Germinal Vesicle Breakdown Research Articles

Could fertility clinics offer a sizable improvement of live birth rates by maturing post-GVBD oocytes in vitro?

The objective of this study is to analyze the potential of immature, denuded, post-GVBD (germinal vesicle breakdown) oocytes (including prometaphase I, metaphase I, and prometaphase II stages) to result live birth after in vitro maturation. Furthermore, we compared two culture media to identify which of them provides better reproductive outcomes when used for in vitro maturation. We performed a retrospective cohort study including 4022 IVF-ICSI (in vitro fertilization-intracytoplasmic sperm injection) cycles between 2011 and 2015. A total of 4450 immature post-GVBD oocytes from 1442 cycles were cultured in vitro; of these, 2364 oocytes reached MII (metaphase II) stage (IVMC oocytes, in vitro meiotic completion) and were fertilized. Overall, 3933 embryo transfers were performed: 3579 were embryos derived from MII oocytes (ET-MII); 264 were embryos derived from MII + IVMC oocytes (ET-MIX), and 90 embryos from IVMC oocytes (ET-IVMC). In total, 399 IVMC embryos were transferred. Maturation rate for immature post-GVBD oocytes was 54.1%. G-2™PLUS (Vitrolife) medium provided significantly higher maturation rate (p < 0.001) than G-IVF™PLUS (Vitrolife) (65.7% vs. 42.5%, p < 0.001). Embryos in ET-IVMC in cleavage stage had an average morphological score of 6.8/10 (7.7 in ET-MII; p < 0.001). Regarding reproductive outcomes, ET-IVMC gave 11.1% biochemical pregnancy rate, 10.0% clinical pregnancy rate, 7.8% ongoing pregnancy rate, and 5.6% live birth rate. Embryos arising from IVMC oocytes resulted in a live birth rate of 5.6%. We suggest that in vitro maturation of denuded immature post-GVBD oocytes should be performed at the very least when few MII are collected, and likely in all patients, as they provide acceptable maturation and fertilization rates, and a sizeable increase in live birth.

Microcystin-LR influences the in vitro oocyte maturation of zebrafish by activating the MAPK pathway

Harmful cyanobacteria and their production of microcystins (MCs) exert significant toxicity on reproduction of fish, especially the process of oogenesis. Our previous studies demonstrated that MCs have negative impacts on the quantity and quality of mature oocytes in female zebrafish. However, the underlying mechanisms of MCs disrupting oocyte maturation (OM) have been rarely reported. In the present study, in vitro oocytes (immature) were separated from zebrafish and treated with 1, 10, 100 μg/L MC-LR. The serine/threonine protein phosphatase 2A (PP2A) activity was downregulated significantly in oocytes exposed to 10 and 100 μg/L MC-LR for both 2 and 4 h. The phosphorylation levels of mitogen-activated protein kinase (MAPK) were detected without noticeable change in all oocytes treated with MC-LR for 2 h, whereas the activated levels of MAPK subtypes (ERK, p38 and JNK) increased remarkably in the 100 μg/L MC-LR treatment of 4 h. In the oocytes exposed to 100 μg/L MC-LR for 4 h, germinal vesicle breakdown (GVBD) rates changed abnormally and maturation-promoting factor (MPF) activity increased significantly, in accordance with the upregulation of Cyclin B protein levels. Moreover, the MAPK inhibitors (10 μM) were applied to explore the role of MAPK subtypes during MC-LR influencing OM and results showed that ERK inhibitor U0126 and p38 inhibitor SB203580 mitigated the effects of 100 μg/L MC-LR-induced MAPK hyper-phosphorylation and elevated GVBD in the oocytes. In conclusion, the present study indicates that microcystins disrupt the meiotic maturation by the pathway of MC-PP2A-MAPK-OM due to the phosphorylation disorder in oocytes.

