Pronounced Trace Element Variation in Follicular Fluids of Subfertile Women Undergoing Assisted Reproduction

Female subfertility is a growing concern, especially in view of an increasing prevalence of polycystic ovary syndrome (PCOS). Assisted reproductive technologies (ART) offer a perspective for pregnancy, but the outcome rate is still suboptimal. The trace elements (TE), copper (Cu), selenium (Se), and zinc (Zn) are essential for fertility and development. We hypothesized that TE concentrations are related to oocyte quality and growth and affect pregnancy outcomes in women undergoing ART. Concentrations of TE were measured by total reflection X-ray fluorescence. Extracellular glutathione peroxidase 3 (GPX3) and selenoprotein P (SELENOP) were determined as additional Se biomarkers. Corresponding serum and follicular fluid (FF) samples were available from women with (n = 20) and without (n = 20) PCOS diagnosis undergoing hormone treatment within the ART procedure, respectively, and FF samples were classified into five groups based on morphological assessment. Serum showed higher TE concentrations than FF, and TE levels correlated positively between both matrices. Individual FF from the same women showed surprisingly high variability in TE concentration, and follicles without oocytes displayed the lowest TE concentrations. Both Se biomarkers GPX3 and SELENOP were present in FF and correlated positively to Se concentrations. Some notable relationships were observed between morphokinetic parameters, TE concentrations, and GPX3 activity. A slightly depressed serum Zn concentration was observed in PCOS. Our results indicate a direct relationship between TE in serum and FF, positive correlations between the three Se biomarkers in FF, and high variability between the FF from the same woman with the lowest TE concentrations in the follicles with the poorest quality. The differences observed in relation to PCOS diagnoses appear relatively minor. Collectively, the data support the notion that TE assessment of follicles may contribute to optimal oocyte selection and subsequently influence ART success.

Early Drosophila Oogenesis: A Tale of Centriolar Asymmetry

Among the morphological processes that characterize the early stages of Drosophila oogenesis, the dynamic of the centrioles deserves particular attention. We re-examined the architecture and the distribution of the centrioles within the germarium and early stages of the vitellarium. We found that most of the germ cell centrioles diverge from the canonical model and display notable variations in size. Moreover, duplication events were frequently observed within the germarium in the absence of DNA replication. Finally, we report the presence of an unusually long centriole that is first detected in the cystoblast and is always associated with the developing oocyte. This centriole is directly inherited after the asymmetric division of the germline stem cells and persists during the process of oocyte selection, thus already representing a marker for oocyte identification at the beginning of its formation and during the ensuing developmental stages.

Symmetry breaking in the female germline cyst

In mammals and flies, only a limited number of cells in a multicellular female germline cyst become oocytes, but how the oocyte is selected is unknown. Here we show that the microtubule minus end-stabilizing protein, Patronin/CAMSAP marks the future Drosophila oocyte and is required for oocyte specification. The spectraplakin, Shot, recruits Patronin to the fusome, a branched structure extending into all cyst cells. Patronin stabilizes more microtubules in the cell with most fusome and this weak asymmetry is amplified by Dynein-dependent transport of Patronin-stabilized microtubules. This forms a polarized microtubule network, along which Dynein transports oocyte determinants into the presumptive oocyte. Thus, Patronin amplifies a weak fusome anisotropy to break cyst symmetry. These findings reveal a molecular mechanism of oocyte selection in the germline cyst.

Mapping parameter spaces of biological switches

Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided our understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. Here, we use a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, we enumerate all phase portraits of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, we locate parameter regions that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches.

β-importins Tnpo-SR and Cadmus and the small GTPase Ran promote ovarian cyst formation in Drosophila

ABSTRACTGerm cells undergo mitotic expansion via incomplete cytokinesis, forming cysts of undifferentiated cells that remain interconnected prior to meiotic initiation, through mechanisms that are not well-defined. In somatic cells, Ras-related nuclear protein (Ran) spatiotemporally regulates mitotic spindle assembly, cleavage furrow formation and abscission. Here, we identify Ran and β-importins as critical regulators of cyst development in the Drosophila ovary. Depletion of Ran or the β-importins Tnpo-SR and cadmus disrupts oocyte selection and results in egg chambers with variable numbers of germ cells, suggesting abnormal cyst development and cyst fragmentation. We demonstrate that Ran, Tnpo-SR, and Cadmus regulate key cellular processes during cyst formation, including cell cycle dynamics, fusome biogenesis, and ring canal stability, yet do so independently of mitotic spindle assembly. Further, Tnpo-SR and Cadmus control cyclin accumulation and suppress cytokinesis independent of Ran-GTP, suggesting that β-importins sequester protein cargos that normally promote the mitotic-to-meiotic transition. Our data demonstrates that Ran and β-importins are critical for germ cell cyst formation, a role that is likely conserved in other organisms.SUMMARY STATEMENTRan and two β-importins function coordinately to promote oocyte selection and cyst development in the Drosophila ovary.

