Full-grown Oocytes Research Articles

Increased DNA damage in full-grown oocytes is correlated with diminished autophagy activation

Unlike mild DNA damage exposure, DNA damage repair (DDR) is reported to be ineffective in full-grown mammalian oocytes exposed to moderate or severe DNA damage. The underlying mechanisms of this weakened DDR are unknown. Here, we show that moderate DNA damage in full-grown oocytes leads to aneuploidy. Our data reveal that DNA-damaged oocytes have an altered, closed, chromatin state, and suggest that the failure to repair damaged DNA could be due to the inability of DDR proteins to access damaged loci. Our data also demonstrate that, unlike somatic cells, mouse and porcine oocytes fail to activate autophagy in response to DNA double-strand break-inducing treatment, which we suggest may be the cause of the altered chromatin conformation and inefficient DDR. Importantly, autophagy activity is further reduced in maternally aged oocytes (which harbor severe DNA damage), and its induction is correlated with reduced DNA damage in maternally aged oocytes. Our findings provide evidence that reduced autophagy activation contributes to weakened DDR in oocytes, especially in those from aged females, offering new possibilities to improve assisted reproductive therapy in women with compromised oocyte quality.

The maternal protein NLRP5 stabilizes UHRF1 in the cytoplasm: implication for the pathogenesis of multilocus imprinting disturbance.

We have recently discovered that the so-called subcortical maternal complex (SCMC) proteins composing of cytoplasmic lattices are destabilized in Uhrf1 knockout murine fully grown oocytes (FGOs). Here we report that human UHRF1 interacts with human NLRP5 and OOEP, which are core components of the SCMC. Moreover, NLRP5 and OOEP interact with DPPA3, which is an essential factor for exporting UHRF1 from the nucleus to the cytoplasm in oocytes. We identify that NLRP5, not OOEP, stabilizes UHRF1 protein in the cytoplasm utilizing specifically engineered cell lines mimicking UHRF1 status in oocytes and preimplantation embryos. Further, UHRF1 is destabilized both in the cytoplasm and nucleus of Nlrp5 knockout murine FGOs. Since pathogenic variants of the SCMC components frequently cause multilocus imprinting disturbance and UHRF1 is essential for maintaining CpG methylation of imprinting control regions during preimplantation development, our results suggest possible pathogenesis behind the disease, which has been a long-standing mystery.

Long-term tributyltin exposure alters behavior, oocyte maturation, and histomorphology of the ovary due to oxidative stress in adult zebrafish

Tributyltin (TBT), an organotin endocrine-disrupting substance, is recognized as one of the important toxic environmental pollutants. The present study was carried out to investigate the toxic effects of TBT on behavior and the ovary of adult zebrafish with a focus on oxidative stress markers and oocyte maturation. Adult zebrafish were exposed to three different concentrations (125, 250, and 500 ng/L of water) of TBT for 28 days. TBT exposure produced a concentration-dependent negative effect on the body weight and behavior (anxiety-like symptoms) of adult zebrafish. Alterations in the activity of superoxide dismutase (SOD) and catalase (CAT), the total antioxidant capacity of ovarian tissue by the highest exposure level of TBT resulted in lipid peroxidation as indicated by increased malondialdehyde (MDA) level. The numbers of early-vitellogenic oocytes were significantly increased in zebrafish exposed to TBT as low as 125 ng/L. However, the numbers and size of fully-grown (mature) oocytes were significantly reduced in the highest exposure group only. Correlation between the MDA level and pre-vitellogenic oocytes in the 500 ng/L group indicated that lipid peroxidation prevented the maturation of pre-vitellogenic oocytes. TBT exposure produced significant histological changes in the ovary as evidenced by disturbed maturation of oocytes. In conclusion, TBT adversely affected the maturation of oocytes in zebrafish ovary through oxidative stress-mediated mechanisms.

