth dynamics. However, more complex agronomic traits such as inflorescence architecture (IA), which predominantly contributes to grain crop yield are more challenging to quantify and hence are relatively less explored. Previous efforts to estimate inflorescence-related traits using image-based phenotyping have been limited to destructive end-point measurements. Development of non-destructive inflorescence phenotyping platforms could accelerate the discovery of the phenotypic variation with respect to inflorescence dynamics and mapping of the underlying genes regulating critical yield components.ResultsThe major objective of this study is to evaluate post-fertilization development and growth dynamics of inflorescence at high spatial and temporal resolution in rice. For this, we developed the Panicle Imaging Platform (PI-Plat) to comprehend multi-dimensional features of IA in a non-destructive manner. We used 11 rice genotypes to capture multi-view images of primary panicle on weekly basis after the fertilization. These images were used to reconstruct a 3D point cloud of the panicle, which enabled us to extract digital traits such as voxel count and color intensity. We found that the voxel count of developing panicles is positively correlated with seed number and weight at maturity. The voxel count from developing panicles projected overall volumes that increased during the grain filling phase, wherein quantification of color intensity estimated the rate of panicle maturation. Our 3D based phenotyping solution showed superior performance compared to conventional 2D based approaches.ConclusionsFor harnessing the potential of the existing genetic resources, we need a comprehensive understanding of the genotype-to-phenotype relationship. Relatively low-cost sequencing platforms have facilitated high-throughput genotyping, while phenotyping, especially for complex traits, has posed major challenges for crop improvement. PI-Plat offers a low cost and high-resolution platform to phenotype inflorescence-related traits using 3D reconstruction-based approach. Further, the non-destructive nature of the platform facilitates analyses of the same panicle at multiple developmental time points, which can be utilized to explore the genetic variation for dynamic inflorescence traits in cereals.