Germinal Vesicle Breakdown In Oocytes Research Articles

Effects of orange peel extract (Citrus sinensis) treatment on zebrafish oocytes (Danio rerio) exposed to heat stress.

Heat stress (HS) can negatively impact oocytes by disrupting mitochondrial activity, increasing the production of reactive oxygen species, and decreasing antioxidant levels. This study investigated the impact of orange peel (OP) exposure on zebrafish oocytes (ZOs) diameter, survival rate, and germinal vesicle breakdown (GVBD) during HS. We investigated the antioxidant effect of flavonoids (concentration = 328.58 ppm) derived from OP (Citrus sinensis) added to in vitro maturation (IVM) media of ZOs (Danio rerio) under non-heat stress (NHS) and HS conditions to mimic in vivo HS conditions due to the global warming phenomenon on females. ZO in stage 3 (n = 1080) was treated with 4 μL of OP extract (not treated/control) under HS: 32°C (Heat stress 32°C solution/Heat stress 32°C orange peel [HS32S/HS32O]) and 34°C (Heat stress 34°C solution/Heat stress 34°C orange peel [HS34S/HS34O]); and NHS: 28°C (Non-heat stress solution/Non-heat stress orange peel [NHSS/NHSO]), during maturation. After 24 h of maturation, we observed the oocyte diameter, survival rate, and GVBD rate. The data were analyzed with IBM Statistics 23 software using two-way analysis of variance and Kruskal-Wallis (p < 0.05). The highest oocyte diameter data were in NHS treated with OP extract (NHSO) group (0.759 ± 0.01; mean ± standard error) compared with HS group using and without OP extract (HS32S [0.583 ± 0.02]; HS32O [0.689 ± 0.02]; HS34S [0.554 ± 0.02]; and HS34O [0.604 ± 0.02]). The survival rate of OP treated group, namely, NHSO (93% ± 3%), HS32O (85% ± 2%), and HS34O (80% ± 2%) was higher than that of the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). ZO treated with OP extract (NHSO [93% ± 3%], HS32O [85% ± 2%], and HS34O [80% ± 2%]) showed a higher GVBD rate than the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). It revealed that OP can enhance the oocyte diameter, survival rate, and GVBD rate of ZO under NHS and HS. Further investigation should be conducted to determine the effect of OP extract (C. sinensis) on in vivo conditions in females as an alternative treatment to face global warming.

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

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

A homozygous stop codon in HORMAD2 in a patient with recurrent digynic triploid miscarriage.

Recurrent miscarriage (RM) affects 1% to 5% of couples trying to conceive. Despite extensive clinical and laboratory testing, half of the RM cases remain unexplained. We report the genetic analysis of a couple with eight miscarriages and the search for their potential genetic etiology. Short tandem repeat (STR) markers, single nucleotide polymorphic (SNP) microarray, and human DNA methylation microarray were used to analyze the genotypes of two miscarriages. Exomes sequencing was performed on DNA from the two partners and identified variants were validated by Sanger sequencing. STR marker genotyping demonstrated that the two available miscarriages are triploid digynic and resulted from the failure of Meiosis II. SNP microarray analysis revealed an additional Meiosis I abnormality that is the segregation of the two maternal homologous chromosomes in one triploid miscarriage. Whole-exome sequencing on DNA from the two partners identified candidate variants only in the female partner in two genes with roles in female reproduction, a missense in EIF4ENIF1 (OMIM 607445) and a stop gain in HORMAD2 (OMIM 618842). EIF4ENIF1 is a eukaryotic translation initiation factor 4E nuclear import factor required for the oocyte germinal vesicle breakdown, and HORMAD2 is part of the synaptonemal complex that was hypothesized to act as a checkpoint mechanism to eliminate oocytes with asynapsis during meiotic prophase I in mice. While both genes may contribute to the phenotype, the Meiosis I abnormalities in the conceptions favor the causal role of HORMAD2 in the etiology of RM in this couple. This report illustrates the importance of comprehensively analyzing the products of conception to guide the search for the genetic causation of RM.

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Melatonin Improves Turbot Oocyte Meiotic Maturation and Antioxidant Capacity, Inhibits Apoptosis-Related Genes mRNAs In Vitro.