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells

BackgroundGenome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs.ResultsWe show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine.ConclusionsThe results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Oocytes orchestrate protein prenylation for mitochondrial function through selective inactivation of cholesterol biosynthesis in murine species

Emerging research and clinical evidence suggest that the metabolic activity of oocytes may play a pivotal role in reproductive anomalies. However, the intrinsic mechanisms governing oocyte development regulated by metabolic enzymes remain largely unknown. Our investigation demonstrates that Ggps1, the crucial enzyme in the mevalonate pathway responsible for synthesizing isoprenoid metabolite GGPP from FPP, is essential for oocyte maturation in mice. Our findings reveal that the deletion of Ggps1 that prevents protein prenylation in fully-grown oocytes leads to subfertility and offspring metabolic defects without affecting follicle development. Oocytes that lack Ggps1 exhibit disrupted mitochondrial homeostasis and the mitochondrial defects arising from oocytes are inherited by the fetal offspring. Mechanistically, the excessive farnesylation of mitochondrial ribosome protein, Dap3, and decreased levels of small G proteins mediate the mitochondrial dysfunction induced by Ggps1 deficiency. Additionally, a significant reduction in Ggps1 levels in oocytes is accompanied by offspring defects when females are exposed to a high-cholesterol diet. Collectively, this study establishes that MVA pathway-protein prenylation is vital for mitochondrial function in oocyte maturation and provides evidence that the disrupted protein prenylation resulting from an imbalance between FPP and GGPP is the major mechanism underlying impairment of oocyte quality induced by high cholesterol.

Stable maternal proteins underlie distinct transcriptome, translatome, and proteome reprogramming during mouse oocyte-to-embryo transition

BackgroundThe oocyte-to-embryo transition (OET) converts terminally differentiated gametes into a totipotent embryo and is critically controlled by maternal mRNAs and proteins, while the genome is silent until zygotic genome activation. How the transcriptome, translatome, and proteome are coordinated during this critical developmental window remains poorly understood.ResultsUtilizing a highly sensitive and quantitative mass spectrometry approach, we obtain high-quality proteome data spanning seven mouse stages, from full-grown oocyte (FGO) to blastocyst, using 100 oocytes/embryos at each stage. Integrative analyses reveal distinct proteome reprogramming compared to that of the transcriptome or translatome. FGO to 8-cell proteomes are dominated by FGO-stockpiled proteins, while the transcriptome and translatome are more dynamic. FGO-originated proteins frequently persist to blastocyst while corresponding transcripts are already downregulated or decayed. Improved concordance between protein and translation or transcription is observed for genes starting translation upon meiotic resumption, as well as those transcribed and translated only in embryos. Concordance between protein and transcription/translation is also observed for proteins with short half-lives. We built a kinetic model that predicts protein dynamics by incorporating both initial protein abundance in FGOs and translation kinetics across developmental stages.ConclusionsThrough integrative analyses of datasets generated by ultrasensitive methods, our study reveals that the proteome shows distinct dynamics compared to the translatome and transcriptome during mouse OET. We propose that the remarkably stable oocyte-originated proteome may help save resources to accommodate the demanding needs of growing embryos. This study will advance our understanding of mammalian OET and the fundamental principles governing gene expression.

Xrcc5/Ku80 is required for the repair of DNA damage in fully grown meiotically arrested mammalian oocytes

Mammalian oocytes spend most of their life in a unique state of cell cycle arrest at meiotic prophase I, during which time they are exposed to countless DNA-damaging events. Recent studies have shown that DNA double-strand break repair occurs predominantly via the homologous recombination (HR) pathway in small non-growing meiotically arrested oocytes (primordial follicle stage). However, the DNA repair mechanisms employed by fully grown meiotically arrested oocytes (GV-stage) have not been studied in detail. Here we established a conditional knockout mouse model to explore the role of Ku80, a critical component of the nonhomologous end joining (NHEJ) pathway, in the repair of DNA damage in GV oocytes. GV oocytes lacking Ku80 failed to repair etoposide-induced DNA damage, even when only low levels of damage were sustained. This indicates Ku80 is needed to resolve DSBs and that HR cannot compensate for a compromised NHEJ pathway in fully-grown oocytes. When higher levels of DNA damage were induced, a severe delay in M-phase entry was observed in oocytes lacking XRCC5 compared to wild-type oocytes, suggesting that Ku80-dependent repair of DNA damage is important for the timely release of oocytes from prophase I and resumption of meiosis. Ku80 was also found to be critical for chromosome integrity during meiotic maturation following etoposide exposure. These data demonstrate that Ku80, and NHEJ, are vital for quality control in mammalian GV stage oocytes and reveal that DNA repair pathway choice differs in meiotically arrested oocytes according to growth status.

