THE ROLE OF CYTOCHROME P450 ISOFORMS OF HEPATOCYTE ENDOPLASMIC RETICULUM IN ETHANOL METABOLISM

Background. Three metabolic pathways that can function simultaneously are known to be involved in ethanol oxidation in the liver: alcohol dehydrogenase pathway, microsomal ethanol-oxidizing system, and catalase pathway. Though the cytochrome P450-dependent microsomal ethanol-oxidizing system plays an insignificant role in metabolism of small amounts of ethanol, it is induced in case of ethanol excess and becomes essential when ethanol is abused. The main components of this system are cytochrome P450 (CYP) isoforms of smooth endoplasmic reticulum. Objective. To characterize the role of the key isoforms of cytochrome P450 in ethanol oxidation. Material and methods. We carried out an analysis of modern literature data on the role of the main isoforms of cytochrome P450 in liver metabolism of ethanol. Results. Data on the primary role of cytochrome CYP2E1 in ethanol metabolism, as well as on the contribution of isoforms CYP1A2, CYP2B1/2, CYP2C, CYP3A4, CYP4B1 to ethanol oxidation are presented. Conclusions. Ethanol is metabolized by many CYPs of endoplasmic reticulum of hepatocytes. The importance of CYP in biotransformation processes in the liver necessitates the study of the role of individual CYP isoforms in ethanol metabolism for predicting changes in the pharmacokinetics of drugs and metabolism of endogenous compounds under the influence of ethanol.

Smooth Endoplasmic Reticulum Clusters in Oocytes From Patients Who Received Intracytoplasmic Sperm Injections Negatively Affect Blastocyst Quality and Speed of Blastocyst Development

Findings regarding the relationship between smooth endoplasmic reticulum clusters (SERCs) in oocytes and blastocyst development have been conflicting. In this study, the effects of SERCs on blastocyst quality and the speed of blastocyst development were evaluated. Patients who received intracytoplasmic sperm injections (ICSI) at our reproductive center from 2016 to 2020 were retrospectively analyzed. SERC (+) oocytes (n = 217) and SERC (–) oocytes (n = 822), as well as SERC (+) cycles (n = 146) and SERC (–) cycles (n = 1,951) were compared. There was no significant difference in embryological, clinical, and neonatal outcomes between the SERC (+) and SERC (–) cycles. The fertilization rate (73.9%), good quality blastocyst rate (26.7%) and the speed of blastocyst development (44.4%) were significantly lower (P < 0.05) in SERC (+) oocytes than in unaffected counterparts (86.2%, 44.1% and 63.4%, respectively). Furthermore, the proportion of blastocysts with trophectoderm (TE) grade C was significantly higher in the SERC (+) oocyte group than in the SERC (–) oocyte group (73.3 vs. 55.9%, P < 0.05). After adjusting for age, years of infertility, endometriosis, stimulation protocols (GnRHa), and male infertility, multiple logistic regression analysis revealed that the presence of SERCs in the oocytes significantly affected the speed of blastocyst development (odds ratio, 2.812; 95% CI, 1.257–6.292; P = 0.012). These findings suggest that the presence of SERCs in oocytes may negatively affect blastocyst quality and the speed of blastocyst development.

Histological and Ultrastructural Characterization of the Gonads of the Grunting Toadfish Allenbatrachus grunniens (Linnaeus, 1758) from the Pranburi River Estuary, Thailand

