The impact of low oocyte maturity ratio on blastocyst euploidy rate: a matched retrospective cohort study.

OBJECTIVE: To investigate the association between a low oocyte maturity ratio from in vitro fertilization cycle and blastocyst euploidy. METHODS: A total of 563 preimplantation genetic testing (PGT) cycles (PGT cycles with chromosomal structural rearrangements were excluded) were performed between January 2021 and November 2022 at our center (average oocyte maturity rate: 86.4% ± 14.6%). Among them, 93 PGT cycles were classified into the low oocyte maturity rate group (group A, < mean - 1 standard deviation [SD]), and 186 PGT cycles were grouped into the average oocyte maturity rate group (group B, mean ± 1 SD). Group B was 2:1 matched with group A. Embryological, blastocyst ploidy, and clinical outcomes were compared between the two groups. RESULTS: The oocyte maturity (metaphase II [MII oocytes]), MII oocyte rate, and two pronuclei (2PN) rates were significantly lower in group A than in group B (5.2 ± 3.0 vs. 8.9 ± 5.0, P = 0.000; 61.6% vs. 93.0%, P = 0.000; 78.7% vs. 84.8%, P = 0.002, respectively). In group A, 106 of 236 blastocysts (44.9%) that underwent PGT for aneuploidy were euploid, which was not significantly different from the rate in group B (336/729, 46.1%, P = 0.753). However, euploid blastocysts were obtained only in 55 cycles in group A (55/93, 59.1%), which was lower than the rate in group B (145/186, 78.0%, P = 0.001). The clinical pregnancy rate in group B (73.9%) was higher than that in group A (58.0%) (P = 0.040). CONCLUSION: Our results suggest that a low oocyte maturity ratio is not associated with blastocyst euploidy but is associated with fewer cycles with euploid blastocysts for transfer, lower 2PN rates, and lower clinical pregnancy rates.

Propylparaben exposure impairs G2/M and metaphase-anaphase transition during mouse oocyte maturation.

Propylparaben (PrPB) is a known endocrine disrupting chemicals that is widely applied as preservative in pharmaceuticals, food and cosmetics. PrPB has been detected in human urine samples and human serum and has been proven to cause functional decline in reproduction. However, the direct effects of PrPB on mammalian oocyte are still unknown. Here, we demonstrationed that exposure to PrPB disturbed mouse oocyte maturation in vitro, causing meiotic resumption arrest and first polar body extrusion failure. Our results indicated that 600 µM PrPB reduced the rate of oocyte germinal vesicle breakdown (GVBD). Further research revealed that PrPB caused mitochondrial dysfunction and oxidative stress, which led to oocyte DNA damage. This damage further disturbed the activity of the maturation promoting factor (MPF) complex Cyclin B1/ Cyclin-dependent kinase 1 (CDK1) and induced G2/M arrest. Subsequent experiments revealed that PrPB exposure can lead to spindle morphology disorder and chromosome misalignment due to unstable microtubules. In addition, PrPB adversely affected the attachment between microtubules and kinetochore, resulting in persistent activation of BUB3 amd BubR1, which are two spindle-assembly checkpoint (SAC) protein. Taken together, our studies indicated that PrPB damaged mouse oocyte maturation via disrupting MPF related G2/M transition and SAC depended metaphase-anaphase transition.

G-Banding and Molecular Cytogenetics Detect Novel Translocations and Cryptic Aberrations in Human Immortal Endothelial Cells.

Endothelial cells (ECs) maintain vessel tone and barrier integrity, regulate blood homeostasis, and prevent the extravasation of leukocytes under normal physiological conditions. Because of the limited lifespans and batch-to-batch differences with respect to the genetic make-up of primary ECs, established immortal EC lines are extensively used for studying endothelial biology. To address this issue, the immortal endothelial cell line EA.hy926 was developed by fusing primary human umbilical vein endothelial cells (HUVECs) with human lung carcinoma A549 cells. EA.hy926 cells share a number of similar endothelial properties with HUVECs and are considered the immortal counterpart to primary HUVECs. However, the cytogenetic integrity of EA.hy926 cells is not fully elucidated. We characterized EA.hy926 cells with conventional G-banding and molecular cytogenetic techniques such as spectral karyotyping and subtelomeric fluorescence in situ hybridization. Cytogenetic analysis revealed an array of numerical and stable structural chromosomal rearrangements including one deletion, one duplication, one isochromosome, seven simple translocations, and five complex translocations in Ea.hy926 cells. These findings will advance comprehension of EA.hy926 cell biology and augment future endothelial studies, specifically in comparison studies between HUVECs and EA.hy926 cells.