High-quality eggs are essential for the sustainability of commercial aquaculture production. Melatonin is a potent candidate for regulating the growth and maturation of oocytes. Therefore, research on the effect of melatonin on marine fish oocytes in vitro has been conducted. The present study successfully established a culture system of turbot (Scophthalmus maximus) oocytes in vitro and investigated the effect of melatonin on oocyte meiotic maturation, antioxidant capacity, and the expression of apoptosis-related genes. The cultures showed that turbot Scophthalmus maximus late-vitellogenic denuded oocytes, with diameters of 0.5-0.7 mm, had a low spontaneous maturation rate and exhibited a sensitive response to 17α, 20β-dihydroxyprogesterone (DHP) treatment in vitro. Melatonin increased by four times the rate of oocyte germinal vesicle breakdown (GVBD) in a concentration- and time-dependent manner. The mRNA of melatonin receptor 1 (mtnr1) was significantly upregulated in the oocyte and follicle after treatment with melatonin (4.3 × 10-9 M) for 24 h in vitro, whereas melatonin receptor 2 (mtnr2) and melatonin receptor 3 (mtnr3) remained unchanged. In addition, melatonin significantly increased the activities of catalase, glutathione peroxidase, and superoxide dismutase, as well as the levels of glutathione, while decreasing the levels of malondialdehyde and reactive oxygen species (ROS) levels in turbot oocytes and follicles cultures in vitro. p53, caspase3, and bax mRNAs were significantly downregulated in oocytes and follicles, whereas bcl2 mRNAs were significantly upregulated. In conclusion, the use of turbot late-vitellogenesis oocytes (0.5-0.7 mm) is suitable for establishing a culture system in vitro. Melatonin promotes oocyte meiotic maturation and antioxidative capacity and inhibits apoptosis via the p53-bax-bcl2 and caspase-dependent pathways, which have important potential to improve the maturation and quality of oocytes.

BPA interferes with granulosa cell development and oocyte meiosis in mouse preantral follicles.

Bisphenol A (BPA) is an established environmental endocrine disruptor and can interfere with the development of female germ cells. However, the underlying mechanisms are still unclear. We investigated the effects of BPA on granulosa cell development and meiosis of oocytes using in vitro culture system of mouse preantral follicles. Preantral follicles from D14 mouse ovary were treated with 10 μg/mL BPA in vitro for 11 days. The adherent area of follicles was measured. On D11, cumulus cell expansion was observed. The meiosis recovery rate was calculated. Western blot detected P53, proliferating cell nuclear antigen (PCNA), estrogen receptor α (ERα), and cyclin B1. ELISA measured estrogen and progesterone levels. Immunofluorescence detected Cx37 on oocyte membrane. Gap junction communication was assessed. We found that BPA significantly promoted the expressions of PCNA and ERα in granulosa cells and the secretion of estrogen and progesterone by granulosa cells on D10 and significantly increased the attachment area of the follicles on D8 and D10. However, it reduced the expansion of cumulus cells, Cx37 expression, and the gap junction communication between cumulus cells and oocytes on D11. BPA promoted the recovery of oocytes from meiosis, interrupted the expression of cyclin B1 protein in arrested germinal vesicle breakdown (GVBD) oocytes, and reduced the in vitro maturation rate of oocytes. These GVBD oocytes were live without apoptosis or death. Conclusively, BPA disturbs the development of granulosa cells and the meiosis progression of oocytes by decreasing gap junction communication between oocytes and the granulosa cells as well as regulating cyclin B1 expression in GVBD oocytes.

The embryology, metamorphosis, and muscle development of Schizocardium karankawa sp. nov. (Enteropneusta) from the Gulf of Mexico