The toxic effects and possible mechanisms of glyphosate on mouse oocytes

Glyphosate is a high-efficiency, low-toxicity, broad-spectrum herbicide. The residues of glyphosate-based herbicides are frequent pollutants in the environment. However, the effects of glyphosate on oocyte maturation, as well as its possible mechanisms, remain unclear. The present study revealed that mouse oocytes had reduced rates of germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) after treatment with 500 μM glyphosate. Reactive oxygen species (ROS) were found in mouse oocytes exposed to glyphosate, as shown by changes in the mRNA expression of related antioxidant enzyme genes (cat, sod2, gpx). After 14 h of exposure to glyphosate, metaphase II (MII) mouse oocytes displayed an abnormal spindle morphology and DNA double-strand breaks (DNA-DSBs). Simultaneously, mitochondria showed an aggregated distribution and decreased membrane potential in mouse oocytes exposed to glyphosate. The protein expression levels of apoptosis factors (Bax, Bcl-2) and the mRNA expression levels of apoptosis-related genes (bax, bcl-2, caspase3) were measured by Western blot and qRT-PCR, respectively. Meanwhile, the expression levels of autophagy-related genes (lc3, atg14, mtor) and proteins (LC3, Atg12) were significantly decreased in the glyphosate treatment group compared with the control group. Collectively, our results indicated that glyphosate exposure could interfere with mouse oocyte maturation by generating oxidative stress and early apoptosis.

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development.

NEK5, a member of never in mitosis-gene A-related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA-mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin-dependent kinase 1 in oocytes, resulting in a decrease of maturation-promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1-cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.

Oocyte maturation and active motility of spermatozoa are triggered by retinoic acid in pen shell &lt;i&gt;Atrina pectinata&lt;/i&gt;

For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.

Subfertility and reduced progestin synthesis in Pgrmc2 knockout zebrafish

Progestin receptor membrane component (Pgrmc1 & 2) is a heme-binding protein. Studies on Pgrmc1 have suggested possible roles in heme binding, activation of steroid-synthesizing P450s, along with binding and transferring of membrane proteins. However, the studies of Pgrmc1’s paralog, Pgrmc2 are still lacking. In order to determine the physiologic function(s) of Pgrmc2, we generated a zebrafish mutant line (pgrmc2−/−). We found a reduction in both spawning frequency and the number of embryos produced in female pgrmc2−/−. This subfertility is caused by reduced oocyte maturation (germinal vesicle breakdown, GVBD) in pgrmc2−/− in vivo. Nonetheless, oocytes from pgrmc2−/− had similar sensitivity to 17α,20β-dihydroxy-4-pregnen-3-one (DHP, a maturation induced progestin in zebrafish) compared with wildtype (wt) in vitro. Therefore, we hypothesized that oocyte maturation tardiness found in vivo, could be due to lack of progestin in pgrmc2−/−. Interestingly, we found significant reduced expression of hormones, receptors, and steroid synthesizing enzymes including lhcgr, egfra, ar, and esr2, cyp11a1 and hsd3b1. In addition, DHP levels in pgrmc2−/− ovaries showed a significant decrease compared to those in wt. In summary, we have provided a plausible molecular mechanism for the physiological functions of Pgrmc2 in the regulation of female fertility, likely via regulation of receptors and steroids in the ovary, which in turn regulates oocyte maturation in zebrafish.

Oogenesis of Lithodes santolla: histological and histochemical characterization

Although Lithodes santolla is a resource with high commercial importance that has aroused interest in controlled reproduction in captivity, currently, little knowledge is available about basic reproductive aspects of this species. This research study describes detailed oogenesis stages at the histological level in adult females of the southern king crab of the Magellan and Chilean Antarctic Region. Oogenesis was quantitatively analyzed from the proliferation of 10-24 μm oogonia and during oocyte growth. Five stages of ovary development were identified: multiplication (0), previtellogenesis (I), vitellogenesis (II), maturity (III) and spawning (IV). Furthermore, 11 oocyte substages were distinguished: oogonia, chromatin nucleolus, early perinucleolus, late perinucleolus, oil globule, early vitellin globule, late vitellin globule, postvitelogenic, germinal vesicle migration and germinal vesicle breakdown. Primary vitellogenesis occurred in oocytes 180-185 μm, containing acidophilic globules and surrounded by a layer of thick follicle cells (20-40 μm). Secondary vitellogenesis was evident in oocytes at 315-321 μm with numerous acidophilic granules in the cytoplasm and surrounded by thin follicle cells; then, cortical crypts appeared, indicating the prematuration stage and preparation for ovulation. This study allows establishing gonadic changes that occur during a reproductive cycle of female L. santolla and help to strengthen aquaculture initiatives and management in the Magellan and Chilean Antarctic Region.