Plasticity of the mitotic spindle in response to karyotype variation.

Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.

Mechanics of spindle orientation in human mitotic cells is determined by pulling forces on astral microtubules and clustering of cortical dynein.

The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle's measured resistance to rotation, the speed at which it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ∼5 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle's resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering.

Optimization of Fluorescence In Situ Hybridization Protocols in the Era of Precision Medicine.

Fluorescence in situ hybridization (FISH) is a cytogenetic assay that is widely used in both clinical and research settings to validate genetic aberrations. Simple in principle, it is based on denaturation and hybridization of a DNA probe and its complementary sequence; however, it is subject to continuous optimization. Here we share how in-house FISH can be optimized using different control tissues to visualize and ultimately validate common and novel genetic abnormalities unearthed by next-generation sequencing (NGS). Seven specific FISH probes were designed and labeled, and conditions for eight tissue types and one patient-derived tumor organoid were optimized. Formalin-fixed paraffin-embedded (FFPE) tissue slides were used for each experiment. Slides were first deparaffinized, then placed in a pretreatment solution followed by a digestion step. In-house FISH probes were then added to the tissue to be denatured and hybridized, and then washed twice. To obtain optimal results, probe concentration, pepsin incubation time, denaturation, and the two post-hybridization washes were optimized for each sample. By modifying the above conditions, all FISH experiments were optimized in separate tissue types to investigate specific genomic alterations in tumors arising in those tissues. Signals were clear and distinct, allowing for visualization of the selected probes. Following this protocol, our lab has quickly optimized 11 directly labeled in-house FISH probes to support genetic aberrations nominated by NGS, including most recent discoveries through whole-genome sequencing analyses. We describe a robust approach of how to advance in-house labeled FISH probes. By following these guidelines, reliable and reproducible FISH results can be obtained to interrogate FFPE slides from benign, tumor tissues, and patient-derived tumor organoid specimens. This is of most relevance in the era of NGS and precision oncology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Metaphase FISH optimization Support Protocol 1: In-house probe labeling and preparation Support Protocol 2: Metaphase spread preparation Basic Protocol 2: Optimization of FISH on formalin-fixed paraffin-embedded tissue.

Effects of flaxseed oil supplementation on metaphase II oocyte rates in IVF cycles with decreased ovarian reserve: a randomized controlled trial.

Background: This study was designed to explore the effects of flaxseed oil on the metaphase II (MII) oocyte rates in women with decreased ovarian reserve (DOR). Methods: The women with DOR were divided into a study group (n = 108, flaxseed oil treatment) and a control group (n = 110, no treatment). All patients were treated with assisted reproductive technology (ART). Subsequently, the ART stimulation cycle parameters, embryo transfer (ET) results, and clinical reproductive outcomes were recorded. The influencing factors affecting the MII oocyte rate were analyzed using univariate analysis and multivariate analysis. Results: Flaxseed oil reduced the recombinant human follicle-stimulating hormone (r-hFSH) dosage and stimulation time and increased the peak estradiol (E2) concentration in DOR women during ART treatment. The MII oocyte rate, fertilization rate, cleavage rate, high-quality embryo rate, and blastocyst formation rate were increased after flaxseed oil intervention. The embryo implantation rate of the study group was higher than that of the control group (p = 0.05). Additionally, the female age [odds ratio (OR): 0.609, 95% confidence interval (CI): 0.52-0.72, p < 0.01] was the hindering factor of MII oocyte rate, while anti-Müllerian hormone (AMH; OR: 100, 95% CI: 20.31-495, p < 0.01), peak E2 concentration (OR: 1.00, 95% CI: 1.00-1.00, p = 0.01), and the intake of flaxseed oil (OR: 2.51, 95% CI: 1.06-5.93, p = 0.04) were the promoting factors for MII oocyte rate. Conclusion: Flaxseed oil improved ovarian response and the quality of oocytes and embryos, thereby increasing the fertilization rate and high-quality embryo rate in DOR patients. The use of flaxseed oil was positively correlated with MII oocyte rate in women with DOR. Clinical trial number: https://www.chictr.org.cn/, identifier ChiCTR2300073785.

