Non-growing Oocytes Research Articles

Chromatin remodeler CHD4 establishes chromatin states required for ovarian reserve formation, maintenance, and germ cell survival.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGO) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotic arrested NGOs. In addition, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes in male germ cells, allowing male germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 μM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. N/A.

LSM14B controls oocyte mRNA storage and stability to ensure female fertility

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

P–437 The ovaries of transgender men indicate effects of high dose testosterone on the primordial and early growing follicle pool

Abstract Study question Does high-dose testosterone therapy affect the stage distribution, morphological health and DNA damage repair capacity of human ovarian follicles and their survival in vitro? Summary answer Testosterone exposure is associated with reduced follicle growth activation, reduced follicle health and increased DNA damage: these further deteriorate after six days of culture. What is known already: Androgens have diverse actions within the ovary, however, there is a lack of information regarding the long-term effects of high-dose testosterone on ovarian function and reproductive potential. Cumulus-oocyte complexes recovered from transgender men have been successfully matured in-vitro but little is known regarding the impact of this gender affirming endocrine therapy on the primordial follicle pool. Study design, size, duration: Whole ovaries were obtained from four transgender men aged 25–36 years with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12–16 week intervals for a minimum of 4 years pre-operatively. Cortical tissues were dissected into small pieces (≈1x1x0.5mm) and either immediately fixed for histological analysis or cultured for 6 days. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (n = 4, age 26–36). Participants/materials, setting, methods: Follicle number, classification of developmental stage and morphology were evaluated by histological analysis of ovarian cortical tissue from day 0 and 6 days post culture. Immunohistochemical analysis included γH2AX as a marker of DNA damage, and meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM) and Rad51 as DNA repair proteins. A total of 3802 follicles from testosterone exposed and 878 from control ovaries were analysed. Main results and the role of chance: At day 0 (D0), transgender tissue had a higher proportion of non-growing follicles (92.7±1.7%) compared to control (85.4±6.2%, p &amp;lt; 0.05) but a lower proportion of morphologically healthy follicles (non-growing 59%, primary 61%, secondary 36%; vs 83%, 75%, 80% in controls, all p &amp;lt; 0.005). After 6 days in culture, the proportion of growing follicles increased (51.3% vs 46.5%) but follicle health further declined (all stages p &amp;lt; 0.005). DNA damage was assessed by expression of γH2AX. At D0, the proportion of oocytes showing DNA damage was significantly higher in transgender non-growing follicles (48.1±12.5%, vs 12.3±0.25%, p &amp;lt; 0.005). After culture, γH2AX expression increased in both transgender (p &amp;lt; 0.005) and controls (p &amp;lt; 0.005) but remained higher in transgender oocytes (non-growing 72.2%, primary 71.7% vs 27.3%, 46.2%, all p &amp;lt; 0.05). At D0, there was no difference in expression of DNA repair enzymes ATM and RAD51 between transgender and control oocytes, and increased expression of MRE11 in control non-growing follicles (p &amp;lt; 0.05). Post-culture, there was a significant increase in ATM expression in transgender non-growing oocytes compared to control (98.5% vs 77.8%, p &amp;lt; 0.05) and a less marked decline in RAD51 expression(p &amp;lt; 0.05). The expression of MRE–11 in control non-growing oocytes dramatically declined (100% to 58.2%, p &amp;lt; 0.05), unlike in transgender tissue where expression was comparable to D0. Limitations, reasons for caution A large number of follicles have been analysed, but only from a small number of ovaries. DNA damage at D0 and after 6 days of culture may not reflect DNA damage and repair capacity at later stages of follicle growth. The effect of duration of testosterone treatment was not investigated. Wider implications of the findings: These data indicate that high circulating concentrations of testosterone have previously unrecognised effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures. Trial registration number n/a

P-437 The ovaries of transgender men indicate effects of high dose testosterone on the primordial and early growing follicle pool