Role of insulin-like growth factor 1 (IGF1) in the regulation of mitochondrial bioenergetics in zebrafish oocytes: lessons from in vivo and in vitro investigations.

Optimal mitochondrial functioning is indispensable for acquiring oocyte competence and meiotic maturation, whilst mitochondrial dysfunction may lead to diminished reproductive potential and impaired fertility. The role of the intra-ovarian IGF system in ovarian follicular dynamics has been implicated earlier. Although several studies have demonstrated the role of the IGF axis in facilitating mitochondrial function over a multitude of cell lines, its role in oocyte energy metabolism remains largely unexplored. Here using zebrafish, the relative importance of IGF1 in modulating oocyte mitochondrial bioenergetics has been investigated. A dramatic increase in ovarian lhcgr and igf1 expression accompanied heightened ATP levels and mitochondrial polarization in full-grown (FG) oocytes resuming meiotic maturation and ovulation in vivo. Concomitant with elevated igf1 expression and IGF1R phosphorylation, hCG (LH analog) stimulation of FG follicles in vitro prompted a sharp increase in NRF-1 and ATP levels, suggesting a positive influence of gonadotropin action on igf1 expression vis-à-vis oocyte bioenergetics. While recombinant IGF1 administration enhanced mitochondrial function, IGF1R immunodepletion or priming with PI3K inhibitor wortmannin could abrogate NRF-1 immunoreactivity, expression of respiratory chain subunits, ΔΨM, and ATP content. Mechanistically, activation of PI3K/Akt signaling in IGF1-treated follicles corroborated well with the rapid phosphorylation of GSK3β at Ser9 (inactive) followed by PGC-1β accumulation. While selective inhibition of GSK3β promoted PGC-1β, Akt inhibition could abrogate IGF1-induced p-GSK3β (Ser9) and PGC-1β immunoreactive protein indicating Akt-mediated GSK3β inactivation and PGC-1β stabilization. The IGF1-depleted follicles showed elevated superoxide anions, subdued steroidogenic potential, and attenuated G2-M1 transition. In summary, this study highlights the importance of IGF1 signaling in oocyte bioenergetics prior to resumption of meiosis.

O-059 ntermetabolites of cholesterol synthesis in granulosa cell and oocyte aging

Abstract With the development of society, there has been a significant delay in female fertility and an increasing desire for childbearing among females of advanced maternal age in recent decades. Nevertheless, women over 35 years old have more risk of infertility and miscarriage compared to young women, due to decreased oocyte quality. These changes are mainly caused by meiotic defects, especially aneuploidy. Therefore, exploring the mechanisms of aging-related meiotic defects and aneuploidy in oocytes is of great significant for improving the pregnancy outcomes of aged women. Oocytes are enveloped by granulosa cells (GCs) to form follicles, which constitute the reproductive units in ovaries. GCs form a metabolic community with oocytes for oocyte growth and maturation. Numerous studies have reported that metabolic coupling between GCs and oocytes could affect the process of oocyte meiosis resumption, spindle assembly, and chromatin arrangement. Maternal aging is related to disruption of oocyte-GC interactions that leads to disorders of the oocyte meiotic process. Therefore, abnormal metabolic coupling between GCs and oocytes is an important reason for decreased quality in aged oocytes. To further clarify the metabolic coupling changes between GCs and oocytes during ovarian aging, we systematically characterized the dynamic changes in the overall transcriptomic landscapes of oocytes and GCs from young and aged mice throughout oocyte meiosis (during the growing oocyte [GO], full-grown oocyte [FGO], metaphase I [MI] oocyte [MIO], and metaphase II [MII] oocyte [MIIO] phases). Data revealed that the mevalonate (MVA) pathway, an essential metabolic pathway that uses acetyl-CoA to produce cholesterol and isoprenoids, was specifically highly expressed in GCs with the resumption of oocyte meiosis, and the expression of MVA pathway in GCs decreased with age. Atorvastatin-mediated inhibition of MVA metabolism in GCs decreased the first polar body extrusion (PBE) rate, and increased oocyte meiotic defects and aneuploidy in young cumulus-oocyte complexes (COCs). Further studies showed that the effect of the MVA pathway on oocyte meiosis was mainly regulated by protein isoprenoylation mediated by the inter-metabolites of cholesterol synthesis. Importantly, upregulation of the MVA pathway in aged GCs could ameliorate the depletion of ovarian reserve, decrease oocyte meiotic defects, and improve oocyte quality. In conclusion, metabolite in granulosa cell is an important factor determining the oocyte quality; MVA pathway in GCs is a critical regulator of meiotic maturation and euploidy in oocytes, and age-associated MVA pathway abnormalities contribute to oocyte meiotic defects and aneuploidy; Supplementation of inter-metabolites of cholesterol synthesis is a new therapeutic target for clinical intervention to improve oocyte quality. Trial registration number XXXX

