Oocyte Development Research Articles

Identification, expression, and function analysis of Rbpms2 splicing variants in Japanese flounder gonad

Rbpms2, an RNA-binding protein with multiple splicing (Rbpms), can interact with RNAs to involve oocyte development, thereby influencing female sex differentiation in vertebrates. Here, two splicing variants of the Rbpms2 gene from Japanese flounder (Paralichthys olivaceus) were identified, namely Rbpms2.1 and Rbpms2.2. The two variants exhibited 98.22 % amino acid homology, both featuring an RNA recognition motif (RRM) domain spanning positions 98–170 amino acids. They were relatively conserved throughout phylogenetic evolution. Differently, the C-terminal region of the Rbpms2.1 contains five additional sequential amino acids (–VRDQP–) compared to Rbpms2.2. The real-time qPCR results demonstrated that Rbpms2.1 and Rbpms2.2 had relatively abundant expression in the gonads of adult Japanese flounder, with higher expression levels in the ovary compared to the testis (P < 0.05). In situ hybridization results showed strong positive expression of Rbpms2 mRNA in oocytes at stages I-III during the V stage of ovarian development. In the testis atstage IV, the expression of Rbpms2 mRNA was mainly concentrated on primary spermatocytes. Importantly, Rbpms2 binding sites were found in the 3′UTR, 5′UTR, and ORF regions of the sex-related genes including dmrt1, sox9, amh, foxl2, and wnt4. siRNA interference and overexpression analysis of Rbpms2.1 and Rbpms2.2 in primary cells of the ovary and testis showed that Rbpms2 can repress the expression of male-related genes (dmrt1, sox9, and amh) and significantly promote the expression of female-related genes (foxl2 and wnt4). Our results revealed that Rbpms2 may play a critical role by targeting the sex-related genes in the gonad development of Japanese flounder.

Effects of Astaxanthin on the Physiological State of Porcine Ovarian Granulose Cells Cultured In Vitro.

The physiological state of Granulosa cells (GCs) is intricately linked to the growth and development of oocytes. Oxidative stress has been found to cause damage to GCs in vitro. Astaxanthin (AST), a well-known natural ketone-type carotenoid, has demonstrated strong antioxidant properties. This study investigates the impact of astaxanthin supplementation on the physiological state of porcine ovarian granulosa cells cultured in vitro. Variations in morphology, apoptosis, reactive oxygen species (ROS) levels, and the expression of apoptosis and anti-oxidation-related genes in porcine GCs from different passages were observed. Significant morphological changes, increases in apoptosis, and decreases in antioxidant capacity resulting from passage were observed. Subsequently, treatment with 5 μmol/L astaxanthin significantly enhanced cell viability, proliferation, antioxidant capacity and mitochondrial function while also regulating the estradiol (E2) and progesterone (P4) levels. Additionally, the gene expression of antioxidation, E2, and P4 synthesis markers was assessed, revealing reduced apoptosis and ROS levels in porcine GCs. In conclusion, supplementation with 5 μmol/L astaxanthin in vitro effectively enhances the physiological condition of porcine GCs and optimizes the culture system for these cells in vitro. Optimizing the culture system of porcine GCs in vitro can simulate the function of granulosa cells in vivo and provide a theoretical reference for further promoting follicular development, which is beneficial to improving sow fertility in actual production.

Folic acids promote in vitro maturation of bovine oocytes by inhibition of ferroptosis via upregulated glutathione and downregulated Fe2+ accumulation

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe2+ levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe2+ accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

Elucidating the Role of Sirtuin 3 in Mammalian Oocyte Aging.