Abstract Study question Does high-dose testosterone therapy affect the stage distribution, morphological health and DNA damage repair capacity of human ovarian follicles and their survival in vitro? Summary answer Testosterone exposure is associated with reduced follicle growth activation, reduced follicle health and increased DNA damage: these further deteriorate after six days of culture. What is known already Androgens have diverse actions within the ovary, however, there is a lack of information regarding the long-term effects of high-dose testosterone on ovarian function and reproductive potential. Cumulus-oocyte complexes recovered from transgender men have been successfully matured in-vitro but little is known regarding the impact of this gender affirming endocrine therapy on the primordial follicle pool Study design, size, duration Whole ovaries were obtained from four transgender men aged 25-36 years with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12-16 week intervals for a minimum of 4 years pre-operatively. Cortical tissues were dissected into small pieces (≈1x1x0.5mm) and either immediately fixed for histological analysis or cultured for 6 days. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (n = 4, age 26-36). Participants/materials, setting, methods Follicle number, classification of developmental stage and morphology were evaluated by histological analysis of ovarian cortical tissue from day 0 and 6 days post culture. Immunohistochemical analysis included γH2AX as a marker of DNA damage, and meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM) and Rad51 as DNA repair proteins. A total of 3802 follicles from testosterone exposed and 878 from control ovaries were analysed. Main results and the role of chance At day 0 (D0), transgender tissue had a higher proportion of non-growing follicles (92.7±1.7%) compared to control (85.4±6.2%, p &amp;lt; 0.05) but a lower proportion of morphologically healthy follicles (non-growing 59%, primary 61%, secondary 36%; vs 83%, 75%, 80% in controls, all p &amp;lt; 0.005). After 6 days in culture, the proportion of growing follicles increased (51.3% vs 46.5%) but follicle health further declined (all stages p &amp;lt; 0.005). DNA damage was assessed by expression of γH2AX. At D0, the proportion of oocytes showing DNA damage was significantly higher in transgender non-growing follicles (48.1±12.5%, vs 12.3±0.25%, p &amp;lt; 0.005). After culture, γH2AX expression increased in both transgender (p &amp;lt; 0.005) and controls (p &amp;lt; 0.005) but remained higher in transgender oocytes (non-growing 72.2%, primary 71.7% vs 27.3%, 46.2%, all p &amp;lt; 0.05). At D0, there was no difference in expression of DNA repair enzymes ATM and RAD51 between transgender and control oocytes, and increased expression of MRE11 in control non-growing follicles (p &amp;lt; 0.05). Post-culture, there was a significant increase in ATM expression in transgender non-growing oocytes compared to control (98.5% vs 77.8%, p &amp;lt; 0.05) and a less marked decline in RAD51 expression(p &amp;lt; 0.05). The expression of MRE-11 in control non-growing oocytes dramatically declined (100% to 58.2%, p &amp;lt; 0.05), unlike in transgender tissue where expression was comparable to D0. Limitations, reasons for caution A large number of follicles have been analysed, but only from a small number of ovaries. DNA damage at D0 and after 6 days of culture may not reflect DNA damage and repair capacity at later stages of follicle growth. The effect of duration of testosterone treatment was not investigated. Wider implications of the findings These data indicate that high circulating concentrations of testosterone have previously unrecognised effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures. Trial registration number n/a

From Mother or Father: Uniparental Embryos Uncover Parent-of-Origin Effects in Humans.

In mammals, both parents make unique contributions to the offspring and maternal and paternal genomes are required for development. Two recent papers in Cell Stem Cell (Leng etal., 2019; Sagi etal., 2019) study uniparental embryos and uniparental embryonic stem cells to interrogate parent-of-origin effects in human embryogenesis.

MLL2 conveys transcription-independent H3K4 trimethylation in oocytes.

Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferasegene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.