222 nm far-UVC efficiently introduces nerve damage in Caenorhabditis elegans.

Far-ultraviolet radiation C light (far-UVC; 222 nm wavelength) has received attention as a safer light for killing pathogenic bacteria and viruses, as no or little DNA damage is observed after irradiation in mammalian skin models. Far-UVC does not penetrate deeply into tissues; therefore, it cannot reach the underlying critical basal cells. However, it was unclear whether far-UVC (222-UVC) irradiation could cause more biological damage at shallower depths than the 254 nm UVC irradiation (254-UVC), which penetrates more deeply. This study investigated the biological effects of 222- and 254-UVC on the small and transparent model organism Caenorhabditis elegans. At the same energy level of irradiation, 222-UVC introduced slightly less cyclobutane pyrimidine dimer damage to naked DNA in solution than 254-UVC. The survival of eggs laid during 0-4 h after irradiation showed a marked decrease with 254-UVC but not 222-UVC. In addition, defect of chromosomal condensation was observed in a full-grown oocyte by 254-UVC irradiation. In contrast, 222-UVC had a significant effect on the loss of motility of C. elegans. The sensory nervous system, which includes dopamine CEP and PVD neurons on the body surface, was severely damaged by 222-UVC, but not by the same dose of 254-UVC. Interestingly, increasing 254-UVC irradiation by about 10-fold causes similar damage to CEP neurons. These results suggest that 222-UVC is less penetrating, so energy transfer occurs more effectively in tissues near the surface, causing more severe damage than 254-UVC.

Embryonic microRNAs are essential for bovine preimplantation embryo development

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

Mammalian oocytes store mRNAs in a mitochondria-associated membraneless compartment.

Full-grown oocytes are transcriptionally silent and must stably maintain the messenger RNAs (mRNAs) needed for oocyte meiotic maturation and early embryonic development. However, where and how mammalian oocytes store maternal mRNAs is unclear. Here, we report that mammalian oocytes accumulate mRNAs in a mitochondria-associated ribonucleoprotein domain (MARDO). MARDO assembly around mitochondria was promoted by the RNA-binding protein ZAR1 and directed by an increase in mitochondrial membrane potential during oocyte growth. MARDO foci coalesced into hydrogel-like matrices that clustered mitochondria. Maternal mRNAs stored in the MARDO were translationally repressed. Loss of ZAR1 disrupted the MARDO, dispersed mitochondria, and caused a premature loss of MARDO-localized mRNAs. Thus, a mitochondria-associated membraneless compartment controls mitochondrial distribution and regulates maternal mRNA storage, translation, and decay to ensure fertility in mammals.

P-440 Impact of electrospun scaffold topology on the performance of in-vitro Folliculogenesis applied to preantral ovine follicles