120 High variation in prediction of developmental competence of bovine oocytes based on visual examination

The quality of an oocyte is often represented by its morphological characteristics. Stringent selection of bovine cumulus–oocyte complexes (COCs) for invitro embryo production (IVP) is regularly based on parameters such as colour and granulation of the ooplasm, density of the surrounding cumulus cells, brightness of the zona pellucida, among other less common parameters. The link between developmental competence and morphology of the COC has been demonstrated in literature in group culture system. However, this has not been yet examined in an individual invitro production system. This study investigates the ability of IVP researchers to predict whether an oocyte will develop into a blastocyst at Day 8 of invitro culture in an individual production system. A set of 29 pictures of immature bovine COCs was presented in duplicates to eight bovine IVP researchers with different institutional backgrounds. The observers were asked if they would select each oocyte for further IVP processing, assuming that only the oocytes with the highest developmental potential can proceed, and thus a stringent selection is warranted. These 29 immature oocytes were matured, fertilized, and cultured individually invitro (using conventional methods) so that their ability to develop into a blastocyst at Day 8 post-fertilization was known. The ability to reach the blastocyst stage was stated as the gold standard and compared with the answers of the observers using kappa statistics and sensitivity (Se) and specificity (Sp) tests. Kappa (κ) for interobserver agreement was κ=0.01 (Se=50%, Sp=53%; positive predictive value=14%, negative predictive value=87%) with 50% accuracy of prediction. The intraobserver agreement was κ=0.69, with κ=1 referring to 100% agreement (Se=87%, Sp=83%), with an accuracy of 85%. The ability of trained embryologists to predict the fate of oocytes’ developmental competence was accurate for only half of the presented oocytes. Additionally, the proportion of false positive (1 − Sp) and false negative (1−Se) predictions was equally distributed. The low positive predictive value might be due to the lower development rates in individual culture systems versus group culture systems, whereas observers are mainly trained to select oocytes for group culture. This study demonstrated the subjective nature of the oocyte selection process that is based on visual examination of COC morphology because interobserver agreement was almost nonexistent. Interpretation of the morphology within an observer remains consequent, represented by the high intraobserver agreement. Consequently, there is a need to develop a new model for the objective determination of oocyte quality for individual IVP. The implementation of machine learning algorithms could objectively enhance oocyte selection and artificial reproductive technologies in extension.

Expression pattern of olfactory receptor genes in human cumulus cells as an indicator for competent oocyte selection

Odorant or olfactory receptors are mainly localized in the olfactory epithelium for the perception of different odors. Interestingly, many ectopic olfactory receptors with low expression levels have recently been found in nonolfactory tissues to involve in local functions. Therefore, we investigated the probable role of the olfactory signaling pathway in the surrounding microenvironment of oocyte. This study included 22 women in intracytoplasmic sperm injection cycle. The expression of olfactory target molecules in cumulus cells surrounding the growing and mature oocytes was evaluated by Western blotting and real-time polymerase chain reaction. Additionally, integrated bioinformatics analyses were carried out and 6 ectopic olfactory receptors were selected for further evaluation. The initiation of olfactory transduction cascade in cumulus cells of competent oocytes was confirmed by analyzing the expression of adenylyl cyclase type 3 and olfactory market protein. Moreover, the expression pattern of the selected olfactory receptors was evaluated and OR10H2 was selected due to a high level of expression in mature fertile oocytes. We suggested that OR10H2 could be considered as a reliable biomarker for oocyte selection in assisted reproduction technique programs. However, further studies are required to elucidate the role of olfactory transduction cascade in embryo quality and implantation.

Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development

SummaryThe aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

178 Evaluation of polar body extrusion following exposure of cattle oocytes to different concentrations of ethylene glycol cryoprotectant

Cryoprotectant (CPA) toxicity has been recognised as a critical barrier to further advancement of cattle oocytes. Furthermore, CPA at high concentration might be toxic to oocytes, leading to osmotic shock and cell death (Arav et al. 1993). Selection of quality oocytes among a heterogeneous pool is mostly done subjectively. The use of Brilliant Cresyl Blue (BCB) stain for oocyte selection before ethylene glycol (EG) CPA toxicity test on cattle oocytes might be a useful tool. The objectives of this study were to elucidate the toxicity of EG penetrating CPA to cattle oocytes and the effectiveness of BCB on oocyte selection. Ovaries from cows of unknown reproductive status were collected from the local slaughterhouse and transported in warmed (37°C) saline water to the laboratory within 2h of slaughter. The oocytes (n=374) were exposed to 26mM BCB for 90min at 38.5°C under an atmosphere of 5% CO2. The other oocytes (n=450) were not exposed to BCB solution or CPA (positive control: no BCB and no CPA exposure). Oocytes were classified as BCB positive (+) with a varying degree of blue cytoplasm or BCB negative (−) with no blue cytoplasm. Oocytes were exposed in EG at different CPA concentrations as follows: Toxicity test 1 (TT1) was 0, 5, 10, and 15%, followed by exposure to TT2 as follows: 10, 20, and 30% (stepwise increased CPA). The oocytes were then invitro matured (IVM) as per treatment groups for 22h. After maturation, oocytes were removed from the maturation medium and denuded of granulosa cells by vortexing. The polar body extrusion was evaluated with the aid of Oosight Imaging System (Hamilton Thorne) connected to an inverted research microscope. Treatment means were compared using Fisher protected t-test least significant difference. There was a drastic decline of oocytes with polar body extrusion as CPA concentration increased (P<0.05): 40.5% (positive control, no BCB and no CPA exposure), 33.3% [(control, CPA exposure (EG 5 + 10%)], 23.1% [BCB− with CPA toxicity test (EG 5 + 10%)], 12.5% [(BCB− with CPA toxicity test (EG 10 + 20%)] and 4.9% [(BCB− with CPA toxicity test (EG 15 + 30%)]. The BCB+ groups (EG 5 + 10% and EG 10 + 20%) had significantly more oocytes with polar body extrusion (68.9% and 51.9%) compared with the positive control (40.5%), respectively (P<0.05). In conclusion, higher EG cryoprotectant concentrations compromise oocyte polar body extrusion following IVM. We recommend that BCB be used for selection of suitable oocytes before the CPA toxicity test because of its ability to stain larger and more competent oocytes from cattle.