Chromatin condensation of Xist genomic loci during oogenesis in mice

Repression of maternal Xist (Xm-Xist) during preimplantation in mouse embryos is essential for establishing imprinted X chromosome inactivation. Nuclear transplantation (NT) studies using nuclei derived from non-growing (ng) and full-grown (fg) oocytes have indicated that maternal-specific repressive modifications are imposed on Xm-Xist during oogenesis, as well as on autosomal imprinted genes. Recent studies have revealed that histone H3 lysine 9 trimethylation (H3K9me3) enrichments on Xm-Xist promoter regions are involved in silencing at the preimplantation stages. However, whether H3K9me3 is imposed on Xm-Xist during oogenesis is not known. Here, we dissected the chromatin states in ng and fg oocytes and early preimplantation stage embryos. Chromatin immunoprecipitation experiments against H3K9me3 revealed that there was no significant enrichment within the Xm-Xist region during oogenesis. However, NT embryos with ng nuclei (ngNT) showed extensive Xm-Xist derepression and H3K9me3 hypomethylation of the promoter region at the 4-cell stage, which corresponds to the onset of paternal Xist expression. We also found that the chromatin state at the Xist genomic locus became markedly condensed as oocyte growth proceeded. Although the condensed Xm-Xist genomic locus relaxed during early preimplantation phases, the extent of the relaxation across Xm-Xist loci derived from normally developed oocytes was significantly smaller than those of paternal-Xist and ngNT-Xist genomic loci. Furthermore, Xm-Xist from 2-cell metaphase nuclei became derepressed following NT. We propose that chromatin condensation is associated with imprinted Xist repression and that skipping of the condensation step by NT leads to Xist activation during the early preimplantation phase.

Forced expression of DNA methyltransferases during oocyte growth accelerates the establishment of methylation imprints but not functional genomic imprinting

In mammals, genomic imprinting governed by DNA methyltransferase DNMT3A and its cofactor DNMT3L is essential for functional gametes. Oocyte-specific methylation imprints are established during oocyte growth concomitant with DNMT3A/DNMT3L expression, although the mechanisms of oocyte-specific imprinting are not fully understood. To determine whether the presence of DNMT3A/DNMT3L in oocytes is sufficient for acquisition of methylation imprints, we produced transgenic mice to induce DNMT3A/DNMT3L expression prematurely in oogenesis and analyzed DNA methylation imprints. The results showed that 2- to 4-fold greater expression of DNMT3A/DNMT3L was achieved in non-growing (ng) oocytes versus fully grown oocytes derived from wild-type mice, but the analyzed imprint domains were not methylated. Thus, the presence of DNMT3A/DNMT3L in ng oocytes is insufficient for methylation imprints, and imprinted regions are resistant to DNMT3A/DNMT3L in ng oocytes. In contrast, excess DNMT3A/DNMT3L accelerated imprint acquisition at Igf2r, Lit1, Zac1 and Impact but not Snrpn and Mest in growing oocytes. Therefore, DNMT3A/DNMT3L quantity is an important factor for imprint acquisition. Transcription at imprinted domains is proposed to be involved in de novo methylation; however, transcription at Lit1, Snrpn and Impact was observed in ng oocytes. Thus, transcription cannot induce DNMT3A catalysis at imprinted regions even if DNMT3A/DNMT3L is present. However, the accelerated methylation imprints in oocytes, with the exception of Igf2r, were erased during embryogenesis. In conclusion, a sufficient amount of DNMT3A/DNMT3L and a shift from the resistant to permissive state are essential to establish oocyte-specific methylation imprints and that maintenance of the acquired DNA methylation imprints is essential for functional imprinting.

Incorporation of Phosphatase Inhibitor in Culture Prompts Growth Initiation of Isolated Non-Growing Oocytes

