The transgenerational effects of maternal low-protein diet during lactation on offspring.

Environment factors such as diet and lifestyle can influence the health of both mothers and offspring. However, its transgenerational transmission and underlying mechanisms remain largely unknown. Here, using a maternal lactation-period low-protein diet (LPD) mouse model, we show that maternal LPD during lactation causes decreased survival and stunted growth, significantly reduces ovulation and litter size, and alters the gut microbiome in the female LPD-F1 offspring. The transcriptome of LPD-F1 metaphase II (MII) oocytes shows that differentially expressed genes are enriched in female pregnancy and multiple metabolic processes. Moreover, maternal LPD causes early stunted growth and impairs metabolic health, which is transmitted for two generations. The methylome alteration of LPD-F1 oocytes can be partly transmitted to the F2 oocytes. Together, our results reveal that LPD during lactation transgenerationally affects offspring health, probably via oocyte epigenetic changes.

Maternal SMC2 is essential for embryonic development via participating chromosome condensation in mice.

During the oocyte growth, maturation and zygote development, chromatin structure keeps changing to regulate different nuclear activities. Here, we reported the role of SMC2, a core component of condensin complex, in oocyte and embryo development. Oocyte-specific conditional knockout of SMC2 caused female infertility. In the absence of SMC2, oocyte meiotic maturation and ovulation occurred normally, but chromosome condensation showed defects and DNA damages were accumulated in oocytes. The pronuclei were abnormally organized and micronuclei were frequently observed in fertilized eggs, their activity was impaired, and embryo development was arrested at the one-cell stage, suggesting that maternal SMC2 is essential for embryonic development.

Supplementation of mitochondria from endometrial mesenchymal stem cells improves oocyte quality in aged mice.

Maternal ageing is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age-related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aged oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. We cultivated endometrial mesenchymal stem cells (EnMSCs) from aged mice and extracted mitochondria from EnMSCs. To improve the quality of oocytes, GV oocytes were supplemented with mitochondria via microinjection. And MII oocytes from aged mice were fertilized by intracytoplasmic sperm injection (ICSI), combining EnMSCs' mitochondrial microinjection. In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aged oocytes. Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aged mice after ICSI. We also found that the birth rate of mitochondria-injected ageing oocytes was significantly increased after embryo transplantation. Our study demonstrates that supplementation with EnMSC-derived mitochondria can improve the quality of oocytes and promote embryo development in ageing mice, which might provide a prospective strategy for clinical treatment.

PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development.

Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here, we show that protein phosphatase 4 catalytic subunit (PPP4C) deletion causes severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibited DNA lesions that inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of the essential HR factor RAD51. On the other hand, when inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods, which couple the checkpoint to HR.

Diabetic Uterine Environment Leads to Disorders in Metabolism of Offspring.

AIMS: Research evidence indicates that epigenetic modifications of gametes in obese or diabetic parents may contribute to metabolic disorders in offspring. In the present study, we sought to address the effect of diabetic uterine environment on the offspring metabolism. METHODS: Type 2 diabetes mouse model was induced by high-fat diet combined with streptozotocin (STZ) administration. We maintained other effect factors constant and changed uterine environment by zygote transfers, and then determined and compared the offspring numbers, symptoms, body weight trajectories, and metabolism indices from different groups. RESULT: We found that maternal type 2 diabetes mice had lower fertility and a higher dystocia rate, accompanying the increased risk of offspring malformations and death. Compared to only a pre-gestational exposure to hyperglycemia, exposure to hyperglycemia both pre- and during pregnancy resulted in offspring growth restriction and impaired metabolism in adulthood. But there was no significant difference between a pre-gestational exposure group and a no exposure group. The deleterious effects, no matter bodyweight or glucose tolerance, could be rescued by transferring the embryos from diabetic mothers into normal uterine environment. CONCLUSION: Our data demonstrate that uterine environment of maternal diabetes makes critical impact on the offspring health.

Type 1 diabetes affects zona pellucida and genome methylation in oocytes and granulosa cells.