Abstract Study question How to improve in-vitro Folliculogenesis (ivF) protocols to address the enlarged demand of fertility preservation? Summary answer Tissue engineering-based approach opens new frontiers for ivF improving 3D-technologies addressed to support immature-ovarian-follicle-growth to obtain an increased number of competent oocytes enrolled in Assisted-Reproductive-Technology. What is known already ivF is a promising Assisted-Reproductive-Technology (ART) for preserving and restoring fertility. This technology potentially reproduces the early stages of folliculogenesis and oogenesis in-vitro allowing to move a large amount of oocyte on individual basis towards the validated protocol of in-vitro maturation/in-vitro fertilization (IVM/IVF). The current availability of biocompatible-supporting materials offers the challenging opportunity to mimic the native organ stroma in order to better reproduce the 3D environmental conditions leading to synergic follicles-oocyte development in-vitro with the aim to improve the performance of ivF in translational large sized mammal models. Study design, size, duration The present research aimed to compare preantral (PA) follicles culture on two different typologies of scaffolds fabricated using PCL(poly(epsilon caprolactone)), respectively made with patterned and randomly aligned fibers (PCL-Patterned/PCL-Randomic) with a standardized-single-follicle scaffold-free-method (3D-oil), widely validated on ovine model (Cecconi et al., 2004). The culture outcomes are compared analyzing follicle/oocyte growth, percentage of antrum differentiation and the incidence of meiotic competence, by exposing ivF growing oocytes to IVM protocol. Participants/materials, setting, methods PA follicles (mean size diameter: 250±4μm), mechanically isolated from slaughterhoused lamb ovaries, were individually cultured on electrospun PCL scaffolds (patterned vs randomic) or using the 3D-oil method. ivF were cultured alphaMEM-Fetal Bovine Serum free medium (5% Knockout Serum Replacement) supplemented with 4 IU/mL of equine Chorionic Gonadotropin (Di Berardino et al., 2021). At the end of ivF (14-days) the fully-grown oocytes isolated from early-antral follicles were tested on IVM. Main results and the role of chance PCL-Patterned electrospun scaffolds were able to strongly support a synergic oocyte and follicular growth. The 3D culture on Patterned electrospun scaffold supported the highest viability of follicles (87.5% vs 63% under 3D-oil conditions). On the contrary, the highest incidence of degenerated follicles was observed in cultures performed using PCL-Randomic materials (55 vs 37% vs 12.5% for PCL-Randomic vs 3D-oil vs PCL-Patterned, respectively; p <0.0004). The greatest follicle diameter increment (74.7±1 vs 70±0.4 vs 60.9±2%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, respectively p <0.0007) and rate of antrum differentiation (87.5% vs 45% and vs 63%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, for both p <0.0001) were observed in PA ovine follicles cultured on PCL-Patterned scaffolds. Furthermore, PCL-Patterned electrospun scaffolds supported a complete functional development of the oocyte compartment. More in detail, the majority of fully grown oocytes isolated from early- antral follicles grown on PCL-Patterned materials reached the metaphase-II stage (MII 80%) at the end of IVM in comparison to the significant lower percentage in 3D-oil (MII 68%, p =0.04) and PCL-Randomic (MII 18%, p <0.0001) protocols, respectively. Limitations, reasons for caution - Wider implications of the findings Tissue engineering scaffold-based approach represents a valid strategy generating a multi-organ in-vitro system, where different compartments may cooperate generating the complexity of paracrine-mechanism controlling early-follicles outcomes. Scaffold topology is essential to control early-follicles development. Indeed, exclusively PCL-Patterned can preserve long-term follicle 3D-microarchitecture supporting in-vitro oogenesis up to a complete meiotic-competence-acquisition. Trial registration number not applicable

Roles of Gonadotropin Receptors in Sexual Development of Medaka.

The gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted from the pituitary and bind to the FSH receptor (FSHR) and LH receptor (LHR) to regulate gonadal development in vertebrates. Previously, using fshr-knockout (KO) medaka (Oryzias latipes), we demonstrated that FSH regulates ovarian development by elevating estrogen levels. However, the lhr-KO phenotype in medaka is poorly characterized. Here, we generated lhr-KO medaka using the transcription activator-like effector nuclease (TALEN) technique. We analyzed its phenotype and that of fshr-KO, lhr;fshr double-heterozygotes (double-hetero), and double-KO fish. All genetically male medaka displayed normal testes and were fertile, whereas fshr-KO and double-KO genetically female fish displayed small ovaries containing many early pre-vitellogenic oocytes and were infertile. Although lhr-KO genetically female fish had normal ovaries with full-grown oocytes, ovulation did not occur. Levels of 17α,20β-dihydroxy-4-pregnen-3-one, which is required for meiotic maturation of oocytes and sperm maturation in teleost fish, were significantly decreased in all KO female medaka ovaries except for double-heteros. Further, 17β-estradiol levels in fshr-KO and double-KO ovaries were significantly lower than those in double-heteros. These findings indicate that LH is necessary for oocyte maturation and FSH is necessary for follicle development, but that neither are essential for spermatogenesis in medaka.

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.