The field of reproductive biology has made significant progress in recent years, identifying specific molecular players that influence oocyte development and function. Among them, sirtuin 3 (SIRT3) has attracted particular attention for its central role in mediating mitochondrial function and cellular stress responses in oocytes. So far, studies have demonstrated that the knockdown of SIRT3 leads to a decrease in blastocyst formation and an increase in oxidative stress within an embryo, underscoring the importance of SIRT3 in maintaining the cellular redox balance critical for embryonic survival and growth. Furthermore, the literature reveals specific signaling pathways, such as the SIRT3- Glycogen synthase kinase-3 beta (GSK3β) deacetylation pathway, crucial for mitigating oxidative stress-related anomalies in oocyte meiosis, particularly under conditions like maternal diabetes. Overall, the emerging role of SIRT3 in regulating oocyte mitochondrial function and development highlights the critical importance of understanding the intricate connections between cellular metabolism, stress response pathways, and overall reproductive health and function. This knowledge could lead to the development of novel strategies to support oocyte quality and fertility, with far-reaching implications for assisted reproductive technologies and women's healthcare. This commentary aims to provide an overview of the importance of SIRT3 in oocytes by synthesizing results from a multitude of studies. The aim is to elucidate the role of SIRT3 in oocyte development, maturation, and aging and to identify areas where further research is needed.

Antidepressants as new endocrine disruptors? – transcriptomic profiling in gonads of Mytilus trossulus exposed to norfluoxetine

In this study an impact of norfluoxetine (NFLU) on Mytilus trossulus gonads was investigated focusing on sex-related differences in hormonal changes, gene expression, and transcriptomic profiling. Sex-specific differences in gonadal serotonin levels were found. NFLU stimulates serotonin synthesis and/or transport in female gonads, potentially accelerating oocyte maturation and gamete release. In males, NFLU decreases serotonin level what likely leads to impeding sperm maturation and thus spawning delay. Transcriptomic analyses highlighted the presence of NFLU-induced changes in gene expression related to gametogenesis and neurotransmission. In females, NFLU upregulated genes associated with oocyte development and downregulated those involved in sperm maturation. NFLU-treated males exhibited mixed effects in their genes in relation to spermatogenesis. Additionally, sex-related differences in the expression of the CYP450 genes responsible for detoxification were found. Overall, norfluoxetine acts as an endocrine-disrupting chemical and impacts gonadal serotonin levels and gene expression, potentially disrupting reproductive success of M. trossulus.

Lactation affects postweaning metabolic profiles, but not follicle size in multiparous sows

Young sows mobilise body reserves to support milk production during lactation, resulting in a negative energy balance (NEB). This NEB affects the development of follicles and oocytes that give rise to the next litter. Decreased IGF1 levels due to a NEB are thought to play a role in this process. As this has hardly been studied in multiparous sows, the current study focused on relations between lactation BW loss (%), metabolic hormones, and follicle development in multiparous sows at Day 0 and Day 4 after weaning. A total of 31 sows of parity 4.7 ± 2.5 were killed at either Day 0 or Day 4 after weaning. Average BW loss during lactation was 3.3 ± 4.5%, while average backfat loss was 4.1 ± 0.3 mm. The metabolic profile confirmed the metabolic impact of lactation as both non-esterified fatty acid (NEFA), and creatinine levels were higher at Day 0 than that at Day 4. Conversely, serum levels of IGF1 and growth differentiation factor 15 levels were lower on Day 0 than on Day 4. A higher BW loss (%) was related to higher NEFA levels on Day 0, but not on Day 4. IGF1 concentrations in serum and follicle fluid were similar at Day 0 and Day 4 and were not related to follicle size on these days. In conclusion, although lactation affected postweaning metabolic profiles in these multiparous sows, follicle size was not related to these profiles, probably due to the relatively mild BW loss of these sows. IGF1 concentrations were less affected by lactation and did not seem to limit follicle development, as it does in sows experiencing high weight loss.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

VPS34 Governs Oocyte Developmental Competence by Regulating Mito/Autophagy: A Novel Insight into the Significance of RAB7 Activity and Its Subcellular Location.