Diabetes affects oocyte nuclear and cytoplasmic quality. In this study, we generated a type 1 diabetes (T1D) mouse model by STZ injection to study the effects of T1D on zona pellucida and genomic DNA methylation of oocytes and granulosa cells. T1D mice showed fewer ovulated oocytes, reduced ovarian reserve, disrupted estrus cycle, and significantly ruptured zona pellucida in 2-cell in vivo embryos compared to controls. Notably, diabetic oocytes displayed thinner zona pellucida and treatment of oocytes with high concentration glucose reduced the zona pellucida thickness. Differential methylation genes in oocytes and granulosa cells were analyzed by methylation sequencing. These genes were significantly enriched in GO terms by GO analysis, and these GO terms were involved in multiple aspects of growth and development. Most notably, the abnormal methylation genes in oocytes may be related to oocyte zona pellucida changes in diabetic mice. These findings provide novel basic data for further understanding and elucidating dysgenesis and epigenetic changes in type 1 diabetes mellitus.

Non-canonical RNA polyadenylation polymerase FAM46C is essential for fastening sperm head and flagellum in mice†.

Family with sequence similarity 46, member C (FAM46C) is a highly conserved non-canonical RNA polyadenylation polymerase that is abundantly expressed in human and mouse testes and is frequently mutated in patients with multiple myeloma. However, its physiological role remains largely unknown. In this study, we found that FAM46C is specifically localized to the manchette of spermatids in mouse testes, a transient microtubule-based structure mainly involved in nuclear shaping and intra-flagellar protein traffic. Gene knockout of FAM46C in mice resulted in male sterility, characterized by the production of headless spermatozoa in testes. Sperm heads were intermittently found in the epididymides of FAM46C knockout mice, but their fertilization ability was severely compromised based on the results of intracytoplasmic sperm injection assays. Interestingly, our RNA-sequencing analyses of FAM46C knockout testes revealed that mRNA levels of only nine genes were significantly altered compared to wild-type ones (q < 0.05). When considering alternate activities for FAM46C, in vitro assays demonstrated that FAM46C does not exhibit protein kinase or AMPylation activity against general substrates. Together, our data show that FAM46C in spermatids is a novel component in fastening the sperm head and flagellum.

Absence of mitochondrial DNA methylation in mouse oocyte maturation, aging and early embryo development.

Mitochondrial DNA (mtDNA) is important for oxidative phosphorylation; dysfunctions can play a role in many mitochondrial diseases and can also affect the aging of cells and individuals. DNA methylation is an important epigenetic modification that plays a critical role in regulating gene expression. While recent studies have revealed the existence of mtDNA methylation there are still controversies about mtDNA methylation due to the special structure of mtDNA. Mitochondria and DNA methylation are both essential for regulating oocyte maturation and early embryo development, but whether mtDNA methylation changes during this process is unknown. By employing bisulfite sequencing, we found that in the process of mouse oocyte maturation, postovulatory oocyte aging, and early embryo development, all analyzed mitochondrial genes, including 16S-CpGI, DCR, ND6, 12S, and ATP8, lacked 5'mC. Thus, mtDNA methylation does not occur in the oocyte and early embryo.

PKCß1 regulates meiotic cell cycle in mouse oocyte.

PKCßI, a member of the classical protein kinase C family, plays key roles in regulating cell cycle transition. Here, we report the expression, localization and functions of PKCßI in mouse oocyte meiotic maturation. PKCßI and p-PKCßI (phosphor-PKCßI) were expressed from germinal vesicle (GV) stage to metaphase II (MII) stage. Confocal microscopy revealed that PKCßI was localized in the GV and evenly distributed in the cytoplasm after GV breakdown (GVBD), and it was concentrated at the midbody at telophase in meiotic oocytes. While, p-PKCßI was concentrated at the spindle poles at the metaphase stages and associated with midbody at telophase. Depletion of PKCßI by specific siRNA injection resulted in defective spindles, accompanied with spindle assembly checkpoint activation, metaphase I arrest and failure of first polar body (PB1) extrusion. Live cell imaging analysis also revealed that knockdown of PKCßI resulted in abnormal spindles, misaligned chromosomes, and meiotic arrest of oocytes arrest at the Pro-MI/MI stage. PKCßI depletion did not affect the G2/M transition, but its overexpression delayed the G2/M transition through regulating Cyclin B1 level and Cdc2 activity. Our findings reveal that PKCßI is a critical regulator of meiotic cell cycle progression in oocytes. Abbreviations: PKC, protein kinase C; COC, cumulus-oocyte complexes; GV, germinal vesicle; GVBD, germinal vesicle breakdown; Pro-MI, first pro-metaphase; MI, first metaphase; Tel I, telophase I; MII, second metaphase; PB1, first polar body; SAC, spindle assembly checkpoint.