Qualitative rather than quantitative phosphoregulation shapes the end of meiosis I in budding yeast.

Exit from mitosis is brought about by dramatic changes in the phosphoproteome landscape. A drop in Cyclin-dependent kinase (Cdk) activity, the master regulatory kinase, and activation of counteracting phosphatases such as Cdc14 in budding yeast, results in ordered substrate dephosphorylation, allowing entry into a new cell cycle and replication licensing. In meiosis however, two cell divisions have to be executed without intermediate DNA replication, implying that global phosphorylation and dephosphorylation have to be adapted to the challenges of meiosis. Using a global time-resolved phosphoproteomics approach in budding yeast, we compared the phosphoproteome landscape between mitotic exit and the transition from meiosis I to meiosis II. We found that unlike exit from mitosis, Cdk phosphomotifs remain mostly stably phosphorylated at the end of meiosis I, whereas a majority of Cdk-unrelated motifs are reset by dephosphorylation. However, inducing an artificial drop of Cdk at metaphase of meiosis I leads to ordered substrate dephosphorylation, comparable to mitosis, indicating that phosphoregulation of substrates at the end of meiosis I is thus mainly qualitatively rather than quantitatively ordered.

Identification and molecular characterization of genes modulating progression of an oocyte from M-I to M-II in rat ovary.

BACKGROUND: To achieve oocyte competence for successful fertilization, bidirectional communication between oocyte and granulosa cells is crucial. The acquisition of meiotic competency in oocyte is facilitated by various regulatory genes however, expression pattern of these genes is not well documented during meiotic transition from Metaphase-I to Metaphase-II stage. Therefore, the present research analyzed the expression pattern of regulatory genes that are involved in the transition from M-I to M-II stages in rat oocyte. METHODS: The analysis of the data was conducted by applying an array of bioinformatic tools. The investigation of gene group interactions was carried out by employing the STRING database, which relies on co-expression information. The gene ontology (GO) analysis was performed utilizing the comparative GO database. Functional annotation for GO and pathway enrichment analysis were performed for genes involved in networking. The GO obtained through computational simulations was subsequently validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. RESULTS: The findings of our study suggest that there is a distinct gene expression pattern in both the oocyte and granulosa cells. This pattern indicates that oocyte-secreted factors, such as BMP15 and GDF9, play a crucial role in regulating the progression of the meiotic cell cycle from the M-I to M-II stages. We have also examined the level of mRNA expression of genes including CYP11A1, CYP19A1, and STAR, which are crucial for the steroidogenesis. CONCLUSIONS: It is fascinating to observe that the oscillatory pattern of specific key genes may hold significance in the process of in vitro oocyte maturation, specifically during the transition from the M-I to M-II stage. It might be useful for determining biomarker genes and potential pathways that play a role in attaining oocyte competency, thereby aiding in the assessment of oocyte quality for the purpose of achieving successful fertilization.

PAK1-Dependent Regulation of Microtubule Organization and Spindle Migration Is Essential for the Metaphase I-Metaphase II Transition in Porcine Oocytes.

P21-activated kinase 1 (PAK1) is a critical downstream target that mediates the effect of small Rho GTPase on the regulation of cytoskeletal kinetics, cell proliferation, and cell migration. PAK1 has been identified as a crucial regulator of spindle assembly during the first meiotic division; however, its roles during the metaphase I (MI) to metaphase II (MII) transition in oocytes remain unclear. In the present study, the potential function of PAK1 in regulating microtubule organization and spindle positioning during the MI-MII transition was addressed in porcine oocytes. The results showed that activated PAK1 was co-localized with α-tubulin, and its expression was increased from the MI to MII stage (p < 0.001). However, inhibiting PAK1 activity with an inhibitor targeting PAK1 activation-3 (IPA-3) at the MI stage decreased the first polar body (PB1) extrusion rate (p < 0.05), with most oocytes arrested at the anaphase-telophase (ATI) stage. IPA-3-treated oocytes displayed a decrease in actin distribution in the plasma membrane (p < 0.001) and an increase in the rate of defects in MII spindle reassembly with abnormal spindle positioning (p < 0.001). Nevertheless, these adverse effects of IPA-3 on oocytes were reversed when the disulfide bond between PAK1 and IPA-3 was reduced by dithiothreitol (DTT). Co-immunoprecipitation revealed that PAK1 could recruit activated Aurora A and transform acidic coiled-coil 3 (TACC3) to regulate spindle assembly and interact with LIM kinase 1 (LIMK1) to facilitate actin filament-mediated spindle migration. Together, PAK1 is essential for microtubule organization and spindle migration during the MI-MII transition in porcine oocytes, which is associated with the activity of p-Aurora A, p-TACC3 and p-LIMK1.

