Stages Of Oocyte Development Research Articles

The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm.

The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm.

Zinc supports transcription and improves meiotic competence of growing bovine oocytes.

In the last years, many studies focused on the understanding of the possible role of zinc in the control of mammalian oogenesis, mainly on oocyte maturation and fertilization. However, little is known about the role of zinc at earlier stages, when the growing oocyte is actively transcribing molecules that will regulate and sustain subsequent stages of oocyte and embryonic development. In this study, we used the bovine model to gain insights into the possible involvement of zinc in oocyte development. We first mined the EmbryoGENE transcriptomic dataset, which revealed that several zinc transporters and methallothionein are impacted by physiological conditions throughout the final phase of oocyte growth and differentiation. We then observed that zinc supplementation during in vitro culture of growing oocytes is beneficial to the acquisition of meiotic competence when subsequently subjected to standard in vitro maturation. Furthermore, we tested the hypothesis that zinc supplementation might support transcription in growing oocytes. This hypothesis was indirectly confirmed by the experimental evidence that the content of labile zinc in the oocyte decreases when a major drop in transcription occurs in vivo. Accordingly, we observed that zinc sequestration with a zinc chelator rapidly reduced global transcription in growing oocytes, which was reversed by zinc supplementation in the culture medium. Finally, zinc supplementation impacted the chromatin state by reducing the level of global DNA methylation, which is consistent with the increased transcription. In conclusion, our study suggests that altering zinc availability by culture-medium supplementation supports global transcription, ultimately enhancing meiotic competence.

Single-Cell Transcriptomic Analysis Reveals Mitochondrial Dynamics in Oocytes of Patients With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS), characterized by polycystic ovarian morphology, ovarian follicular maturation arrest, and hormonal disorders, affects numerous women in the reproductive age worldwide. A recent study has found that mitochondria are likely to play an essential role in oocyte quality. However, it is still unclear whether oocyte development failure is associated with mitochondria in patients with PCOS. We analyzed the single-cell RNA sequencing data from the previous study, including data from 14 oocytes from 7 healthy fertile women and 20 oocytes from 9 patients with PCOS at the germinal vesicle (GV) stage, metaphase I (MI) stage, and metaphase II (MII) stage. We revealed the transcriptomic dynamics by weighted gene co-expression network analysis (WGCNA) and investigated the differences between stages using PCA and Deseq2 analyses to identify the differential expression genes (DEGs). Gene ontology (GO) was performed using clusterProfiler R package and Metascape. Our results indicated that specific gene modules were related to different stages of oocyte development using WGCNA. Functional enrichment analysis and gene co-expression network analysis found significant enrichment of the mitochondrial regulation genes at the GV stage. PCA (principal component analysis) and differential gene expression analysis suggested that GV was significantly different from the MI and MII stages between the two groups. Further analysis demonstrated that the upregulated differentially expressed genes at the GV stage of patients with PCOS mainly related to mitochondrial function, such as COX6B1, COX8A, COX4l1, and NDUFB9. Meanwhile, these genes tended to be activated at the MII stage in healthy cells, suggesting that some mitochondrial functions may be prematurely activated at the GV stage of PCOS oocytes, whereas this process occurs at the MII stage in healthy oocytes. Collectively, our study showed that aberrant mitochondrial function at the GV stage may contribute to a decline in oocyte quality of PCOS patients.

Cloning and Expression Analysis of Cdc25 Gene of <i>Sipunculus nudus</i> in Oocytes