Fipronil induces apoptosis and cell cycle arrest in porcine oocytes during in vitro maturation.

Fipronil (FPN) is a widely used phenylpyrazole pesticide that can kill pests by blocking γ-aminobutyric acid (GABA)-gated chloride channels. In addition, there are lack of studies on the effects of FPN on the female mammalian gametes. In this study, porcine oocytes were used to investigate the effects of FPN on the oocyte maturation process. The results showed that the first polar body extrusion rate significantly decreased (100μM FPN vs. control, 18.64 ± 2.95% vs. 74.90 ± 1.50%, respectively), and oocytes were arrested at the germinal vesicle stage in 100μM FPN group. Meanwhile, the FPN caused a significant increase in reactive oxygen species(ROS) levels and severe DNA damage inside the oocytes. Furthermore, apoptosis was enhanced along with decreases in mitochondrial membrane potential, BCL-xL, and the release of cytochrome C in FPN-treated group. Additionally, low CDK1 activity and delayed cyclin B1 degradation during germinal vesicle breakdown were found in the FPN-treated group, which resulted from the activation of ATM-P53-P21 pathway. In conclusion, FPN induces apoptosis and cell cycle arrest in porcine oocyte maturation because of increased ROS levels and DNA damage. This suggests that the FPN in the environment may have potential detrimental effects on the female mammalian reproductive system.

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes.

The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20μM) for 24hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5μM D4476, the results of quantitative real-time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF-4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β-catenin, TCF-4, Cx43, MAPK, PTGS-2, PTX-3, TGS-6, Bax and Bcl-2 in CCs. Western blot analysis revealed that the addition of 5μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12hr and a higher expression of Cx43 protein at 24hr compared to the group without D4476. These results indicate that adding optimum D4476 (5μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine 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.

HASPIN kinase mediates histone deacetylation to regulate oocyte meiotic maturation in pigs.

HASPIN kinase-catalyzed phosphorylation of histone H3 on threonine 3 (H3T3p) directs the activity and localization of chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC) to regulate chromosome condensation and segregation in both mitosis and meiosis. However, the function of HASPIN kinase in the meiotic maturation of porcine oocytes is not yet known. Here, we found that HASPIN mRNA is constantly expressed in porcine oocyte maturation and subsequent early embryo development. H3T3p is highly enriched on chromosomes at germinal vesicle breakdown (GVBD) stage and thereafter maintains a low level in progression through metaphase I (MI) to metaphase II (MII). Correspondingly, H3T3p was completely abolished in oocytes treated with an inhibitor of HASPIN kinase. Functionally, inhibition of HASPIN activity led to a significant reduction in the rate of oocyte meiotic maturation and the limited cumulus expansion. Additionally, HASPIN inhibition caused both spindle disorganization and chromosome misalignment in oocytes at MI and MII stage. Importantly, HASPIN inhibition severely prevented deacetylation of several highly conserved lysine (K) residues of histone H3 and H4 including H3K9, H3K14, H4K5, H4K8, H4K12 and H4K16 on the metaphase chromosomes during oocyte meiotic maturation. Taken together, these results demonstrate that HASPIN kinase regulates porcine oocyte meiotic maturation via modulating histone deacetylation.

Acentriolar microtubule organization centers and Ran-mediated microtubule formation pathways are both required in porcine oocytes.

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran-mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC- and Ran-mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo-like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the RanGTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC- and Ran-mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.

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.

Nucleus, Cytoskeleton, and Mitogen-Activated Protein Kinase p38 Dynamics during In Vitro Maturation of Porcine Oocytes.