Reproduction and fecundity of invader of internal insemination Trachelyopterus galeatus in a Neotropical floodplain

After the formation of Itaipu Reservoir, the invader Trachelyopterus galeatus colonized the upper Paraná River. Light microscopy was used to describe gametogenesis and the reproductive phases of females and males. The following data were verified: diameter of the oocytes, spawning type, batch fecundity by ovary weight, standard length, and total weight of the fish, along with the regions where this species reproduced in the upper Paraná River floodplain. A total of 470 specimens were collected quarterly in 2016, 2018 and 2019, and bimonthly in 2017. The gonads were fixed in a Karnovsky solution, dehydrated, infiltrated, and embedded in historesin. The histological slides were stained using PAS + iron hematoxylin + metanil yellow, analyzed and photographed under an image-capturing microscope. As regards diameter of the oocytes and fecundity estimates, ovaries whose oocytes were measured under a stereomicroscope were sampled. In the oogenesis, undifferentiated and differentiated oogonia, early primary growth oocytes, secondary growth oocytes, full-grown oocytes and maturing oocytes were recorded. In the spermatogenesis, primary and secondary spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa were recorded. The reproductive phases found for females and males were: immature, early development, late development, spawning/sperm-releasing capable, regression, and regeneration. Trachelyopterus galeatus prefers to occupy and reproduce in the Ventura, Patos, Guaraná, Fechada, Garças, and Pau Véio lagoons. The diameter of the oocytes varied from 0.4 to 2.9 mm. Females spawn, on average, 113 oocytes per batch. Batch fecundity variation shows that the larger the ovary, standard length, and total weight, the larger the number of oocytes to be spawned. This invader possesses reproductive success in the upper Paraná River floodplain, especially in lagoons.

Alternative Transmission Patterns in Independently Acquired Nutritional Cosymbionts of Dictyopharidae Planthoppers.

ABSTRACTSap-sucking hemipterans host specialized, heritable microorganisms that supplement their diet with essential nutrients. These microbes show unusual features that provide a unique perspective on the coevolution of host-symbiont systems but are still poorly understood. Here, we combine microscopy with high-throughput sequencing to revisit 80-year-old reports on the diversity of symbiont transmission modes in a broadly distributed planthopper family, Dictyopharidae. We show that in seven species examined, the ancestral nutritional symbionts Sulcia and Vidania producing essential amino acids are complemented by co-primary symbionts, either Arsenophonus or Sodalis, acquired several times independently by different host lineages and contributing to the biosynthesis of B vitamins. These symbionts reside within separate bacteriomes within the abdominal cavity, although in females Vidania also occupies bacteriocytes in the rectal organ. Notably, the symbionts are transovarially transmitted from mothers to offspring in two alternative ways. In most examined species, all nutritional symbionts simultaneously infect the posterior end of the full-grown oocytes and next gather in their perivitelline space. In contrast, in other species, Sodalis colonizes the cytoplasm of the anterior pole of young oocytes, forming a cluster separate from the “symbiont ball” formed by late-invading Sulcia and Vidania. Our results show how newly arriving microbes may utilize different strategies to establish long-term heritable symbiosis.

P–533 Effects of sex chromosomal complement, XX, XO, or XY, on the transcriptome and development of mouse oocytes during follicular growth