Cell division cycle 25 (Cdc25) is an important dual specificity phosphatase, which plays an important role in regulating the process of oocyte meiosis and embryo development. In this study, the full-length cDNA of Sn-Cdc25 was cloned from S. nudus using RACE technology. The results show that Sn-Cdc25 is 4 130 bp in length, including 3′ UTR 1 849 bp and 5′ UTR 427 bp. The Open reading frame (ORF) is 1 854 bp and encodes 617 amino acids. Sequence analysis shows that the molecular weight of Sn-Cdc25 protein is 69.58 kD, with two typical Cdc25 protein domains: M-phase inducer phosphatase domain and Rhodanese-like domain, and the active site sequence HCX 5 R that can catalyze the dephosphorylation process. Multi-sequence alignment finds that the C-terminal homology is higher than the N-terminal. The tertiary structure prediction shows that the spatial conformation of Cdc25 homologous protein and its active site are highly conservative. A total of 5 Motifs are found in Motif analysis, of which Motif 1 and Motif 2 are Paxillin LD motif and MYND domain binding motif, respectively. Phylogenetic tree analysis shows that Cdc25 is clustered into two branches: invertebrates and vertebrates. RT-PCR results show that the expression of Sn-Cdc25 , with two peaks, is significantly different in different developmental stages of oocytes. The increase in the expression of Sn-Cdc25 from primary vitellogenic stage to the late of active vitellogenic stage (O1-O3) may be related to the process of Sn-Cdc25 promoting DNA replication. When the oocytes entering metanephridium from coelomic fluid, the rapid rise of Sn-Cdc25 expression may be beneficial to the activation of maturation promoting factor (MPF). The above results have accumulated basic data for further understanding of the developmental mechanism of Sipuncula oocytes and for optimization of artificial breeding techniques.

GDF9 polymorphisms: influence on ovarian response in women undergoing controlled ovarian hyperstimulation.

Objective:The study looked into the possible influence of GDF9 polymorphisms on ovarian response in women with a normal ovarian reserve undergoing controlled ovarian hyperstimulation for in vitro fertilization (IVF).Methods:This cross-sectional study included 67 women with normal ovarian reserve aged 30-39 years submitted to controlled ovarian hyperstimulation for IVF. We sequenced four polymorphisms in the GDF9 gene (C398G, C447T, G546A, and G646A) and analyzed their influence on follicular and oocyte outcomes.Results:The mutant allele C398G decreased the total number of follicles >17mm (6.49 vs. 4.33, p=0.001), total number of follicles (10.11 vs. 7.33, p=0.032), number of MII oocytes retrieved, and serum progesterone levels on trigger day. The C447T polymorphism was associated with a greater number of follicles between 12 and 14 mm on the day of r-hCG, while the G546A polymorphism was associated with lower serum progesterone levels on trigger day.Conclusions:GDF9 gene polymorphisms C398G and C447T adversely affected ovarian response in women undergoing controlled ovarian hyperstimulation. These findings show that in addition to playing a role in the early stages of folliculogenesis, GDF9 polymorphisms have an important impact on the final stage of oocyte development.

New insights in oocyte dynamics shed light on the complexities associated with fish reproductive strategies

Information on temporal variations in stock reproductive potential (SRP) is essential in fisheries management. Despite this relevance, fundamental understanding of egg production variability remains largely unclear due to difficulties in tracking the underlying complex fluctuations in early oocyte recruitment that determines fecundity. We applied advanced oocyte packing density theory to get in-depth, quantitative insights across oocyte stages and seasons, selecting the commercially valuable European hake (Merluccius merluccius) as a case study. Our work evidenced sophisticated seasonal oocyte recruitment dynamics and patterns, mostly driven by a low-cost predefinition of fecundity as a function of fish body size, likely influenced also by environmental cues. Fecundity seems to be defined at a much earlier stage of oocyte development than previously thought, implying a quasi-determinate – rather than indeterminate – fecundity type in hake. These results imply a major change in the conceptual approach to reproductive strategies in teleosts. These findings not only question the current binary classification of fecundity as either determinate or indeterminate, but also suggest that current practices regarding potential fecundity estimation in fishes should be complemented with studies on primary oocyte dynamics. Accordingly, the methodology and approach adopted in this study may be profitably applied for unravelling some of the complexities associated with oocyte recruitment and thereby SRP variability.

PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development.

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. PTPN11 (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.