Asynchronous nuclear and cytoplasmic maturation in human oocytes is believed to cause morphological anomalies after controlled ovarian hyperstimulation. Vacuolar protein sorting 34 (VPS34) is renowned for its pivotal role in regulating autophagy and endocytic trafficking. To investigate its impact on oocyte development, oocyte-specific knockout mice (ZcKO) are generated, and these mice are completely found infertile, with embryonic development halted at 2- to 4-cell stage. This infertility is related with a disruption on autophagic/mitophagic flux in ZcKO oocytes, leading to subsequent failure of zygotic genome activation (ZGA) in derived 2-cell embryos. The findings further elucidated the regulation of VPS34 on the activity and subcellular translocation of RAS-related GTP-binding protein 7 (RAB7), which is critical not only for the maturation of late endosomes and lysosomes, but also for initiating mitophagy via retrograde trafficking. VPS34 binds directly with RAB7 and facilitates its activity conversion through TBC1 domain family member 5 (TBC1D5). Consistent with the cytoplasmic vacuolation observed in ZcKO oocytes, defects in multiple vesicle trafficking systems are also identified in vacuolated human oocytes. Furthermore, activating VPS34 with corynoxin B (CB) treatment improved oocyte quality in aged mice. Hence, VPS34 activation may represent a novel approach to enhance oocyte quality in human artificial reproduction.

Comparative transcriptome analysis reveals molecular mechanisms of the effects of light intensity and photoperiod on ovarian development in Procambarus clarkii (Girard, 1852)

Procambarus clarkii (Girard, 1852) has important economic value in China and internationally. In this research, the comparative transcriptome analysis was used to reveal molecular mechanisms of influences of photoperiod and light intensity on ovarian development in P. clarkii for the first time. Some genes (such as laminin, collagen, integrin beta, catenin) and pathways (including TGF-beta signaling pathway, focal adhesion, ECM–receptor interaction) associated with ovarian development and oocyte maturation were significantly upregulated. Some genes related to circadian clock (such as CLK, PER) were identified in this research. The results indicated that when light intensity or photoperiod increased, P. clarkii could up-regulate the expression levels of the laminin and collagen, thereby synthesizing related proteins, promoting meiosis of the oocytes, thus increasing the number of oocytes in the ovary. At the same time, P. clarkii could up-regulate the expression levels of integrin beta, integrin alpha 6, and diacylglycerol to synthesize related proteins, thereby promoting the formation of proteins and fats such as triglycerides, these proteins and fats can provide material basis for maturation and development of oocytes, resulting in oocyte maturation and ovarian development. P. clarkii could synthesize related proteins by upregulating expression levels of genes (such as catenin), these proteins or hormones can adhere to other actins (such as integrins), thereby stabilizing the morphology of the oocytes and ensuring normal development. Meantime, the increase in light intensity or photoperiod could cause release GSH and VTG, resulting in oocytes development and maturation. The data in this research can reveal molecular mechanisms of impacts of photoperiod and light intensity on oocyte maturation and ovarian development in P. clarkii, can offer crucial genomic data for studying developmental mechanisms of ovary and oocyte in crustacean.

Optimization of lipofection protocols for CRISPR/Cas9 delivery in porcine zona pellucida intact oocytes: A study of coincubation duration and reagent efficacy

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h.

Molecular characterization and CRISPR/Cas9 validation of the precursor of egg yolk protein gene, vitellogenin of Leucinodes orbonalis Guenée (Lepidoptera: Crambidae)

Vitellogenin (Vg), a yolk protein precursor, plays an important role in the oocyte development of insects and is an important target of genetic pest management. Vg is synthesized in the fat body, transported through haemolymph and accumulates in developing oocytes. In this regard, the eggplant shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major pest in South and South East Asia and a serious concern for farmers. Therefore, in the present study, we have cloned and characterized Vg from L. orbonalis (LoVg) for further applications. The cloned Vg consisted of 5,370 base pairs encoding 1,790 amino acid residues long protein. Further, sequence alignment revealed that LoVg has three conserved domains: a Vitellogenin N domain (LPD-N), a domain of unknown function protein families (DUF1943), and a von Willebrand factor type D domain (VWD). Using phylogenetic analysis, it was found that LoVg evolved alongside homologous proteins from different insects. The real-time expression levels of LoVg were significantly greater in female adults followed by the pupal stage. This suggests that Vg production and absorption in L. orbonalis occurs in the later pupal stage. Our studies showed that editing LoVg using CRISPR/Cas9 did not affect the total number of eggs laid but affected egg hatchability. These studies help us to design newer approaches in insect pest management through genetic suppression for sustainable pest management.