Schizocardium karankawa sp. nov. has been collected from subtidal muds of the Laguna Madre, Texas, and the Mississippi coast, Gulf of Mexico. The Texas population is reproductive from early February to mid-April. Gametes are liberated by a small incision in a gonad. Oocyte germinal vesicle breakdown is increased in the presence of sperm, and the highest fertilization success was in the artificial seawater Jamarin U. Manually dechorionated embryos develop normally. Development was asynchronous via a tornaria larva, metamorphosis and maintained to the juvenile worm 6 gill-pore stage. Phalloidin-labeled late-stage tornaria revealed retractor muscles that connect the pericardial sac with the apical tuft anteriorly, the oesophagus ventrally, and muscle cells of the early mesocoels. The muscle development of early juvenile worms began with dorso-lateral trunk muscles, lateral trunk bands, and sphincters around the gill pores and anus. Adult worms are characterized by a stomochord that bifurcates anteriorly into paired vermiform processes, gill bars that extend almost the entire dorsal to ventral branchial region resulting in a narrow ventral hypobranchial ridge, and an elaborate epibranchial organ with six zones of discrete cell types. The trunk has up to three rows of liver sacs, and lateral gonads. The acorn worm evo-devo model species Saccoglossus kowalevskii, Ptychodera flava, and Schizocardium californicum are phylogenetically distant with disparate life histories. S. karnakawa from S. californicum are phylogenetically close, and differences between them that become apparent as adult worms include the number of gill pores and hepatic sacs, and elaborations of the heart–kidney–stomochord complex. An important challenge for evolutionary developmental biology is to form links from phylogenetically distant and large-scale differences to phylogenetically close and small-scale differences. This description of the embryology, development, and adult morphology of S. karankawa permits investigations into how acorn worm development evolves at fine scales.

ZP3 and AIPL1 participate in GVBD of mouse oocytes by affecting the nuclear membrane localization and maturation of farnesylated prelamin A.

The low maturation rate of oocytes is an important reason for female infertility and failure of assisted pregnancy. The germinal vesicle breakdown (GVBD) is a landmark event of oocyte maturation. In our previous studies, we found that zona pellucida 3 (ZP3) was strongly concentrated in the nuclear region of germinal vesicle (GV) oocytes and interacted with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) and lamin A to promote GVBD. In the current study, we found that lamin A is mainly concentrated in the nuclear membrane. When ZP3 is knocked down, lamin A will be partially transferred to the nucleus of oocytes. The prelamin A is increased in both the nuclear membrane and nucleus, while phosphorylated lamin A (p-lamin A) is significantly reduced. AIPL1 was also proved to accumulate in the GV region of oocytes, and ZP3 deletion can significantly inhibit the aggregation of AIPL1 in the nuclear region. Similar to ZP3 knockdown, the absence of AIPL1 resulted in a decrease in the occurrence of GVBD, an increase in the amount of prelamin A, and a significant decrease in p-lamin A in oocytes developed in vitro. Finally, we propose the hypothesis that ZP3 can stabilize farnesylated prelamin A on the nuclear membrane of AIPL1, and promote its further processing into mature lamin A, therefore promoting the occurrence of GVBD. This study may be an important supplement for the mechanism of oocyte meiotic resumption and provide new diagnostic targets and treatment clues for infertility patients with oocyte maturation disorder.

Granulosa Cell Specific Loss of Adar in Mice Delays Ovulation, Oocyte Maturation and Leads to Infertility.

Adenosine deaminases acting on RNA-(ADAR) comprise one family of RNA editing enzymes that specifically catalyze adenosine to inosine (A-to-I) editing. A granulosa cell (GC) specific Adar depleted mouse model [Adar flox/flox:Cyp19a1-Cre/+ (gcAdarKO)] was used to evaluate the role of ADAR1 during the periovulatory period. Loss of Adar in GCs led to failure to ovulate at 16 h post-hCG, delayed oocyte germinal vesicle breakdown and severe infertility. RNAseq analysis of GC collected from gcAdarKO and littermate control mice at 0 and 4 h post-hCG following a super-ovulatory dose of eCG (48 h), revealed minimal differences after eCG treatment alone (0 h), consistent with normal folliculogenesis observed histologically and uterine estrogenic responses. In contrast, 300 differential expressed genes (DEGs; >1.5-fold change and FDRP < 0.1) were altered at 4 h post-hCG. Ingenuity pathway analysis identified many downstream targets of estrogen and progesterone pathways, while multiple genes involved in inflammatory responses were upregulated in the gcAdarKO GCs. Temporal expression analysis of GCs at 0, 4, 8, and 12 h post-hCG of Ifi44, Ifit1, Ifit3b, and Oas1g and Ovgp1 confirmed upregulation of these inflammatory and interferon genes and downregulation of Ovgp1 a glycoprotein involved in oocyte zona pellucida stability. Thus, loss of ADAR1 in GCs leads to increased expression of inflammatory and interferon response genes which are temporally linked to ovulation failure, alterations in oocyte developmental progression and infertility.