Simple SummaryThe exact roles played by p38 during oocyte maturation are not completely known and is the main theme of this study. The subcellular localization of p-p38 was increased along the progression of porcine oocyte maturation. Alterations in p38 expression and activation appeared to participate in regulating oocyte maturation, along with the progressive reorganization of the cytoskeleton and redistribution of cytoplasmic p-p38.The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.

Identification and Characterization of a Luteinizing Hormone Receptor (LHR) Homolog from the Chinese Mitten Crab Eriocheir sinensis.

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

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.

In Vitro Growth of Preantral Follicle and Maturation of Intrafollicular Oocyte from Aged Mice

This study aimed to recover the ovarian function through in vitro culture of preantral follicles from aged mice. First, we isolated the preantral follicles from ovaries of sixty-seven-week old B6D2F1 mice with decreased fecundity to know how many follicles were present in them, which was 6 preantral follicles including 2 primary, 2 early secondary and late secondary follicles from 8 aged mice. It was confirmed that a few follicles (~2) were present in aged mice through histological analysis compared to adult mice as control. The 9 days of in vitro culture of preantal follicles showed in vitro growth and induced maturation after treatment with hCG (2.5 IU/mL) and EGF (5 ng/mL). Cumulus cells in the cumulus-oocyte complexes (COCs) were removed using hyaluronidase and oocytes at the germinal vesicle (GV) and GV breakdown (GVBD) were obtained from preantral follicle culture of aged mice in vitro. In conclusion, these observations demonstrated that there still were a few preantral follicles in the ovaries of 67 week-old mice, which we were able to culture in vitro and oocytes were obtained from them. This study proposed an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation in humans for assisted reproductive medicine.

Role of Type 1 Inositol 1,4,5-triphosphate Receptors in Mammalian Oocytes.

The ability of oocytes to undergo normal fertilization and embryo development is acquired during oocyte maturation which is transition from the germinal vesicle stage (GV), germinal vesicle breakdown (GVBD) to metaphase of meiosis II (MII). Part of this process includes redistribution of inositol 1,4, 5-triphosphate receptor (IP3R), a predominant Ca2+ channel on the endoplasmic reticulum membrane. Type 1 IP3R (IP3R1) is expressed in mouse oocytes dominantly. At GV stage, IP3R1 are arranged as a network throughout the cytoplasm with minute accumulation around the nucleus. At MII stage, IP3R1 diffuses to the entire cytoplasm in a more reticular manner, and obvious clusters of IP3R1 are observed at the cortex of the egg. This structural reorganization provides acquisition of [Ca2+]i oscillatory activity during fertilization. In this review, general properties of IP3R1 in somatic cells and mammalian oocyte are introduced.

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.

Aquaculture Breeding Enhancement: Maturation and Spawning in Sea Cucumbers Using a Recombinant Relaxin-Like Gonad-Stimulating Peptide.

Wild sea cucumber resources have been rapidly exhausted and therefore there is an urgent need to develop approaches that will help restocking. Currently, there is a lack of information regarding the genes involved in sea cucumber reproductive processes. The neurohormone relaxin-like gonad-stimulating peptide (RGP) has been identified as the active gonad-stimulating peptide in sea stars (Asteroidea), which could also be present in other echinoderm groups. In this study, a sea cucumber RGP was identified and confirmed by phylogenetic analysis. A recombinant Holothuria scabra RGP was produced in the yeast Pichia pastoris and confirmed by mass spectrometry. To assess bioactivity, four levels of purification were tested in an in vitro germinal vesicle breakdown (GVBD) bioassay. The most pure form induced 98.56 ± 1.19% GVBD in H. scabra and 89.57 ± 1.19% GVBD in Holothuria leucospilota. Cruder levels of purification still resulted in some GVBD. Upon single injection into female H. scabra, the recombinant RGP induced head waving behavior followed by spawning within 90–170 min. Spawned oocytes were fertilized successfully, larvae settled and developed into juveniles. Our results provide a key finding for the development of a break-through new artificial breeding approach in sea cucumber aquaculture.