Neocortical neurogenesis in development and evolution-Human-specific features.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

An artificial intelligence-based approach for selecting the optimal day for triggering in antagonist protocol cycles.

RESEARCH QUESTION: Can a machine-learning model suggest an optimal trigger day (or days), analysing three consecutive days, to maximize the number of total and mature (metaphase II [MII]) oocytes retrieved during an antagonist protocol cycle? DESIGN: This retrospective cohort study included 9622 antagonist cycles between 2018 and 2022. The dataset was divided into training, validation and test sets. An XGBoost machine-learning algorithm, based on the cycles' data, suggested optimal trigger days for maximizing the number of MII oocytes retrieved by considering the MII predictions, prediction errors and outlier detection results. Evaluation of the algorithm was conducted using a test dataset including three quality groups: 'Freeze-all oocytes', 'Fertilize-all' and 'ICSI-only' cycles. The model suggested 1, 2 or 3 days as trigger options, depending on the difference in potential outcomes. The suggested days were compared with the actual trigger day chosen by the physician and were labelled 'concordant' or 'discordant' in terms of agreement. RESULTS: In the 'freeze-all' test-set, the concordant group showed an average increase of 4.8 oocytes and 3.4 MII oocytes. In the 'ICSI-only' test set there was an average increase of 3.8 MII oocytes and 1.1 embryos, and in the 'fertilize-all' test set an average increase of 3.6 oocytes and 0.9 embryos was observed (P < 0.001 for all parameters in all groups). CONCLUSIONS: Utilizing a machine-learning model for determining the optimal trigger days may improve antagonist protocol cycle outcomes across all age groups in freeze-all or fresh transfer cycles. Implementation of these models may more accurately predict the number of oocytes retrieved, thus optimizing physicians' decisions, balancing workloads and creating more standardized, yet patient-specific, protocols.

Metaphase chromosomes of five Neotropical species of the genus Drosophila (Diptera, Drosophilidae).

The mitotic metaphases of five Andean species of genus Drosophila are described for the first time. The evolutionary and interspecific genetic relationships within three Neotropical Drosophila species groups are analyzed. The diploid chromosome number for each species is as follows: D.cashapamba Céspedes et Rafael, 2012 2n = 6 (2V, 1J) (X = J, Y = R), D.ecuatoriana Vela et Rafael, 2004 2n = 10 (3R, 2V) (X = V, Y = R), D.ninarumi Vela et Rafael, 2005 2n = 10 (3R, 1V, 1D) (X = V, Y = R), D.urcu Vela et Rafael, 2005 2n = 12 (4R, 2V) (X = V, Y = R), D.valenteae Llangarí-Arizo et Rafael, 2018 2n = 8 (3R, 1J) (X = J, Y = R).

Alloprevotella Can be Considered as a Potential Oral Biomarker in Intestinal Metaphase of Gastric Patients.

Gastric cancer is a malignant tumor with high incidence and death rate. Every year, Approximately 950,000 new cases of gastric cancer occur globally with nearly 700000 deaths,so gastric precancerous lesions(GPL) was crucial and important.At present, the effective diagnostic methods for gastric precancerous lesions are generally gastroscope and pathological changes of gastric mucosal, but those methods were invasive and would bring some pains to patients and not suitable for frequent and large-scale screening of gastric cancer or GPL.This study aimed to look for a sensitive,effective and non-invasive diagnostic method to improve the early diagnosis rate of GLP, and thereby reduce the incidence and death rate of gastric cancer.Tongue diagnosis is one of the classic diagnostic methods in traditional Chinese medicine(TCM).The tongue was closely related to the spleen and stomach.In the study, we collected 133 patients with chronic gastritis, including 53 cases in inflammatory group, 31 cases in atrophic group, and 49 cases in intestinal metaplasia group. and we analyzed the correlation between tongue,microbiota of tongue coating and clinical symptoms of GLP.The results showed that greasy coating was closely related to the intestinal metaphase of patients, indicating that greasy coating was closed link with intestinal metaphase phase of patients.Abundance of 209 genus were significant differences between greasy and non-greasy coating in intestinal metaphase phase of patients, Top10 were Streptococcus,norank_p__Saccharibacteria,Alloprevotella, Atopobium, Megasphaera, Gemella, Moraxella,unclassified_f__Prevotellaceae, Solobacterium and Stomatobaculum. Alloprevotella and Streptococcus were important genus markers and Alloprevotella was selected as a potential oral biomarker to diagnose intestinal metaphase phase of patients, the AUC value is 0.74.