Neuregulin 1 (NRG1) modulates oocyte nuclear maturation during IVM and improves post-IVF embryo development

Oocyte in vitro maturation (IVM) is still a major challenge in human and animal assisted reproduction. Gradual instead of abrupt activation of the ovulatory cascade during IVM has been proposed to enhance nuclear-cytoplasmic synchrony and cumulus-oocyte communication, thus favoring oocyte developmental competence. Herein, we assessed the effects of neuregulin 1 (NRG1), an EGF-like factor that modulates EGFR signaling, on oocyte nuclear maturation dynamics, cumulus expansion and expression of mRNAs regulating these processes during IVM, as well as on post-IVF embryo development following AREG-stimulated IVM in cattle. In experiment 1, cumulus-oocyte complexes (COCs) were subjected to IVM with graded doses of NRG1 (1, 10 or 100 ng/mL) for 6, 9, 12, 20, and 24 h, after which oocyte nuclear status and cumulus mRNA expression were assessed. At 6 h of IVM, NRG1 at 1 ng/mL significantly decreased the percentage of GVBD (germinal vesicle breakdown) oocytes without altering later meiotic dynamics or the percentage of oocytes achieving meiosis II. In experiment 2, adding NRG1 (1 ng/mL) to the IVM medium did not affect cumulus expansion but increased the percentage of expanded and hatched blastocysts, and blastocyst total cell number following IVF/IVC. NRG1 decreased EGFR mRNA abundance while increasing NPR2 and PTX3 mRNA levels at 9 h, and TNFAIP6 mRNA abundance at 20 h of IVM. This is the first study that reports the modulatory effect of NGR1 during oocyte maturation in a mono-ovulatory species and demonstrates that this action may be applied during IVM to improve post-IVF embryo development.

Octocrylene exposure impairs mouse oocyte quality by inducing spindle defects and mitochondria dysfunction

One of organic ultraviolet (UV) filters, Octocrylene (OCL), is mainly used in various cosmetic products, which is being frequently detected in soil, sediment, aquatic systems and food chain. There is evidence confirmed the reproductive toxicity of OCL in Japanese medaka. However, less was known about the effects of OCL exposure on oocyte quality. Here, we investigated the impacts of OCL on mouse oocyte maturation and quality by exposing oocytes to OCL in vitro at concentrations of 8, 22, 30, 40 and 50 nM. The results showed that OCL markedly reduced mouse oocyte germinal vesicle breakdown (GVBD) at 50 nM and polar body extrusion (PBE) rates at 40 and 50 nM. OCL exposure further disrupted spindle assembly and chromosome alignment, finally inducing aneuploid. Mitochondrial function was also damaged by OCL exposure, leading to ROS overproduction and apoptosis in oocytes. Moreover, OCL treatment impaired the distribution of cortical granules and sperm binding ability of oocytes. In summary, these data demonstrated that OCL could disturb the oocyte meiotic maturation and reduce oocyte quality.

The insulin-like growth factor 1 stimulates ovarian steroidogenesis and oocyte maturation in spotted steed Hemibarbus maculatus

The insulin-like growth factor 1 (IGF1) plays important roles in fish reproduction. To understand the role of IGF1 in steroidogenesis and oocyte maturation, we investigated expression pattern and functions of Igf1 during in vitro maturation of spotted steed (Hemibarbus maculatus) oocytes. Igf1 expression quantified by qPCR and western blotting revealed that both Igf1 mRNA and protein was significantly increased in mature follicles. The immunofluorescence analyses showed IGF1 protein signal was detected in the theca cells of ovarian follicles during vitellogenesis stage, and in both theca cells and granulosa cells of matured oocytes. Incubation of ovarian follicles with recombinant IGF1 (rIGF1) significantly enhanced the rate of spotted steed oocyte germinal vesicle breakdown (GVBD), a marker of oocyte maturation. qPCR analysis showed that the transcription of steroidogenesis-related genes (3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a) were influenced after IGF1 treatment. In addition, the expression profiles of receptors of Igf1, Fsh and Lh indicated that a cross talk response to IGF1 induced oocyte maturation. Furthermore, IGF1 treatment did not affect ERK protein expression but significantly upregulated CyclinB and AKT protein expression. All together, these findings help us to understand the molecular mechanism of IGF1 in regulation ovarian steroidogenesis and oocyte maturation of spotted steed.

Approaches to oocyte meiotic arrest in vitro and impact on oocyte developmental competence.