EB-SUN, a New Microtubule Plus-End Tracking Protein in Drosophila.

Microtubule (MT) regulation is essential for oocyte development. In Drosophila, MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins (MAPs) that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, encoding a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing microtubule plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in a decrease in MT density at the onset of mid-oogenesis (Stage 7) and delays oocyte growth during late mid-oogenesis (Stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduced MT density at Stage 7. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, unlike single gene depletion, suggesting a functional redundancy between these two EB proteins during embryogenesis. Our findings indicate that EB-SUN and EB1 play distinct roles during early embryogenesis.

The global phosphorylation landscape of mouse oocytes during meiotic maturation.

Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells.

Rejuvenation of aged oocyte through exposure to young follicular microenvironment.

Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and developmental potential. A possible cause is aging of the surrounding follicular somatic cells that support oocyte growth and development by providing nutrients and regulatory factors. Here, by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed, we show that young oocytes cultured in aged follicles exhibited impeded meiotic maturation and developmental potential, whereas aged oocytes cultured within young follicles were significantly improved in rates of maturation, blastocyst formation and live birth after in vitro fertilization and embryo implantation. This rejuvenation of aged oocytes was associated with enhanced interaction with somatic cells, transcriptomic and metabolomic remodeling, improved mitochondrial function and higher fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat female infertility.

Angiotensin II type-1 receptor autoantibody positively correlates with the rate of metaphase I oocytes in infertility with ovulatory disorder

The renin-angiotensin system (RAS) plays an important role in reproductive function. Our previous study identified that angiotensin II type-1 receptor autoantibody (AT1-AA), an autoantibody that activates RAS, was closely associated with infertility. However, its distribution in different types of infertility remained unclear. This study was designed to explore the distribution of AT1-AA in infertile patients and the connections between AT1-AA and oocyte development and pregnancy outcome. A total of 184 infertile women participated, with samples collected from peripheral venous blood. ELISA was used to detect AT1-AA levels in their sera. It was observed that the proportion of ovulation-disorder factors in AT1-AA-positive group was significantly higher than that in negative group (P=0.001). In 59 infertile women with ovulatory disorders, compared with negative group, AT1-AA-positive group had lower rate of retrieval (P=0.032) and metaphase II (MII) oocytes (P=0.011) but higher proportion of metaphase I (MI) oocytes (P=0.019). A negative correlation was found between the levels of AT1-AA and rate of retrieval and MII oocytes (P=0.027; P=0.043), whereas a positive correlation was observed with the proportion of MI oocytes (P=0.002). Moreover, a specific predictive value for proportion of reaching MII and MI oocytes was exhibited by AT1-AA (P < 0.01; P < 0.05). But no significant difference in embryonic parameters or pregnancy outcomes between two groups was observed (P > 0.05). This study revealed that serum AT1-AA levels were significantly increased in infertile women with ovulatory disorders and positively correlated with proportion of MI oocytes, but not associated with outcomes of assisted reproduction.

An integrated analysis of multiple datasets reveals novel gene signatures in human granulosa cells

Granulosa cells (GCs) play crucial roles in oocyte maturation. Through gap junctions and extracellular vesicles, they mediate the exchange of molecules such as microRNAs and messenger RNAs. Different ovarian cell types exhibit unique gene expression profiles, reflecting their specialized functions and stages. By combining RNA-seq data from various cell types forming the follicle, we aimed at capturing a wide range of expression patterns, offering insights into the functional diversity and complexity of the transcriptome regulation across GCs. Herein, we performed an integrated bioinformatics analysis of RNA sequencing datasets present in public databases, with a unique and standardized workflow., By combining the data from different studies, we successfully increased the robustness and reliability of our findings and discovered novel genes, miRNAs, and signaling pathways associated with GCs function and oocyte maturation. Moreover, our results provide a valuable resource for further wet-lab research on GCs biology and their impact on oocyte development and competence.