Human Egg Maturity Assessment and Its Clinical Application.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg's developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg's readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte's developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

Fatty Acid Profile of Hepatopancreas and Oocyte of Tiger Shrimp, Penaeus monodon, Fed Modified-commercial Diet by Supplementing Vitamin C and E

Vitamin C and E have important roles in the process of reproduction, immune system, and anti-oxidants to protect cell membrane lipid, and the circulation of essential fatty acid in the body of the aquatic organism including shrimp. The aims of this study was to analyze the effect of vitamin C and E supplementation in modified-commercial diet on the fatty acids profile of hepatopancreas and oocyte of tiger shrimp, Penaeus monodon . The experimental treatments were: modified-commercial diet by supplementing with vitamin C (500 mg/kg) and E (300 mg/kg) (PS), and modified-commercial diet without vitamins supplementation (PK). Cultured tiger shrimp were used as the animal with initial weight of 43.1 ± 5.1 g (mean±STD) for females and 41.9 ± 4.4 g for males. The shrimps were cultured in two of 1000 m 2 concrete ponds with a density of 100 shrimp / pond. During three months culture in the pond, they were fed the test at rate of 3% biomass/day. When the tiger shrimp reached the weight over 90 g for females and > 70 g for males, then the shrimp were transferred to concrete tanks for oocyte maturation evaluation. In maturation tank, the shrimp fed test diets (pellet) combined with fresh food (squid) as much as 3% (dry basis) at combination ratio of 50% : 50%. Shrimp reaching oocyte development stage III were dissected and sampled for hepatopancreas and oocytes for analysis of fatty acid profile. The results showed that the hepatopancrea and oocyte fatty acids contents were higher in the tiger shrimp fed PS compared to the tiger shrimp fed PK. The poly unsaturated fatty acid (PUFA) especially for arachidonic, docosahexaenoic, and eicosapentaenoic acid were higher in shrimp fed with PS than the shrimp fed PK. Despite higher total fatty acid contents in hepatopancreas than in oocyte for the both treatments, the esensial fatty acid arachidonic, docosahexaenoic, and eicosapentaenoic acid were higher in oocyte than in hepatopancreas of the shrimp. This suggested that these three types of PUFA played important role for oocyte development for female reproduction. Supplementation of vitamin C and E in the test diet had an effect on increasing PUFA content in hepatopancreas and oocytes of tiger shrimp.

Comparative impact of coumaphos, amitraz and plant extract of Ageratum conyzoides on the oogenesis of Rhipicephalus microplus

The present experiment was conducted to evaluate and compare the impact of Ageratum conyzoides plant extract (ACE) with routinely used synthetic acaricides i.e., amitraz and coumaphos on the oogenesis of engorged adult females of Rhipicephalus microplus tick. On the day of dropping from the host, panoistic ovary of R. microplus appeared white in colour, horseshoe shaped, hollow tubular organ with immature oocytes predominantly in dorsal groove. Different developmental stages of oocytes (I–V) proceed simultaneously and asynchronously. Oocytes showed gradual increase in size, deep brown colored with accumulation of eggs in oviduct during 24–72 hours of development.At LC90 concentration a highly significant (p < 0.001) cessation of egg laying after exposure to amitraz and ACE while significant reduction (p < 0.01) of egg laying in coumaphos treated ticks was observed. Upon dissection of treated ticks, uterus and oviduct packed with eggs, which failed to pass out was observed. The histo-architectural alterations including presence of extensive vacuolation, alteration of oocyte morphology, deformation of chorion and disorganization of yolk granules were observed in the treated ovaries. Histochemically, low level of storage or synthesis of essential elements viz., proteins, polysaccharides and lipids in treated oocytes responsible for reduction of fertility and inhibition of progress of vitellogenesis was observed.

In Situ Hybridization for Detecting Mature MicroRNAs In Vivo at Single-Cell Resolution.

MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate. We present an in situ hybridization protocol to map miRNA subcellular expression in single cells in vivo in four days. Using this protocol, we mapped distinct miRNAs that accumulate in the cytoplasm of one sibling oocyte but not another, dependent on the oocyte developmental stage. Thus, this method provides spatial and temporal resolution of the heterogeneity in expression of miRNAs during Caenorhabditis elegans oogenesis. This protocol can generally be adapted to any tissue amenable to dissection and fixation. © 2019 by John Wiley & Sons, Inc.