In vitro folliculogenesis of primordial and early preantral follicles is necessary for increment of reproductive efficiency in domestic animals, humans and endangered species. Recent study in phosphatase and tensin homolog (Pten) -knockout mice has revealed that this phosphatase acts as an inhibitory factor in follicle activation of primordial pool with the resultant inhibition of oocyte growth. To test in vitro effect of a phosphatase inhibitor on growth initiation of isolated non-growing oocytes in neonatal ovaries, we applied a specific inhibitor (bpV (HOpic)) for PTEN in culturing system. Non-growing oocytes isolated from the ovaries of newborn BDF1 (C57BL/6 × DBA/2) pups were divided to four culture groups. Five days after culture, the oocytes in 14 μmol/l bpV only, 14 μmol/l bpV plus 100 ng/ml Kit Ligand (KL), and 100 ng/ml KL groups showed significantly (P<0.05) growth (19.3±0.55, 25.8±0.53 and 21.6±0.29 μm, respectively) compared with that of the control (no additive) (16.9±0.53 μm). In addition, western blotting in those groups showed enhanced expression of phosphorylated Akt. In conclusion, we clearly demonstrate that isolated non-growing oocytes develop in phosphatase inhibitor, especially to PTEN, incorporated culturing system, and show first as we know that oocytes with zona Pellucidae can be obtained in vitro from isolated non-growing oocytes.

Long exposure to mature ooplasm can alter DNA methylation at imprinted loci in non-growing oocytes but not in prospermatogonia

DNA methylation imprints that are established in spermatogenesis and oogenesis are essential for functional gametes. However, the mechanisms underlying gamete-specific imprinting remain unclear. In this study, we investigated whether male and female gametes derived from newborn mice are epigenetically plastic and whether DNA methylation imprints are influenced by the niche surrounding the nuclei of the gametes. When prospermatogonia possessing sperm-specific DNA methylation imprints were fused with enucleated fully grown oocytes and exposed to the ooplasm for 5–6 days, the DNA methylation status of the reconstituted oocytes remained identical to that of prospermatogonia for all the imprinted regions analysed. These results suggest that the imprinting status of prospermatogonia is stable and that the epigenome of prospermatogonia loses sexual plasticity. By contrast, when non-growing oocytes lacking oocyte-specific DNA methylation imprints were fused with enucleated fully grown oocytes and the reconstituted oocytes were then cultured for 5–6 days, the Igf2r, Kcnq1ot1 and, unexpectedly, H19/Igf2 differentially methylated regions (DMRs) were methylated. Methylation imprints were entirely absent in oocytes derived from 5-day-old mice, and H19/Igf2 DMR is usually methylated only in spermatogenesis. These findings indicate that in the nuclei of non-growing oocytes the chromatin conformation changes and becomes permissive to DNA methyltransferases in some DMRs and that mechanisms for maintaining non-methylated status at the H19/Igf2 DMR are lost upon long exposure to mature ooplasm.

Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases

DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.

Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey

Widespread use of the endocrine disrupting chemical bisphenol A (BPA) in consumer products has resulted in nearly continuous human exposure. In rodents, low-dose exposures have been reported to adversely affect two distinct stages of oogenesis in the developing ovary: the events of prophase at the onset of meiosis in the fetal ovary and the formation of follicles in the perinatal ovary. Because these effects could influence the reproductive longevity and success of the exposed individual, we conducted studies in the rhesus monkey to determine whether BPA induces similar disturbances in the developing primate ovary. The routes and levels of human exposure are unclear; hence, two different exposure protocols were used: single daily oral doses and continuous exposure via subdermal implant. Our analyses of second trimester fetuses exposed at the time of meiotic onset suggest that, as in mice, BPA induces subtle disturbances in the prophase events that set the stage for chromosome segregation at the first meiotic division. Our analyses of third-trimester fetuses exposed to single daily oral doses during the time of follicle formation revealed an increase in multioocyte follicles analogous to that reported in rodents. However, two unique phenotypes were evident in continuously exposed animals: persistent unenclosed oocytes in the medullary region and small, nongrowing oocytes in secondary and antral follicles. Because effects on both stages of oogenesis were elicited using doses that yield circulating levels of BPA analogous to those reported in humans, these findings raise concerns for human reproductive health.