Abstract Study question How does the sex chromosome complement affect the transcriptome and development of oocytes during follicular growth in the mouse ovary? Summary answer Highly expressed X-linked genes adjust their transcript levels according to the X dosage. Y-linked genes affect the transcript levels of some X-linked and autosomal genes. What is known already Female mice carrying XO and XY chromosomes on the C57BL/6J (B6) genetic background are healthy but encounter subfertility and infertility, respectively. Our previous results have shown that the XY oocyte is defective in its cytoplasm; its replacement with that of an XX oocyte at the GV stage allows for production of healthy offspring after fertilization. Since transcription is shut down in the oocyte by the end of growth phase, the mRNAs and proteins necessary for meiotic progression and early embryonic development are accumulated during follicular growth. Study design, size, duration 30 oocytes of 50–59 μm diameter were pooled for each genotype in biological triplicate and subjected to RNA-Sequencing. Total RNA extracted from 10–30 pooled oocytes of each size range and genotype in biological triplicate were subjected to qRT-PCR. All experiments were performed between 2019–2021. Participants/materials, setting, methods XY and XO females were generated by cross between B6 females with B6.YTIR and B6.XPafY males, respectively. Oocytes in the growth phase were collected at 8–18 days postpartum (dpp), whereas fully-grown oocytes were collected at 27–29 dpp after injection with equine chorionic gonadotropin. Oocytes of 50–59 μm diameter were subjected to RNA-Sequencing using a version of SmartSeq2, followed by DEG analyses. Transcript levels in the oocytes of various diameters were determined by qRT-PCR. Main results and the role of chance Chromatin configuration, mitochondrial distribution, and de novo transcription were largely comparable among the XX, XO, and XY oocytes smaller than 60 µm. Three way comparisons of RNA-Seq data in the oocytes of 50–59 μm revealed; (1) 13.8% of X-linked DEGs showed the transcript levels in correspond to the X chromosome dosage; (2) 9 genes on the Y short arm and 2 genes near the distal end of the Y long arm were highly expressed in XY oocytes; (3) transcript levels of X- or autosomal homologs were affected by the XY complement compared to XX and XO oocytes; and (4) 54 and 39 X-linked and autosomal genes show higher and lower transcript levels, respectively, in XY oocytes compared to XX and XO oocytes. The results of qRT-PCR of selected genes revealed distinct dynamic changes in transcript levels in the oocyte during follicular growth. Data of RNA-Seq were statistically analyzed using R Bioconductor limma package for differentially expressed genes having Benjamini-Hochberg adjusted P values lower than 0.01 and log2 fold change higher than 1. All data of qRT-PCR were statistically analyzed by one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test. Limitations, reasons for caution In humans, most XO females die in utero and those who reach the term suffer from congenital abnormalities and infertility (Turner’s syndrome). However, the severer phenotype can be attributed to somatic cells with a greater number of genes that escape from X chromosome inactivation in humans than mice. Wider implications of the findings: XO and XY mice provide animal models for investigating the consequence of X haplodeficiency in the female germline, independent of somatic defects. Furthermore, XY female mice provide a unique opportunity for examining whether and how Y-linked genes are transcribed outside the male germline. Trial registration number Not applicable

Septin 4 controls CCNB1 stabilization via APC/CCDC20 during meiotic G2/M transition in mouse oocytes.

In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/CCDC20 . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.

Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice.

Completion of the first meiosis is an essential prerequisite for producing a functionally normal egg for fertilization and embryogenesis, but the precise mechanisms governing oocyte meiotic progression remains largely unclear. Here, we report that echinoderm microtubule associated protein (EMAP) like 1 (EML1), a member of the conserved EMAP family proteins, plays a crucial role in the control of oocyte meiotic progression in the mouse. Female mice carrying an ENU-induced nonsense mutation (c.1956T > A; p.Tyr652∗) of Eml1 are infertile, and the majority of their ovulated oocytes contain abnormal spindles and misaligned chromosomes. In accordance with the mutant oocyte phenotype, we find that EML1 is colocalized with spindle microtubules during the process of normal oocyte meiotic maturation, and knockdown (KD) of EML1 by specific morpholinos in the fully grown oocytes (FGOs) disrupts the integrity of spindles, and delays meiotic progression. Moreover, EML1-KD oocytes fail to progress to metaphase II (MII) stage after extrusion of the first polar body, but enter into interphase and form a pronucleus containing decondensed chromatins. Further analysis shows that EML1-KD impairs the recruitment of γ-tubulin and pericentrin to the spindle poles, as well as the attachment of kinetochores to microtubules and the proper inactivation of spindle assembly checkpoint at metaphase I (MI). The loss of EML1 also compromises the activation of maturation promoting factor around the time of oocyte resumption and completion of the first meiosis, which, when corrected by WEE1/2 inhibitor PD166285, efficiently rescues the phenotype of oocyte delay of meiotic resumption and inability of reaching MII. Through IP- mass spectrometry analysis, we identified that EML1 interacts with nuclear distribution gene C (NUDC), a critical mitotic regulator in somatic cells, and EML1-KD disrupts the specific localization of NUDC at oocyte spindles. Taken together, these data suggest that EML1 regulates acentrosomal spindle formation and the progression of meiosis to MII in mammalian oocytes, which is likely mediated by distinct mechanisms.