A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human.

The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown to be possibly related to oocyte abnormality and female infertility; few reports have confirmed the functions of these mutations in living animal models. Here, we identified a novel heterozygous missense mutation (NM_001376231.1:c.1616C>T, p.Thr539Met) in ZP2 from a primary infertile female. We showed that the mutation reduced ZP2 expression and impeded ZP2 secretion in cell lines. Furthermore, we constructed the mouse model with the mutation (Zp2T541M) using CRISPR-Cas9. Zp2WT/T541M female mice had normal fertility though generated oocytes with the thin ZP, whereas Zp2T541M female mice were completely infertile due to degeneration of oocytes without ZP. Additionally, ZP deletion impaired folliculogenesis and caused female infertility in Zp2T541M mice. Our study not only expands the spectrum of ZP2 mutation sites but also, more importantly, increases the understanding of pathogenic mechanisms of ZP2 mutations.

NLRP14 Safeguards Calcium Homeostasis via Regulating the K27 Ubiquitination of Nclx in Oocyte-to-Embryo Transition.

Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.

Septin 9 controls CCNB1 stabilization via APC/CCDC20 during meiotic metaphase I/anaphase I transition in mouse oocytes.

The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time-lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI-AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5 h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5 h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI-AI transition by regulating the kinetochore-microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes.

Roles of NEK family in cell cycle regulation.

As a serine/threonine kinase, NIMA-related kinases (NEKs) play important roles in the regulation of cell cycle, and involve in several cellular activities such as centrosome separation, spindle assembly, chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signaling, cytokinesis, cilia formation and DNA damage response. In this review, we summarize the component, structural characteristics and functions of NEK family in mitosis and meiosis based on the relevant researches in recent years, providing a reference for the further study on the roles of NEKs in the regulation of cell cycle and a theoretical basis for the clinical diagnosis and treatment of tumors.

Sperm-carried RNAs play critical roles in mouse embryonic development.

Recently, numerous studies have reported that the mature sperm contains both coding and non-coding RNAs and the sperm delivers some RNAs to the oocyte at fertilization. However, the functions of the RNAs carried to the oocyte by sperm at fertilization in embryonic development remains a mystery. In this study, the mature spermatozoa were treated with lysolecithin, pronase and RNases (RNase A and RNase H) to remove the sperm-carried RNAs, and then injected into normal mature oocyte. The results showed that after the treatment, the content of the sperm RNAs was decreased by about 90%. The blastocyst formation rate and the live birth rate of the embryos from intracytoplasmic sperm injection (ICSI) using the treated sperm were significantly decreased (P<0.01), while these effects were partially rescued by injecting total wide-type sperm RNAs. The reproductive capacity of offspring (F0) in sperm-treated group was similar with that in control group (P>0.05), but the body weight of F1 mice from sperm-treated group was lower than that in control group after two weeks of birth (P<0.05). These results demonstrated that the sperm-carried RNAs have important roles in embryonic development.

Is embryonic hypothermia tolerance common in birds?

Avian incubation temperatures oscillate within narrow limits to ensure proper embryonic development. However, field observations and experimental studies have found that some species can tolerate very low incubation temperatures, either regularly or occasionally. We artificially incubated eggs from five domestic species, which represent a range of egg sizes, to examine whether a diversity of avian species could exhibit an unusual hypothermia tolerance, as observed in the field. We found that eggs of the chicken (Gallus gallus domesticus), pigeon (Columba livia domestica), Japanese quail (Coturnix japonica) and budgerigar (Melopsittacus undulatus) survived the incubation period and hatched after experiencing 10°C hypothermia for 6 h each day. However, embryos of white-rumped munia (Lonchura striata) died after 10 days of hypothermia. Our results showed that unusual hypothermia tolerance occurs in several avian species. This phenomenon might have been selected through the evolutionary history of birds. Future research should identify the importance of phylogeny, egg size and embryonic stage in tolerance to hypothermia.