Information on the reproductive biology of toadfish remains limited. In this study, we examined the structure and development of gonads in the grunting toadfish Allenbatrachus grunniens (Linnaeus, 1758) using morphological, histological and ultrastructural methods. The fish were collected from the Pranburi River Estuary, Thailand, during the annual reproductive period for this species (January to December 2018). The ovary of this species was paired and had elongated sac-like structures parallel to the kidneys and the digestive tract. In females, we identified 4 oocyte differentiation phases in the ovary including oogonia proliferation phase, a primary growth phase that was further classified into 2 steps (perinucleolar and oil droplets-cortical alveolar steps), and a secondary growth phase that also contained 2 steps (secondary growth and full-grown oocyte steps) and post-ovulatory phases, indicating an asynchronous pattern in ovarian development for this species. Transmission electron microscopy showed the 4 layers including the zona pellucida, basement membrane, granulosa cells and theca cells, all of which initially appeared in the oil droplets-cortical alveolar stage. The zona pellucida and the granulosa cells were highly developed during the secondary growth stage. The granulosa cells contained abundant smooth endoplasmic reticulum near the mitochondria. In males, the spermatogenesis was classified into spermatogonium to spermatozoa. Finally, we associated the morphological gonadal developments (stage I - IV) and the gonadosomatic index (GSI value) with the cellular developmental processes in both sexes. These results help integrate various levels of reproductive observations, which will be applied to understanding the reproductive cycle and development for aquaculture.

Pure Smooth Endoplasmic Reticulum (SER) Inclusions in Granulocytes of MYH9-Related Disorder Patients

MYH9-related disorders are a group of disorders (May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome) characterized by macrothrombocytopenia and ribosome inclusions in granulocytes. They are caused by mutations in the MYH9 gene and, depending on the location of the mutation, are associated with extrahematological pathologies (sensorineural hearing loss, progressive nephropathy, presenile cataracts) of variable intensity. The inclusions are formed by single and clustered ribosomes and are partially surrounded by segments of rough endoplasmic reticulum (RER). Typical inclusions in May-Hegglin anomaly are spindle-shaped, well-defined, and contain longitudinal filaments. Those found in Sebastian and Fechter syndromes are round or oval, have no filaments and, sometimes, show cross-striated arrangement of the ribosome aggregates. When reviewing the electron micrographs of the granulocyte inclusions of 10 patients with MYH9-related disorders previously published (Pujol-Moix et al. Haematologica 2004;89:330-337) we observed a special type of inclusion not described in the article. This inclusion has the typical appearance of smooth endoplasmic reticulum (SER) clusters, that is, a meshwork of branching tubules of SER without limiting membranes and no other structures such as ribosomes or RER. In 3 of the patients reviewed, in addition to the typical inclusions of MYH9-related disorder, some granulocytes with SER clusters were observed. These clusters were rounded or oval in shape and, when present, were 1 or 2 per cell (Fig. 1). The patients who presented granulocyte SER clusters are those identified as B4, C6 and D8 in the Haematologica article and are briefly described in Table 1. The patients presenting with SER clusters showed different characteristics such as age, bleeding, platelet counts, and proportion of giant platelets. Therefore, we cannot relate the presence of SER clusters to any clinical or biological characteristic of patients. It should be noticed that we did not find SER clusters in any patient with May-Hegglin anomaly. However, the small number of cases studied does not allow any conclusions to be drawn. SER clusters have been described in different types of cells such as hepatocytes or kidney tubular epitelial cells (Ghadially FN. Ultrastructural Pathology of the Cell and Matrix. Butterwords. London, 1988, pp 422-427). Normal granulocytes (neutrophils, eosinophils, and basophils) besides their own specific granules, also contain other general subcellular structures, including dispersed SER membranes. However, SER membranes forming clusters in these cells have not been previously described. In addition, SER clusters do not seem to correspond to evolved forms of typical MYH9 inclusions, for example by degranulation of the RER, based on the observations that typical inclusions show abundant ribosomes but few RER segments, no appreciable ribosomes are observed in the SER clusters, and the morphology of the tubules forming the SER and RER is different. We suggest that SER clusters could be considered a new type of inclusion in MYH9-related disorders. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Oocytes with smooth endoplasmic reticulum aggregates do not impact blastocyst euploidy rate