Cyclic variations of gonad development of snow trout, Schizopyge niger from the river Jhelum of Kashmir Himalaya

The aim of the present study was to know the cyclic changes in gonadal maturation and to investigate the various developmental stages of oocytes and testicular germ cells of snow trout, Schizopyge niger. A total of 316 fish specimens were procured from three sites of river Jhelum during November 2016 to October 2017 and the histological analysis of their gonad was carried out. The results of the present study indicates that the male population was found significantly (p < 0.05) higher compared to female population and were reported as (1.6:1; M:F). The highest mean GSI value was recorded in male (12.24 ± 0.89) during the month of March and for female (13.74 ± 0.92) also in the month of March, which indicated that the gonadal development reached its peak during this month. The highest mean oocyte diameter was 1.04 ± 0.20 mm observed in March, showing that the oocyte reached its maturity during this month. Histological study of ovary revealed the evidence of vitellogenic yolk granule stage and ripe mature stage from December to April. While in case of male, four stages of spermatogenesis were distinguished and spermatozoa were highly abundant in the months of March and April. Mean ± SE of absolute fecundity was 3,224 ± 421 eggs and also, the relative fecundity was 69 egg/g body weight. Based on the results of GSI and the analysis of gonad histology, it was observed that the peak breeding season of S. niger occurred in the month of March. It was also noted that spawning period of this fish begins in the month of February and lasts till April with a peak breeding season in the month of March. The present study will provide a baseline information about the appropriate peak breeding of the fish, which will be useful for artificial breeding as well for prevention of fishing from natural habitat.

Intracellular localization of vitellogenin receptor mRNA and protein during oogenesis of a parthenogenetic tick, Haemaphysalis longicornis

BackgroundVitellogenin (Vg), a key molecule for oocyte development synthesized in the fat body during blood-feeding, is released into the hemolymph and then taken into the oocytes via Vg receptor (VgR) in ticks. Previously, we showed that VgR mRNA is expressed in the ovary at the adult stage of parthenogenetic Haemaphysalis longicornis ticks and its expression increases after blood-feeding. However, intracellular localization of VgR mRNA and protein at each developmental stage of oocytes during oogenesis remains largely unclear.MethodsmRNA and protein expression profiles of H. longicornis VgR (HlVgR) in the oocytes from the unfed to oviposition periods were analyzed by real-time PCR, in situ hybridization, and immunostaining. To elucidate the timing of the onset of Vg uptake, RNA interference (RNAi)-mediated gene silencing of HlVgR was performed.ResultsIn situ hybridization revealed that HlVgR mRNA was detected in the cytoplasm of stage I-III oocytes, and weaker positive signals for HlVgR mRNA were found in the cell periphery of stage IV and V oocytes. Likewise, HlVgR protein was detected by immunostaining in the cytoplasm of stage I-III oocytes and in the cell periphery of stage IV and V oocytes. Each developmental stage of the oocytes showed distinct patterns of mRNA and protein expression of HlVgR. Moreover, RNAi of HlVgR caused delayed or arrested development in the oocytes. The ovaries of control ticks showed all developmental stages of oocytes, whereas stage I-III oocytes were found in the ovaries of HlVgR-RNAi ticks at 5 days after engorgement.ConclusionsThese results suggest that active uptake of Vg is required for development from stage III to stage IV during oogenesis. Our data clearly revealed an apparent shift in the intracellular localization of VgR for both mRNA and protein level in oocytes during oogenesis.

Oocyte-specific deletion of Hdac8 in mice reveals stage-specific effects on fertility.

Eighteen histone deacetylases exist in mammals. The class 1 histone deacetylases HDAC1 and HDAC2 are important for oogenesis and fertility in mice, likely via their effects on histones. The reproductive function of HDAC8, another class 1 enzyme, has not been explored. One key target of HDAC8 is the SMC3 subunit of cohesin, an essential complex mediating sister chromatid cohesion and chromosome segregation. In current models, HDAC8 activity is required for SMC3 recycling, but this function should be dispensable in oocytes since cohesion is established during pre-meiotic S phase and maintained until meiotic resumption during ovulation. Whether other oocyte-specific HDAC8-mediated deacetylation events are required for oogenesis and female fertility is unknown. We used two Cre drivers to remove Hdac8 at specific stages of oocyte development to address whether HDAC8 is required for female fertility in mice. When HDAC8 was knocked out in oocytes in primary and later stage follicles (Zp3-Cre), oogenesis and folliculogenesis appeared normal and mice were fertile. However, females were subfertile when HDAC8 was knocked out prior to pre-meiotic S phase and cohesion establishment (Vasa-Cre). This subfertility was independent of chromosome segregation errors during meiosis but rather appeared to be the result of defects in oogenesis that resulted in smaller fully grown oocytes with a reduced ability to resume meiosis. In all cases, we did not observe compensatory changes in HDAC1, HDAC2 and HDAC3 levels. Thus, although oocyte-specific expression of HDAC8 is not essential for mouse oogenesis after meiotic S phase, it contributes to optimal fertility. We infer that oocyte-specific expression of the deacetylase HDAC8 is required early in oogenesis for optimal fertility.