Maternal obesity and diabetes may cause DNA methylation alteration in the spermatozoa of offspring in mice.

BACKGROUND: The adverse effects on offspring of diabetic and/or obese mothers can be passed to the next generation. However, the mechanisms behind this are still unclear. Epigenetics may play a key role during this process. METHODS: To confirm the hypothesis, we investigated the DNA methylation of several imprinted genes in spermatozoa of offspring from diabetic and/or obese mothers utilizing streptozotocin (STZ)- and high-fat-diet (HFD)-induced mouse models. RESULTS: We found that the DNA methylation of Peg3 was significantly increased in spermatozoa of offspring of obese mothers compared to that in spermatozoa of offspring of normal mothers. The DNA methylation of H19 was significantly higher in spermatozoa of offspring of diabetic mothers than that in spermatozoa of offspring of non-diabetic mothers. CONCLUSIONS: These results indicate that pre-gestational diabetes and/or obesity can alter DNA methylation in offspring spermatozoa.

DNA methylation in oocytes and liver of female mice and their offspring: effects of high-fat-diet-induced obesity.

BACKGROUND: Maternal obesity has adverse effects on oocyte quality, embryo development, and the health of the offspring. OBJECTIVES: To understand the underlying mechanisms responsible for the negative effects of maternal obesity, we investigated the DNA methylation status of several imprinted genes and metabolism-related genes. METHODS: Using a high-fat-diet (HFD)-induced mouse model of obesity, we analyzed the DNA methylation of several imprinted genes and metabolism-related genes in oocytes from control and obese dams and in oocytes and liver from their offspring. Analysis was performed using combined bisulfite restriction analysis (COBRA) and bisulfite sequencing. RESULTS: DNA methylation of imprinted genes in oocytes was not altered in either obese dams or their offspring; however, DNA methylation of metabolism-related genes was changed. In oocytes of obese mice, the DNA methylation level of the leptin (Lep) promoter was significantly increased and that of the Ppar-α promoter was reduced. Increased methylation of Lep and decreased methylation of Ppar-α was also observed in the liver of female offspring from dams fed the high-fat diet (OHFD). mRNA expression of Lep and Ppar-α was also significantly altered in the liver of these OHFD. In OHFD oocytes, the DNA methylation level of Ppar-α promoter was increased. CONCLUSIONS: Our results indicate that DNA methylation patterns of several metabolism-related genes are changed not only in oocytes of obese mice but also in oocytes and liver of their offspring. These data may contribute to the understanding of adverse effects of maternal obesity on reproduction and health of the offspring.

Maternal diabetes causes alterations of DNA methylation statuses of some imprinted genes in murine oocytes.

Maternal diabetes has adverse effects not only on oocyte quality but also on embryo development. However, it is still unknown whether the DNA imprinting in oocytes is altered by diabetes. By using streptozotocin (STZ)-induced and nonobese diabetic (NOD) mouse models we investigated the effect of maternal diabetes on DNA methylation of imprinted genes in oocytes. Mice which were judged as being diabetic 4 days after STZ injection were used for experiments. In superovulated oocytes of diabetic mice, the methylation pattern of Peg3 differential methylation regions (DMR) was affected in a time-dependent manner, and evident demethylation was observed on Day 35 after STZ injection. The expression level of DNA methyltransferases (DNMTs) was also decreased in a time-dependent manner in diabetic oocytes. However, the methylation patterns of H19 and Snrpn DMRs were not significantly altered by maternal diabetes, although there were some changes in Snrpn. In NOD mice, the methylation pattern of Peg3 was similar to that of STZ-induced mice. Embryo development was adversely affected by maternal diabetes; however, no evident imprinting abnormality was observed in oocytes from female offspring derived from a diabetic mother. These results indicate that maternal diabetes has adverse effects on DNA methylation of maternally imprinted gene Peg3 in oocytes of a diabetic female in a time-dependent manner, but methylation in offspring's oocytes is normal.