Objective: To investigate whether the euploidy rate of blastocysts derived from smooth endoplasmic reticulum (SERa) positive cycles and oocytes are impacted.Design: Retrospective cohort study.Setting: A tertiary hospital-based reproductive medicine center.Patient(s): A total of 601 preimplantation genetic testing (PGT) cycles with obtained oocytes in our center between April 2017 and May 2021 were included in the study. Intervention(s): Women＞35 years and PGT cycles with chromosomal structural rearrangements (PGT-SR) were excluded. Embryological and blastocyst ploidy outcomes were compared between SERa+ oocyte, sibling SERa- oocytes and oocytes in SERa- cycles.Main Outcome Measure(s): Embryological outcomes and blastocyst euploidy rate.Results: No significant difference was observed in the normal fertilization rate (82.1 % vs. 77.8 % vs. 83.1 %, respectively, P=0.061), blastocyst formation rate (71.0 % vs. 72.5 % vs. 68.4 %, respectively, P=0.393), good quality blastocyst formation rate (46.4 % vs. 48.3 % vs. 42.6 %, respectively, P=0.198) between the SERa+ oocyte group, sibling SERa- oocyte group and SERa- oocyte group. No significant difference was observed in the euploidy rate (50.0 % vs. 62.5 % vs. 63.3 %, respectively, P=0.324), mosaic rate (12.5 % vs. 9.7 % vs. 13.4 %, respectively, P=0.506) and aneuploidy rate (37.5 % vs. 27.8% vs. 23.2 %, respectively, P=0.137) between the three groups.Conclusion: Our results suggest that the euploidy rate of blastocysts derived from SERa+ cycles and oocytes are not impacted.

Stabilization of spine Synaptopodin by mGluR1 is required for mGluR-LTD

Dendritic spines, actin-rich protrusions forming the postsynaptic sites of excitatory synapses, undergo activity-dependent molecular and structural remodeling. Activation of group 1 metabotropic glutamate receptors - mGluR1 and mGluR5 - by synaptic or pharmacological stimulation, induces LTD but whether this is accompanied with spine elimination remains unresolved. A subset of telencephalic mushroom spines contains the spine apparatus (SA), an enigmatic organelle composed of stacks of smooth endoplasmic reticulum, whose formation depends on the expression of the actin-bundling protein Synaptopodin. Allocation of Synaptopodin to spines appears governed by cell-intrinsic mechanisms as the relative frequency of spines harboring Synaptopodin is conserved in vivo and in vitro. Here we show that expression of Synaptopodin/SA in spines is required for induction of mGluR-LTD at Schaffer collateral-CA1 synapses. Post-mGluR-LTD, mushroom spines lacking Synaptopodin/SA are selectively lost whereas spines harboring it are preserved, a process dependent on activation of mGluR1 but not mGluR5. Mechanistically, we find that mGluR1 supports physical retention of Synaptopodin within excitatory spine synapses during LTD while triggering lysosome-dependent degradation of the protein residing in dendritic shafts. Together, these results reveal a cellular mechanism, dependent on mGluR1, which enables selective preservation of stronger spines containing Synaptopodin/SA while eliminating weaker ones and potentially countering spurious strengthening by de novo recruitment of Synaptopodin. Overall our results identify spines with Synaptopodin/SA as the locus of mGluR-LTD and underscore the importance of the molecular microanatomy of spines in synaptic plasticity.

Oocytes With Smooth Endoplasmic Reticulum Aggregates Are Not Associated With Impaired Reproductive Outcomes: A Matched Retrospective Cohort Study

ObjectiveTo investigate whether the reproductive outcomes of oocytes with smooth endoplasmic reticulum aggregates (SERa) are impaired.MethodsA total of 2893 intracytoplasmic sperm injection (ICSI) cycles were performed between January 2010 and December 2019 in our center. In 43 transfer cycles, transferred embryos were totally derived from SERa+ oocytes. Each of the 43 cycles was matched with a separate control subject from SERa- patient of the same age ( ± 1 year), embryo condition, main causes of infertility, type of protocols used for fresh or frozen embryo transfer cycles. The clinical pregnancy, implantation, ectopic pregnancy and live birth rate were compared between the two groups.Results43 embryo transfer cycles from SERa- patient were matched to the 43 transferred cycles with pure SERa+ oocytes derived embryos. No significant difference was observed in clinical pregnancy rate (55.81% vs. 65.11%, p=0.5081), implantation rate (47.89% vs. 50.70%, p=0.8667) and live birth rate (48.84% vs. 55.81%, p=0.6659) between the SERa+ oocyte group and the matched group. No congenital birth defects were found in the two groups.ConclusionOur results suggest that the implantation, clinical pregnancy, live birth and birth defects rate of embryos derived from oocytes with SERa are not impaired.

Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase

The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After high-pressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell.

O-209 The impact of sperm DNA fragmentation on pregnancy outcomes depends on oocyte dimorphisms

Study question Does the impact of sperm DNA fragmentation (SDF) on Intracytoplasmic sperm injection (ICSI) outcomes depend on the presence of oocyte dimorphisms? Summary answer There is a significant influence of oocyte quality on the impact of SDF on pregnancy outcomes. What is known already Sperm DNA fragmentation has been associated with ICSI outcomes. DNA damage is commonly encountered in human spermatozoa and it has been widely accepted that the oocyte assumes responsibility for the repair and remodelling of both the maternal and paternal genomes during the oocyte-embryo transition. Indeed, spermatozoa with DNA damage can fertilise oocytes and still lead to embryo development due to the oocyte DNA repair capacity. Considering the vital role played by the oocyte in the developmental process, it could be hypostatised that the oocyte quality, translated as oocyte morphology, would influence the machinery responsible for sperm DNA repair after fertilization. Study design, size, duration This cohort study included 3,035 oocytes from 525 patients undergoing ICSI cycles in a university-affiliated IVF-center, between June/2016 and July/2019. Oocytes were split into groups according to the SDF index of the sample used for ICSI: low-fragmentation (<30% SDF, n = 2,277) and high-fragmentation (≥30% SDF, n = 758). Oocytes were evaluated before sperm injection and the dimorphisms were recorded. The influence of SDF index on ICSI outcomes, depending on the presence of oocytes dimorphisms was evaluated. Participants/materials, setting, methods Data was evaluated using generalized linear models (GZLM) followed by Bonferroni post hoc. The results are expressed as mean ± standard error for continuous variables or percentages for dichotomous variables, and p-values. The sample size calculation suggested that a sample of at least 504 subjects had 95% power to detect a 20% effect with a significance level of 5% (α). The study was performed in a private university–affiliated in vitro fertilization (IVF) center. Main results and the role of chance The association of both factors: the presence of oocyte dimorphisms (dark cytoplasm, vacuoles in the ooplasm, and resistant membrane) and high SDF index resulted in the lowest fertilization rate among groups, while oocytes free of these dimorphisms injected with samples with <30% SDF had the highest fertilization rate (p = 0.05, p < 0.01 and p < 0.01 for dark cytoplasm, vacuoles in the ooplasm and resistant membrane respectively). The impact of SDF index on high quality embryos rate on cleavage stage was also influence by the presence smooth endoplasmic reticulum clusters and resistant membrane oocytes (p = 0.013 and p = 0.018). As for the clinical outcomes, the impact of SDF index on the implantation rate was influenced by the presence of vacuoles in the ooplasm (p < 0.01), smooth endoplasmic reticulum clusters (p < 0.01), large perivitelline space (p < 0.01), resistant membrane (p < 0.01), and non-resistant membrane (p < 0.01), while the influence of SDF index on the pregnancy rate was influenced by the presence large perivitelline space (p < 0.01), resistant membrane (p = 0.018) and non-resistant membrane (p < 0.01). The effect of SDF on the miscarriage rate was also increased in the presence of large perivitelline space (p = 0.045), non-resistant membrane (0.037) and centrally located cytoplasmic granular area (p = 0.025). Limitations, reasons for caution The retrospective nature is a limitation. It could be argued that using samples with high SDF index does not necessarily mean that a sperm cell with a fragmented DNA was injected, however, the higher the SDF index, the higher the chance of selecting one with fragmented DNA. Wider implications of the findings The findings presented here highlight the crucial role of male and female factors when facing assisted reproduction. The association of low oocyte quality and high SDF index may lead to impaired results. As the oocyte defect cannot be modified, in vivo upgrading of spermatozoa before the treatment should be encouraged. Trial registration number Not applicable