Helical chromonema coiling is conserved in eukaryotes.

Efficient chromatin condensation is required to transport chromosomes during mitosis and meiosis, forming daughter cells. While it is well accepted that these processes follow fundamental rules, there has been a controversial debate for more than 140 years on whether the higher-order chromatin organization in chromosomes is evolutionarily conserved. Here, we summarize historical and recent investigations based on classical and modern methods. In particular, classical light microscopy observations based on living, fixed, and treated chromosomes covering a wide range of plant and animal species, and even in single-cell eukaryotes suggest that the chromatids of large chromosomes are formed by a coiled chromatin thread, named the chromonema. More recently, these findings were confirmed by electron and super-resolution microscopy, oligo-FISH, molecular interaction data, and polymer simulation. Altogether, we describe common and divergent features of coiled chromonemata in different species. We hypothesize that chromonema coiling in large chromosomes is a fundamental feature established early during the evolution of eukaryotes to handle increasing genome sizes.

Cytogenetic profile of Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia in Northern Pakistan.

Objective: To determine the frequencies of different cytogenetic abnormalities in patients of Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia in Northern Pakistan. Methods: It was descriptive cross-sectional study conducted in Hematology Department of a Tertiary care referral institute from June 2015 to July 2017. All newly diagnosed cases of Acute Leukemia were analyzed. Cytogenetic analysis was performed on bone marrow aspirate samples using Giemsa-trypsin banding technique. Karyotypes were identified and interpreted according to ISCN criteria. Results: A total of 355 newly diagnosed patients of Acute Leukemia were analyzed. Out of these, 180 patients had AML and 175 had ALL. In Acute Myeloid Leukemia chromosomal abnormalities were detected in 28.2 % cases. Of these the common ones included t(8;21),t(15;17),+8, Inversion 16 and Monosomy 7. Other abnormalities included Complex karyotype, Down's syndrome related AML, Hyperdiploidy, del 16q,-8,+Y and t(3p;17q)del 10. In Acute Lymphoblastic Leukemia chromosomal abnormalities were detected in 40% cases. Common ones included Hyperdiploidy, Tetraploidy and t(9;22). Other abnormalities included t(1;19) and t(2;8)t(8;14). Conclusion: Cytogenetically favorable abnormalities are commonest occurring chromosomal defects in both Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia in Northern Pakistan, i.e., t(8;21) in AML and Hyperdiploidy in ALL.

Clustering of cortical dynein regulates the mechanics of spindle orientation in human mitotic cells.

The forces which orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle's measured resistance to rotation, the speed it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ~1 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle's resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering.

Chemoprevention curcumin analog 1.1 promotes metaphase arrest and enhances intracellular reactive oxygen species levels on TNBC MDA-MB-231 and HER2-positive HCC1954 cells.