Describing gonad development and gametogenesis in southern Africa’s endemic box jellyfish Carybdea branchi (Cubozoa, Carybdeidae)

Information on the reproductive development of species of box jellyfish (class Cubozoa) is poor globally, despite their significance as potent stingers and worrying projections about range expansions. While most species are confined to tropical and subtropical waters, the South African box jellyfish Carybdea branchi is commonly found in the cold-water (especially southern) Benguela ecosystem. Its biology is unknown. Here, we examine reproductive development and describe gametogenesis in this dioecious species. Four and five maturity classes were determined for males and females, respectively, as well as five oocyte developmental stages. Oocyte sizes differed significantly between developmental stages. Mature and immature medusae differed significantly in size, but males and females did not. Individuals matured at approximately 30 mm average bell dimensions. Carybdea branchi displayed gonadal development characteristic of a semelparous organism.

MicroRNAs: potential targets and agents of endocrine disruption in female reproduction.

MicroRNAs are short non-coding RNAs that have been widely recognized as key mediators in the epigenetic control of gene expression and which are present in virtually all cells and tissues studied. These regulatory molecules are generated in multiple steps in a process called microRNA biogenesis. Distinct microRNA expression patterns during the different stages of oocyte and embryo development suggest important regulatory roles for these small RNAs. Moreover, studies antagonizing specific microRNAs and enzymes in microRNA biogenesis pathways have demonstrated that interference with normal miRNA function leads to infertility and is associated with some reproductive abnormalities. Endocrine disrupting chemicals such as Bisphenol A (BPA) are synthetic hormone mimics that have been found to negatively impact reproductive health. In addition to their direct effects on gene expression, these chemicals are widely implicated in the disruption of epigenetic pathways, including the expression and activity of miRNAs, thereby altering gene expression. In this review, the roles of microRNAs during mammalian oocyte and embryo development are outlined and the different mechanisms by which endocrine disruptors such as BPA interfere with these epigenetic regulators to cause reproductive problems is explored.

All Oocytes Attach to the Dorsal Ovarian Epithelium in the Ovary of Medaka, Oryzias latipes.

In the teleost fish medaka, an adult ovary simultaneously contains developing oocytes at all phases of oogenesis during the breeding season. However, it remains unclear where oocytes at each developmental stage are located in the ovary by the time of ovulation. To examine the relationship between the developmental stage of oocytes and their positions in the ovary of vertebrate medaka during oogenesis, the stage of oocyte development was determined from the diameter of the oocytes and the cellular morphological characteristics, such as the germinal vesicle and micropyle at the animal pole and attaching filaments at the vegetal pole, and the positions of developing oocytes in the ovary in all sections were observed. Furthermore, to investigate the characteristics of the dorsal ovarian epithelium to which the oocytes attach themselves, the dorsal and vegetal ovarian epithelia were observed. The dorsal ovarian epithelium invaginated in spots. When all serial sections of the oocytes were observed, all oocytes at stages I-VIII were attached to the dorsal ovarian epithelium, regardless of whether it invaginated or not.

Effects of Different Light Spectra on the Oocyte Maturation in Grass Puffer Takifugu niphobles.