Cyclin O regulates germinal vesicle breakdown in mouse oocytes.

It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

Diabetic uterus environment may play a key role in alterations of DNA methylation of several imprinted genes at mid-gestation in mice.

BACKGROUND: Maternal diabetes mellitus not only has severe deleterious effects on fetal development, but also it affects transmission to the next generation. However, the underlying mechanisms for these effects are still not clear. METHODS: We investigated the methylation patterns and expressions of the imprinted genes Peg3, Snrpn, and H19 in mid-gestational placental tissues and on the whole fetus utilizing the streptozotocin (STZ)-induced hyperglycemic mouse model for quantitative analysis of methylation by PCR and quantitative real-time PCR. The protein expression of Peg3 was evaluated by Western blot. RESULTS: We found that the expression of H19 was significantly increased, while the expression of Peg3 was significantly decreased in dpc10.5 placentas of diabetic mice. We further found that the methylation level of Peg3 was increased and that of H19 was reduced in dpc10.5 placentas of diabetic mice. When pronuclear embryos of normal females were transferred to normal/diabetic (NN/ND) pseudopregnant females, the methylation and expression of Peg3 in placentas was also clearly altered in the ND group compared to the NN group. However, when the pronuclear embryos of diabetic female were transferred to normal pesudopregnant female mice (DN), the methylation and expression of Peg3 and H19 in dpc10.5 placentas was similar between the two groups. CONCLUSIONS: We suggest that the effects of maternal diabetes on imprinted genes may primarily be caused by the adverse uterus environment.

Effect of postovulatory oocyte aging on DNA methylation imprinting acquisition in offspring oocytes.

OBJECTIVE: To investigate whether postovulatory aging of oocytes in the mother affects DNA methylation acquisition of imprinted genes in oocytes from the offspring. DESIGN: Randomized research experimental study. SETTING: Academic basic research laboratory. ANIMAL(S): Mice. INTERVENTION(S): Fresh oocytes and aged oocytes from mothers were artificially inseminated, and oocytes were collected from the resultant offspring. MAIN OUTCOME MEASURE(S): Methylation status was evaluated at differentially methylated regions (DMRs) in oocytes of maternally imprinted genes Peg3, Snrpn, and Peg1 and paternally imprinted gene H19. RESULT(S): Our results showed that methylation patterns at DMRs of Peg3, Snrpn, Peg1, and H19 in oocytes from aged-oocyte offspring were mainly normal, with only a small number of oocytes showing aberrant methylation in the DMR of Peg3. CONCLUSION(S): Postovulatory oocyte aging causes a decline in reproductive outcomes but does not evidently lead to defects in DNA methylation imprinting acquisition in the oocytes from viable offspring.

The effects of postovulatory aging of mouse oocytes on methylation and expression of imprinted genes at mid-term gestation.

Previous studies by others and ourselves have suggested that the methylation pattern of imprinted genes in oocytes is altered during postovulatory aging. The purpose of the current study was to evaluate the effects of postovulatory aging of mouse oocytes on methylation and expression of imprinted genes at the mid-gestation development stages. Proestrous females were artificially inseminated at 13 h (fresh-oocyte group) or 22 h (aged-oocyte group) post-hCG. Estrous females were mated with males as a control group. On dpc (day post coitus) 10.5 of development, embryos and placentas were collected and DNA and RNA were extracted, respectively. Methylation and total expression of Igf2r and H19 was investigated by quantitative analysis of methylation by PCR and quantitative real-time RT-PCR, respectively. Our results showed no significant changes of methylation in the differentially methylated region (DMR) and total expression of Igf2r in embryos and placentas, and no significant changes in methylation of H19 in embryos at dpc 10.5 of development compared with the control group regardless of artificial insemination of fresh or aged oocytes. In contrast, placentas of the aged-oocyte group exhibited significantly lower methylation levels in the H19 DMR. Furthermore, we observed that the increased expression of H19 in placentas of the aged-oocyte group was associated with significant hypomethylation of H19 DMR. These results suggest that postovulatory aging of mouse oocytes has adversed effects on methylation and expression of H19 in placentas at the mid-gestation development stage.