Oocytes are maintained in a state of meiotic arrest following the first meiotic division until ovulation is triggered. Within the antral follicle, meiotic arrest is actively suppressed in a process facilitated by the cyclic nucleotides cGMP and cAMP. If removed from this inhibitory follicular environment and cultured in vitro, mammalian oocytes undergo spontaneous meiotic resumption in the absence of the usual stimulatory follicular stimuli, leading to asynchronicity with oocyte cytoplasmic maturation and lower developmental competence. For more than 50years, pharmacological agents have been used to attenuate oocyte germinal vesicle (GV) breakdown in vitro. Agents that increase intra-oocyte cAMP or prevent its degradation have been predominantly used; however, agents such as kinase and protein synthesis inhibitors have also been trialed. Twenty years of research demonstrates that maintaining GV arrest for a period before in vitro maturation (IVM) improves oocyte developmental competence, and is likely attributed to maintenance of bidirectional communication with cumulus cells leading to improved oocyte metabolic function. However, outcomes are influenced by various factors including the mode of action of the modulators, dose, treatment duration, species, and the degree of hormonal priming of the oocyte donor. Cyclic GMP and/or cAMP modulation in a prematuration step (called pre-IVM) prior to IVM has shown the greatest consistency in improving oocyte developmental competence, whereas kinase and protein synthesis inhibitors have proven less effective at improving IVM outcomes. Such pre-IVM approaches have shown potential to alter current use of artificial reproductive technologies in medical and veterinary practice.

Azole pharmaceuticals induce germinal vesicle breakdown (GVBD) in preovulatory oocytes of zebrafish (Danio rerio): an in vitro study.

Azoles, the antifungal pharmaceuticals are emerging as a new class of water contaminants with a potential to influence the endocrine physiology of surrounding aquatic fauna. In this study, we made an attempt to assess the relative efficacy of widely used azoles belonging to two subclasses, i.e., (i) triazoles (letrozole, fluconazole, itraconazole) and (ii) imidazoles (ketaconazole, ornidazole, clotrimazole), on the onset of germinal vesicle breakdown (GVBD) (an initial step in the final maturation of oocytes) in fully grown preovulatory oocytes of zebrafish (Danio rerio) using an in vitro model. Oocytes (> 650 μm) isolated manually from gravid ovaries were exposed to (i) 0.01 and/or 0.1, 1.0, 5.0, 10, 15, and 20 ng/ml and (ii) 1.0, 2.0, 3.0, 4.0, and 5.0 μg/ml of drugs. Zebrafish Ringer's solution (vehicle) and 0.01%ethyl alcohol (solvent) were used as negative controls. 17α, 20 β-Dihydroxy-4-pregnen-3-one (17α-DHP) and diethylstibestrol (DES), potent inducers of GVBD in fish, were used as positive controls. GVBD was scored hourly from 0-6 h. In negative controls, there were no indications of GVBD even at the 6th hour, while in 17α-DHP- and DES-exposed oocytes, GVBD was initiated from the 1st hour, reaching 80% and 76% respectively at the 6th hour. Among azoles, letrozole induced GVBD in 73-85%, fluconazole (30-33%), itraconazole (23-33%), ketaconazole (46-53%), ornidazole (36-40%), and clotrimazole (30-33%) of oocytes. These results suggest that azole pharmaceuticals induce GVBD in fish oocytes that may be attributed to their variable degree of cytochrome P450 enzyme inhibitor activity.

Microcystin-LR promotes zebrafish (Danio rerio) oocyte (in vivo) maturation by activating ERK1/2-MPF signaling pathways, and cAMP is involved in this process