In order to examine the effects of four different light spectra (white, red, green, and blue) on the oocyte maturation, the change of reproductive parameters, via brain-pituitary-gonad (BPG) axis in grass puffer, were investigated. After exposure four different light spectra for 7 weeks, the abundance of gonadotropin-releasing hormone (GnRH) mRNA which is a type of seabream (sbGnRH) and two different subunit of gonadotropin hormones mRNAs, follicle-stimulating hormone (fshβ) mRNA and luteinizing hormone (lhβ) mRNA, were analyzed in the brain and pituitary. Histological analysis showed that the mature oocyte ratio in the green spectrum was higher than other light spectra-exposed groups. Gonadosomatic index (GSI) and oocyte developmental stage were also investigated in the gonad based on histological observations. GSI value with the presence of yolk stage oocytes was significantly increased in the green spectrum-exposed group when compared to that of the other light-exposed groups (white, red, and blue) (p˂0.05). The abundances of sbGnRH mRNA and fshβ mRNA in the green spectrum-exposed group were also significant higher than those of the other light spectra-exposed groups (p˂0.05). These results indicate that the maturation of oocyte in grass puffer can be accelerated by exposure to the spectrum of green. To better understand the molecular mechanism for the maturation of oocyte in grass puffer, further study examining the relationship between oocyte development and its related genes is required.

Ovarian morphology and development of 3 to 8 months old African catfish, Clarias gariepinus

Thirty female African catfish of 3, 4, 5, 6, 7, and 8 months (n = 5 per age) cultured in south-east Nigeria were used to investigate ovarian morphology and development using gross anatomical and histological techniques. The results illustrated that the gonadosomatic indices of the African catfish peaked at 7 months of age (20.05 ± 1.45). Histologically, the ovaries of 3-month-old fish exhibited only pre-vitellogenic oocytes, while 4- to 8-month-old fish showed all stages of oocyte development, with predominance of pre-vitellogenic at 4 and 5 months old, and mature oocytes at 6 to 8 months old. The African catfish demonstrated an asynchronous mode of ovarian development. Oocytes representing the primary, secondary, and maturation growth phases were observed. Based on the gonadosomatic index and the histological evaluation of these ovaries, it was suggested that the African catfish mature for spawning at 7 months of age; moreover, its asynchronous mode of ovarian development may indicate that oocyte development and onset of sexual maturity of the catfish may be influenced by the amount and quality of food available in the culture environment. The study has provided essential information on the oogenesis and ovarian development of C. gariepinus, which may be useful for effective production and management of the fish in captivity.

Manipulating Cohesin Levels in Live Mouse Oocytes.

The cohesin complex is essential for chromosome segregation in mitosis and meiosis. Cohesin is a tripartite protein complex that holds sister chromatids together from DNA replication until anaphase. In mammals, meiotic DNA replication occurs in oogonia of embryos and chromosome segregation occurs in oocytes of sexually mature females. Sister chromatid cohesion establishment and chromosome segregation are thus separated by months in the mouse and decades in the human. The meiotic cohesin complex that maintains sister chromatid cohesion must therefore hold replicated sisters together for a long time in oocytes. Remarkably, this is achieved by establishing cohesion exclusively in prenatal oocytes. Meiotic cohesion in females ismaintained without detectable turnover and cohesinis therefore thought to be a long-lived protein complex. Nevertheless, the lifespan of cohesin molecules is limited as chromosomal cohesin levelsdecline with maternal age. The age-related loss of cohesin and weakened cohesion correlate with an age-related increase in chromosome missegregation of meiosis I oocytes that results in aneuploid eggs. Therefore, loss of chromosomal cohesin has been proposed to be a leading cause of the maternal age effect. To better understand cohesin deterioration in oocytes, it is crucial to gain insights into mammalian cohesion establishment and maintenance mechanisms by manipulating cohesin in live oocytes.This chapter describes techniques that address the manipulation of meiotic cohesin levels in mouse oocytes. First, we describe how cohesin can be efficiently removed from meiotic chromosomes by injecting mRNA encoding TEV protease in live oocytes expressing cohesin with engineered TEV recognition sites, followed by imaging. Secondly, we describe how cohesin expression can be induced during different stages of oocyte development using genetically modified mouse strains. In particular, we describe how to determine the deletion timing of germline-specific Cre recombinases using β-galactosidase staining of fetal ovaries. Lastly, we provide guidance on how to quantify cohesin levels on metaphase I chromosome spreads.