[Effect of DNA methylation and histone modification during the development of cloned animals.].

Somatic cell nuclear transfer (SCNT) has great potential for agricultural applications, generation of medical model animals, transgenic farm animals or generating human embryonic stem cells for treatment of human diseases. Cloned animals derived from somatic cells have been generated in several mammal species, but there are still some unsolved problems with current cloning technology, for example, the low efficiency of animal cloning and the abnormal development of cloned animals. One critical factor of these developmental failures of cloned embryos is the aberrant epigenetic reprogramming. This review focuses on DNA methylation and histone modifications and the relationship between these two epigenetic modifications and the development of cloned embryos. Understanding the mechanisms of epigenetic regulation will be useful to solve the technical problems of SCNT and enable better applications of this technology.

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development.

Mitochondrial DNA heteroplasmy in calves cloned by using adult somatic cell.

Adult somatic cell cloned calves were produced by somatic cell nuclear transfer prepared by fusion of cultured ear fibroblast from a Holstein cow into enucleated oocytes of Luxi Yellow cow. In order to determinate the source of mitochondrial DNA of cloned calves, we designed the breed-specific PCR primers by aligning the known D-loop sequences of Bos taurus and analyzed the displacement loop sequences of five live cloned calves by breed-specific primers PCR. The results demonstrated that mtDNA originated from Holstein breed and that from Luxi breed co-exist in all five live calves.

In vitro development and mitochondrial fate of macaca-rabbit cloned embryos.

Interspecies cloning may be used as an effective method to conserve highly endangered species and to support the development of non-human primate animal models for studying therapeutic cloning and nuclear-cytoplasm interaction. The use of the monkey model for biomedical research can avoid legal, ethical, and experimental limitations encountered in a clinical situation. We describe in this study the in vitro development of macaca-rabbit embryos produced by fusing macaca fibroblasts with enucleated rabbit oocytes and examine the fate of mitochondrial DNA in these embryos. We show that macaca-rabbit cloned embryos can develop to the blastocyst stage when cultured in vitro in HECM(10) +10% FBS and that mitochondrial DNA derived from donor somatic cells was detectable in cloned embryos throughout preimplantation development. These results suggest that (1) macaca fibroblast nuclei can dedifferentiate in enucleated metaphase II rabbit oocytes; (2) HECM(10) +10% FBS can break through the development block and support the development of macaca-rabbit cloned embryos to blastocysts; and (3) donor-cell-derived mitochondrial DNA is not eliminated until blastocyst stage.

Flow cytometric cell-cycle analysis of cultured fibroblasts from the giant panda, Ailuropoda melanoleuca L.

In animal cloning, it is generally believed that the inactive diploid G(0)or G(1)stage of the cell cycle is beneficial to initiate cell-cycle coordination and reprogramming following transfer of the donor nucleus. Previous experiments have demonstrated that serum starvation results in quiescent cell stage. Some experiments show that the majority of cells in a fully confluent cell culture are also in an inactive G(1)stage. In order to provide more G(0)/G(1)stage cells for giant panda cloning, we carried out a flow cytometric analysis of the cell cycle of fibroblasts from the abdominal muscle of a giant panda at different passage numbers under different growth conditions, and after different periods of serum starvation. The percentage of G(0)+G(1)stage cells differed significantly under different growth conditions. Serum starvation effectively increased the percentage of G(0)+G(1)stage cells, and the cell cycle characteristics following serum starvation for varying periods of time differed with this and the initial confluency of the cultures. The data should help in choosing the optimal stage for preparing donor cells as well as increasing the potential cloning efficiency in our study of giant panda cloning.

Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes.

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.