Cyanobacterial blooms and their secondary metabolites, microcystins (MCs), are not only toxic to aquatic organisms, but also to humans. MCs exert reproductive toxicity in female fish by affecting the oocyte development. However, the mechanism behind MC-LR interference in oocyte development remains largely unknown. In our study, adult female zebrafish were exposed to MC-LR (0, 1, 5, 20 μg/L) for 30 d. After exposure to MC-LR for 30 d, fertilized eggs from the treated females and healthy males were collected and cultured in water without MC-LR. Histomorphological observations showed pathological damage in the ovary after MC-LR exposure, which was mainly characterized by enlarged intercellular spaces, detachment of follicular cells from oocytes, and vacuolation of parenchymal tissues. The 20 μg/L MC-LR treatment caused a remarkable increase in the rate of the zebrafish oocytes germinal vesicle breakdown (GVBD) and a significant decrease in the levels of cyclic adenosine monophosphate (cAMP) and vitellogenin (VTG). In addition, the phosphorylation levels of the extracellular signal-regulated kinases (ERK) were elevated in ovaries from zebrafish exposed to 5 and 20 μg/L MC-LR, and cyclinB phosphorylation levels were also upregulated notably in the 20 μg/L MC-LR group. However, MC-LR exposure did not cause any change in the levels of cAMP-dependent protein kinase (PKA) protein and cdc2 phosphorylation in all the treatments. All the doses of MC-LR reduced the number of eggs, prematurely hatched the fertilized eggs and increased the abnormal rate of offspring generation. In summary, the present study demonstrates that MC-LR promotes oocyte maturation by activating the ERK1/2 and MPF signaling pathways, and cAMP is involved in this process.

Effects of melatonin on the in vitro growth of early antral follicles and maturation of ovine oocytes

This study aimed to evaluate the effect of melatonin on the in vitro culture and maturation of isolated sheep early antral follicles. Isolated early antral follicles were cultured for 12 d in α-minimum essential medium (MEM+) alone (control) or α-MEM+ added with fixed different concentrations (100, 500, or 1,000 pg/mL) or a sequential concentration of melatonin (MelSeq; day 6 = 100; day 12 = 500 pg/mL). The percentage of morphologically normal follicles was higher (P < 0.05) in 500 pg/mL melatonin than the other treatments at 6 d. Mel 500 also showed a higher rate of fully grown oocytes (P < 0.05) than other treatments. After in vitro culture, reactive oxygen species (ROS) levels in oocytes were similar between Mel 500 and MelSeq, with both being lower (P < 0.05) than other treatments. Oocytes cultured in both Mel 500 and Mel 1000 showed glutathione peroxidase levels similar (P > 0.05) to the control group and higher (P < 0.05) than other treatments. Mitochondrial activity was similar (P > 0.05) among control, Mel 500, and Mel 1000 treatments. Mel 500 treatment presented a higher percentage of germinal vesicle breakdown oocytes than the control group and similar percentages to the other treatments. Follicles cultured in melatonin followed by oocyte maturation with the addition of 500 pg/mL melatonin in maturation medium showed increased (P < 0.05) levels of mitochondrial activity compared to α-MEM+ alone. In conclusion, the concentration of 500 pg/mL of melatonin promotes development and decreases ROS levels of ovine oocytes from in vitro grown early antral follicles. Moreover, melatonin increases mitochondrial activity and promotes the acquisition of meiotic competence of these oocytes.

Serotonergic Mechanisms of Oocyte Germinal Vesicle Breakdown in the Mud Crab, Scylla paramamosain.

The mechanism of serotonin (5-HT)-induced oocyte germinal vesicle breakdown (GVBD) in the mud crab, Scylla paramamosain, was investigated in this study. Histological staining showed that there were two meiotic arrests in oocyte, appearing at prophase I and metaphase I. This result indicated that meiosis I arrest at prophase I in S. paramamosain was similar to that of vertebrates, but meiosis II arrest at metaphase I was different from that of vertebrates. Resumption of oocytes arrest at meiosis prophase I could be induced by 5-HT rapidly within 5 min in S. paramamosain. We obtained the sequence of the 5-HT receptor type 1A (5-HTR1A) from the NCBI database, and found that 5-HTR1A was expressed in oocytes and follicle cells. In addition, we found that an agonist 8-OH-DPAT which binds 5-HTR1A induced GVBD and an antagonist WAY100635 which inhibited 5-HT induced GVBD in S. paramamosain. This result showed that 5-HTR1A mediated the regulation of oocyte GVBD by 5-HT. To explore the functional mechanism of 5-HT in inducing oocyte GVBD, forskolin, a cAMP agonist was used. Results showed that, forskolin significantly blocked 5-HT-induced GVBD, and there was a negative correlation between GVBD rate and cAMP level. Our data indicate that there are two meiotic arrests in S. paramamosain, and the resumption of prophase I arrest can be induced by 5-HT, which binds to 5-HTR1A, and this process is mediated by cAMP, which acts as negative regulator via cAMP signaling pathway.