Background and purpose: Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By utilizing immortalized MDA-MB-231 and HCC1954 cells, we evaluated the anticancer properties of CCA-1.1 and its mediated activity to promote cellular death. Experimental approach: Cytotoxicity and anti-proliferation were assayed using trypan blue exclusion. The cell cycle profile after CCA-1.1 treatment was established through flow cytometry. May-Grünwald-Giemsa and Hoechst staining were performed to determine the cell cycle arrest upon CCA-1.1 treatment. The involvement of CCA-1.1 in mitotic kinases (aurora A, p-aurora A, p-PLK1, and p-cyclin B1) expression was investigated by immunoblotting. CCA-1.1-treated cells were stained with the X-gal solution to examine the effect on senescence. ROS level and mitochondrial respiration were assessed by DCFDA assay and mitochondrial oxygen consumption rate, respectively. Findings/Results: CCA-1.1 exerted cytotoxic activity and inhibited cell proliferation with an irreversible effect, and the flow cytometry analysis demonstrated that CCA-1.1 significantly halted during the G2/M phase, and further assessment revealed that CCA-1.1 caused metaphase arrest. Immunoblot assays confirmed CCA-1.1 suppressed aurora A kinase in MDA-MB-231 cells. The ROS level was elevated after treatment with CCA-1.1, which might promote cellular senescence and suppress basal mitochondrial respiration in MDA-MB-231 cells. Conclusion and implications: Our data suggested the in vitro proof-of-concept that supports the involvement in cell cycle regulation and ROS generation as contributors to the effectiveness of CCA-1.1 in suppressing breast cancer cell growth.

Predicting nuclear maturation speed of oocytes from Japanese Black beef heifers through non-invasive observations during IVM: An attempt using machine learning algorithms.

Nuclear maturation is an essential process in which oocytes acquire the competence to develop further. However, the time required for nuclear maturation during IVM varies among oocytes. Therefore, predicting nuclear maturation speed (NMS) could help identify the optimal timing for IVF and maximize the developmental competence of each oocyte. This study aimed to establish machine learning-based prediction models for NMS using non-invasive indicators during the individual IVM of Japanese Black (JB) beef heifer oocytes. We collected ovaries from abattoirs and aspirated cumulus-oocyte complexes (COCs) from follicles with diameters ranging between 2 and 8 mm. The COCs were matured individually for 18 h, and photographs of each COC were taken at the beginning and every 3 h from 12 h to the end of maturation. After IVM culture, we denuded COCs and stained oocytes to confirm the progression of meiosis. Only oocytes that reached the metaphase II (MII) stage were considered to have a fast NMS. Morphological features, including COC area, cumulus expansion ratio, expansion rate per hour, and expansion pattern, were extracted from the recorded photos and applied to develop prediction models for NMS using machine learning algorithms. The MII rates of oocytes with fast- and slow-predicted NMS differed when the decision tree (DT) and random forest (RF) models were employed (P < 0.05). To evaluate the relationship between predicted NMS by DT and RF models and fertilization dynamics during individual IVF, sperm penetration and pronuclear formation were evaluated at 3, 6, 9, and 12 h after IVF start, following 24 h of IVM. The association between predicted NMS and embryo development was investigated by performing IVC for seven days using microwell culture dishes after 24 h of IVM and 6 h of IVF. Predicted NMS did not show a significant association with fertilization dynamics. However, oocytes with fast-predicted NMS by the RF model exhibited a tendency towards a higher cleavage rate 48 h after IVF start (P = 0.08); no other relationship was found between predicted NMS and embryo development. These findings demonstrate the feasibility of using non-invasive indicators during IVM to develop prediction models for NMS of JB beef heifer oocytes. Although the effect of predicted NMS on embryo development remains unclear, customized treatments based on NMS predictions have the potential to improve the efficiency of in vitro embryo production following individual IVM culture.

Establishment of Primary Adult Skin Fibroblast Cell Lines from African Savanna Elephants (Loxodonta africana).

Following population declines of the African savanna elephant (Loxodonta africana) across the African continent, the establishment of primary cell lines of endangered wildlife species is paramount for the preservation of their genetic resources. In addition, it allows molecular and functional studies on the cancer suppression mechanisms of elephants, which have previously been linked to a redundancy of tumor suppressor gene TP53. This methodology describes the establishment of primary elephant dermal fibroblast (EDF) cell lines from skin punch biopsy samples (diameter: ±4 mm) of African savanna elephants (n = 4, 14-35 years). The applied tissue collection technique is minimally invasive and paves the way for future remote biopsy darting. On average, the first explant outgrowth was observed after 15.75 ± 6.30 days. The average doubling time (Td) was 93.02 ± 16.94 h and 52.39 ± 0.46 h at passage 1 and 4, respectively. Metaphase spreads confirmed the diploid number of 56 chromosomes. The successful establishment of EDF cell lines allows for future elephant cell characterization studies and for research on the cancer resistance mechanisms of elephants, which can be harnessed for human cancer prevention and treatment and contributes to the conservation of their genetic material.