Type 2 diabetes increases oocyte mtDNA mutations which are eliminated in the offspring by bottleneck effect.

BACKGROUND: Diabetes induces many complications including reduced fertility and low oocyte quality, but whether it causes increased mtDNA mutations is unknown. METHODS: We generated a T2D mouse model by using high-fat-diet (HFD) and Streptozotocin (STZ) injection. We examined mtDNA mutations in oocytes of diabetic mice by high-throughput sequencing techniques. RESULTS: T2D mice showed glucose intolerance, insulin resistance, low fecundity compared to the control group. T2D oocytes showed increased mtDNA mutation sites and mutation numbers compared to the control counterparts. mtDNA mutation examination in F1 mice showed that the mitochondrial bottleneck could eliminate mtDNA mutations. CONCLUSIONS: T2D mice have increased mtDNA mutation sites and mtDNA mutation numbers in oocytes compared to the counterparts, while these adverse effects can be eliminated by the bottleneck effect in their offspring. This is the first study using a small number of oocytes to examine mtDNA mutations in diabetic mothers and offspring.

Transfer of autologous mitochondria from adipose tissue-derived stem cells rescues oocyte quality and infertility in aged mice.

Elder women suffer from low or loss of fertility because of decreasing oocyte quality as maternal aging. As energy resource, mitochondria play pivotal roles in oocyte development, determining oocyte quality. With advanced maternal age, increased dysfunctions emerge in oocyte mitochondria, which decrease oocyte quality and its developmental potential. Mitochondria supplement as a possible strategy for improving egg quality has been in debate due to ethnic problems. Heterogeneity is an intractable problem even transfer of germinal vesicle, spindle, pronuclei or polar body is employed. We proposed that the autologous adipose tissue-derived stem cell (ADSC) mitochondria could improve the fertility in aged mice. We found that autologous ADSC mitochondria could promote oocyte quality, embryo development and fertility in aged mice, which may provide a promising strategy for treatment of low fertility or infertility in elder women.

Essential role for SUN5 in anchoring sperm head to the tail.

SUN (Sad1 and UNC84 domain containing)-domain proteins are reported to reside on the nuclear membrane playing distinct roles in nuclear dynamics. SUN5 is a new member of the SUN family, with little knowledge regarding its function. Here, we generated Sun5-/- mice and found that male mice were infertile. Most Sun5-null spermatozoa displayed a globozoospermia-like phenotype but they were actually acephalic spermatozoa. Additional studies revealed that SUN5 was located in the neck of the spermatozoa, anchoring sperm head to the tail, and without functional SUN5 the sperm head to tail coupling apparatus was detached from nucleus during spermatid elongation. Finally, we found that healthy heterozygous offspring could be obtained via intracytoplasmic injection of Sun5-mutated sperm heads for both male mice and patients. Our studies reveal the essential role of SUN5 in anchoring sperm head to the tail and provide a promising way to treat this kind of acephalic spermatozoa-associated male infertility.

Removal of mouse ovary fat pad affects sex hormones, folliculogenesis and fertility.

As a fat storage organ, adipose tissue is distributed widely all over the body and is important for energy supply, body temperature maintenance, organ protection, immune regulation and so on. In humans, both underweight and overweight women find it hard to become pregnant, which suggests that appropriate fat storage can guarantee the female reproductive capacity. In fact, a large mass of adipose tissue distributes around the reproductive system both in the male and female. However, the functions of ovary fat pad (the nearest adipose tissue to ovary) are not known. In our study, we found that the ovary fat pad-removed female mice showed decreased fertility and less ovulated mature eggs. We further identified that only a small proportion of follicles developed to antral follicle, and many follicles were blocked at the secondary follicle stage. The overall secretion levels of estrogen and FSH were lower in the whole estrus cycle (especially at proestrus); however, the LH level was higher in ovary fat pad-removed mice than that in control groups. Moreover, the estrus cycle of ovary fat pad-removed mice showed significant disorder. Besides, the expression of FSH receptor decreased, but the LH receptor increased in ovary fat pad-removed mice. These results suggest that ovary fat pad is important for mouse reproduction.

Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity.

Mammalian oocytes are arrested at prophase of the first meiotic division in the primordial follicle pool for months, even years, after birth depending on species, and only a limited number of oocytes resume meiosis, complete maturation, and ovulate with each reproductive cycle. We recently reported that protein phosphatase 6 (PP6), a member of the PP2A-like subfamily, which accounts for cellular serine/threonine phosphatase activity, functions in completing the second meiosis. Here, we generated mutant mice with a specific deletion of Ppp6c in oocytes from the primordial follicle stage by crossing Ppp6cF/F mice with Gdf9-Cre mice and found that Ppp6cF/F; GCre+ mice are infertile. Depletion of PP6c caused folliculogenesis defects and germ cell loss independent of the traditional AKT/mTOR pathway, but due to persistent phosphorylation of H2AX (a marker of double strand breaks), increased susceptibility to DNA damage and defective DNA repair, which led to massive oocyte elimination and eventually premature ovarian failure (POF). Our findings uncover an important role for PP6 as an indispensable guardian of genomic integrity of the lengthy prophase I oocyte arrest, maintenance of primordial follicle pool, and thus female fertility.

Oocyte-specific deletion of N-WASP does not affect oocyte polarity, but causes failure of meiosis II completion.

STUDY QUESTION: There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. SUMMARY ANSWER: In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. WHAT IS KNOWN ALREADY: N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P < 0.001 compared to control). Further analysis showed that a defective midbody may be responsible for the failure of cytokinesis completion. LIMITATIONS, REASONS FOR CAUTION: The present study did not include a detailed analysis of the mechanisms underlying the results, which will require more extensive further investigations. WIDER IMPLICATIONS OF THE FINDINGS: N-WASP may play an important role in mediating and co-ordinating the activity of the spindle (midbody) and actin (contractile ring constriction) when cell division occurs. The findings are important for understanding the regulation of oocyte meiosis completion and failures in this process that affect oocyte quality. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the National Basic Research Program of China (No. 2012CB944404) and the National Natural Science Foundation of China (Nos 30930065, 31371451, 31272260 and 31530049). There are no potential conflicts of interests.

[Effect of long-term fertilization on lignin accumulation in typical subtropical upland soil].

To investigate the effect of long-term fertilization on lignin accumulation and clarify its influencing factors in subtropical agricultural upland soils, alkaline CuO oxidation and gas chromatography was performed to quantify the amount of lignin and its monomers components (V, S and C). The soil samples were collected from the fertilization treatments of NPK and NPKS (NPK combined with straw) in Huanjiang County, Guangxi Province (limestone soil) and Taoyuan County, Hunan Province (red soil). The results showed that NPK had no significant effect on the lignin content (Sumvsc) of limestone soil, whereas the content in red soil significantly increased by (55 ± 1)%. For the NPKS treatment, the lignin content in limestone and red soil increased by (328 ± 4)% and (456 ± 9)%, respectively. After the same fertilization treatment, the proportion of cinnamyl (C)-type significantly increased in red soil, while a significant increase of vanillyl (V)-type monomers occurred in limestone soil, indicating that lignin degradation in agricultural soils was monomer specific. Furthermore, the acid-to-aldehyde ratios of syringyl-type [(Ac/Al)] or vanillyl-type [(Ac/Al)v] monomers tended to decrease after long-term fertilization with the higher value for limestone soil, suggesting the degree of lignin degradation in limestone was higher than that in red soil. Soil organic matter and total nitrogen were not correlated with lignin content, but were significantly correlated with the composition of VSC monomers. Meanwhile, the available nutrient content in the soil (available nitrogen, phosphorus, and potassium) was closely related to the contents and components of V, S, and C-type monomers (P<0.05). It indicated that the availability of soil nutrition should be considered as a key factor for the accumulation of lignin.