A Crown-of-Thorns Seastar recombinant relaxin-like gonad-stimulating peptide triggers oocyte maturation and ovulation

The Acanthaster planci species-complex [Crown-of-Thorns Seastar (COTS)] are highly fecund echinoderms that exhibit population outbreaks on coral reef ecosystems worldwide, including the Australian Great Barrier Reef. A better understanding of the COTS molecular biology is critical towards efforts in controlling outbreaks and assisting reef recovery. In seastars, the heterodimeric relaxin-like gonad stimulating peptide (RGP) is responsible for triggering a neuroendocrine cascade that regulates resumption of oocyte meiosis prior to spawning. Our comparative RNA-seq analysis indicates a general increase in RGP gene expression in the female radial nerve cord during the reproductive season. Also, the sensory tentacles demonstrate a significantly higher expression level than radial nerve cord. A recombinant COTS RGP, generated in a yeast expression system, is highly effective in inducing oocyte germinal vesicle breakdown (GVBD), followed by ovulation from ovarian fragments. The findings of this study provide a foundation for more in-depth molecular analysis of the reproductive neuroendocrine physiology of the COTS and the RGP.

Plasminogen activator, tissue type regulates germinal vesicle breakdown and cumulus expansion of bovine cumulus-oocyte complex in vitro†.

Plasminogen activator, tissue type (PLAT) and its inhibitor serpin family E member 1 (SERPINE1) cooperatively regulate PLAT activity in various reproductive processes. However, it is unknown whether this includes bovine oocyte maturation. We addressed this question in the present study by evaluating PLAT and SERPINE1 protein localization in immature cumulus-oocyte complexes (COCs), as well as PLAT mRNA and protein expression in cultured COCs after 0, 8, 16, and 24 h of in vitro maturation (IVM). We also examined the effects of PLAT and SERPINE1 on germinal vesicle breakdown (GVBD) and oocyte cyclic 3' 5' adenosine monophosphate (cAMP) levels, cumulus expansion index, and expansion-related gene expression in oocytes derived from bovine COCs cultured for 4, 8, and 12 h and in COCs cultured for 16 h. Both PLAT and SERPINE1 localized in cumulus cells but only the latter was detected in oocytes. PLAT and SERPINE1 transcript levels increased during IVM; however, from 8 to 16 h, the levels of PLAT remained stable whereas those of SERPINE1 increased, resulting in a decline in PLAT concentration. Additionally, PLAT delayed GVBD, increased oocyte cAMP levels, and blocked cumulus expansion and associated gene expression, which was reversed by SERPINE1 supplemented. Thus, PLAT delays bovine oocyte GVBD by enhancing oocyte cAMP levels during the first 8 h of IVM; suppression of PLAT activity via accumulation of SERPINE1 in COCs results in cumulus expansion from 8 to 16 h of IVM. These findings provide novel insights into the molecular mechanisms underlying in vitro bovine oocyte maturation.

Stimulatory effects of NESFATIN-1 on meiotic and developmental competence of porcine oocytes.

NESFATIN-1 acts as a neuroendocrine hormone to suppress gonadotropin secretion in the female goldfish and to prevent germinal vesicle breakdown of oocytes in the zebrafish. However, the expression and function of NESFATIN-1 in meiotic maturation and development of porcine oocytes remains elusive. Genomic structure of porcine NESFATIN-1 precursor nucleobindin 2 (NUCB2) is first characterized in detail and an evolutionally closer relationship of NESFATIN-1 between pig and rat is shown by phylogenetic analysis of multiple species. Additionally, immunofluorescence analysis revealed that NESFATIN-1 is predominantly expressed and localizes on the membrane of both theca cells and granulosa cells, but not expressed in oocytes. Real-time quantitative polymerase chain reaction showed that the abundance of NESFATIN-1 transcripts in granulosa cells progressively decreases during the developmental transition from small follicles to large follicles. Correspondingly, NESFATIN-1 could significantly enhance both the cleavage and blastocyst rate of parthenogenetically activated oocytes from small follicles (p < 0.05), whereas it did not affect meiotic maturation and development of oocytes from large follicles. Interestingly, we found that NESFATIN-1 significantly improves meiotic maturation of oocytes cultured in chemically defined medium in the absence of pyruvate compared with the control group (p < 0.05), suggesting that the NESFATIN-1 as a substitute for pyruvate exerts beneficial effects on porcine oocyte maturation. In conclusion, these results demonstrate that NESFATIN-1 facilitates both meiotic maturation and development of porcine oocytes.