A novel heterozygous missense variant of PANX1 causes human oocyte death and female infertility

Pannexin1 (PANX1) is a highly glycosylated membrane channel-forming protein, which has been found to implicate in multiple physiological and pathophysiological functions. Variants in the PANX1 gene have been reported to be associated with oocyte death and recurrent in vitro fertilization failure. In this study, we identified a novel heterozygous PANX1 variant (NM_015368.4 c.410 C > T (p.Ser137Leu)) associated with the phenotype of oocyte death in a non-consanguineous family, followed by an autosomal dominant (AD) mode. We explored the molecular mechanism of the novel variant and the variant c.976_978del (p.Asn326del) that we reported previously. Both of the variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, affected ATP release and membrane electrophysiological properties, which resulted in mouse and human oocyte death in vitro. For the first time, we presented the direct evidence of the effect of the PANX1 variants on human oocyte development. Our findings expand the variant spectrum of PANX1 genes associated with oocyte death and provide new support for the genetic diagnosis of female infertility.

Effects of combination of melatonin and L-carnitine on in vitro maturation in mouse oocytes: An experimental study.

Melatonin and L-carnitine are free radical scavengers with antiapoptotic and antioxidant properties that improve oocyte development. This study aimed to find the possible effect of combining 2 antioxidant agents of melatonin and L-carnitine on oocyte morphology, maturation, apoptosis, and expression of bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) genes in a mice model. To overstimulation, 60 female NMRI mice were injected intraperitoneally using mare serum gonadotropin. On day 2 post-injection, 70 cumulus-oocyte complexes were collected from each mouse. The collected oocytes randomly were then divided into 4 groups including, the control, melatonin, L-carnitine, and melatonin + L-carnitine groups. The morphology and maturation rate of the oocytes was evaluated using a light microscope. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and the expression of BMP-15 and growth and differentiation factor GDF-9 genes was also evaluated by real-time polymerase chain reaction. Oocyte diameter significantly was increased in combination treatment of L-carnitine and melatonin compared to other groups (p 0.05). L-carnitine group showed the highest mean percentage of oocyte cytoplasmic pattern. Results of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling indicated that the lowest apoptosis rate belonged to the melatonin + L-carnitine group. Moreover, the combination groups showed the highest number of oocytes and maturation rate. The BMP-15 and GDF-9 genes were significantly upregulated in all treatment groups compared to the control group. Our results suggested a combination of melatonin + L-carnitine administration as a more effective choice for in vitro promotion of oocyte maturation.

New Immunological Indexes for the Effect of Systemic Inflammation on Oocyte and Embryo Development in Women With Unexplained Infertility: Systemic Immune Response Index and Pan-Immune-Inflammation Value.

Predicting the impact of systemic inflammation on oocyte and embryonic development in unexplained infertile women using the new immunological indexes. This retrospective cohort study was conducted using the records of the In Vitro Fertilization Department of Ankara Gülhane Training and Research Hospital. After reviewing the records of patients who had undergone in vitro fertilization (IVF) for unexplained infertility (UI) and excluding all known factors that could cause systemic immune inflammation, the systemic immune response index (SIRI), and pan-immune score were calculated from the pre-treatment hemogram parameters between the embryo arrest (EA) group and the embryo transfer group. It was investigated whether there was a statistical difference between the two groups and whether an SIRI value affecting embryo quality was found. A receiver operating characteristic (ROC) curve analysis was performed to determine the optimal cut-off values for inflammatory markers to predict EA. The 108 EA group (embryos that were arrested during their development and could not be transferred) and the 140 embryo transfer group showed statistically significant differences in the parameters of systemic inflammatory index (SII), SIRI, pan-immune inflammation value (PIV), and neutrophil/lymphocyte ratio (NLR) (p < 0.05). These inflammatory parameters, which were examined before ovulation induction, also correlated positively with the required total dose of gonadotropin and negatively with the ovarian sensitivity index (OSI). SII, SIRI, PIV, and NLR have specific cut-off values with ROC analysis and determine the effect of the inflammatory status of the environment in which the oocyte develops on EA (p < 0.005). In women with UI, high levels of systemic immune inflammation have a negative impact on oocyte and embryo development, and treatments to suppress inflammation may improve IVF success.