Roles of Genes Regulated by Two Paternally Methylated Imprinted Regions on Chromosomes 7 and 12 in Mouse Ontogeny

We studied the longevity of mice produced without sperm using the genomes of oocytes that are already committed to a germline cell lineage. The first sperm-free mouse "KAGUYA", which we term 'bi-maternal mouse', was born on 3 February, 2003. Bi-maternal embryos were generated using 2 sets of female genomes--one derived from fully grown oocytes from normal adults and the other from non-growing oocytes from newborn pups. These genomes were combined by nuclear transfer. We refined the technique for generating bi-maternal mice and found that genetic manipulations in only 2 regions--the imprinting centres of Igf2-H19 and Dlk1-Gtl2--on chromosomes 7 and 12 of the newborn pups allowed us to generate bi-maternal mice at a high rate. Studying bi-maternal conceptuses and mice provides further insight into the mechanisms by which paternally methylated imprinted genes regulate mammalian ontogenesis.

Longevity in mice without a father

Females live longer than males in many mammalian species, including humans. It has been observed that women are at an advantage over men with regard to the lifespan; however, the reason for this sex difference in longevity is unclear. Bi-maternal mice (BM), which are produced in a 'sperm-free' manner, could provide an opportunity to analyse the longevity of animals lacking paternal genomes. We studied the longevity of BM, which were generated using two sets of female genomes--one derived from fully grown oocytes from normal adults and the other from non-growing oocytes from newborn pups. These newborn pups were also genetically manipulated in two regions--the imprinting centres of Igf2-H19 and Dlk1-Gtl2--on chromosomes 7 and 12. We determined lifespan of the control (n = 13) and BM (n = 13). Our results revealed that the bi-maternal genotype clearly shifted the entire survival curve to the right, suggesting a delay in the expression of all causes of mortality. BM survived 186 days longer than controls. Furthermore, the body weight was significantly lower in the BM as compared with the controls at 20 months after birth (P < 0.05), and leukocyte composition analysis at 8 weeks revealed that the eosinophil count was significantly increased in the BM as compared with the controls (P < 0.05, n = 6). These findings demonstrate that the maternal genome may play a role in ontogenetic longevity. Our results further suggested sex differences in longevity, originating at the genome level, implying that the sperm genome has a detrimental effect on longevity in mammals.

Large-scale production of growing oocytes in vitro from neonatal mouse ovaries

Although fetal or neonatal mammalian ovaries contain many non-growing oocytes within primordial follicles, most degenerate and only a few contribute to the oocyte pool in the mature ovary. Here, we report a follicle-free culture system that allows a large number of these arrested oocytes to enter the growth phase in vitro. As many as 800 oocytes from a newborn mouse, corresponding to more than 10(4) oocytes in large animals, continued to develop, formed a zona pellucida, and were able to fuse with spermatozoa. Some oocytes reached the size of those in normal antral follicles and entered metaphase I, indicating the completion of the growth phase. The key to success was the sequential provision of essential nutrients and growth factors to the oocytes, while preventing the apoptosis that normally occurs in the majority of growing oocytes in vivo. Importantly, maternal genomic imprinting, which is necessary for normal embryonic development, was imposed correctly on their genomes autonomously. Thus, arrested primordial oocytes can be rescued effectively in vitro and can undergo the morphological and genomic modifications necessary for fertilization and subsequent embryonic development. This culture system may provide a significant impetus to the development of new techniques for the efficient production of oocytes from fetal or neonatal ovaries, for research, clinical, and zoological purposes.

Generation and characterization of mouse parthenogenetic embryonic stem cells containing genomes from non‐growing and fully grown oocytes

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem cells. Unfortunately, differentiation capacity of the parthenogenetic embryonic stem cells was rather lower than fertilized embryos derived ES cells, which might be the result of the absence of male genome. It had been found that some maternally expressed genes were repressed and some paternally expressed genes were expressed in the non-growing oocytes. Therefore, maternal genome from non-growing oocytes can partially act as "sperm genome". In the present study, parthenogenetic blastocysts containing genome from non-growing and fully grown oocytes (named as NF-pBlastocysts) were produced by germinal vesicle transfer, and three newly established parthenogenetic embryonic stem (named as NF-pES) cell lines were derived from the resulting parthenogenetic blastocysts. All three NF-pES cell lines were positive for ES cell markers, such as alkaline phosphatase (AKP), stage-specific embryonic antigen 1 (SSEA-1) and octamer-binding transcription factor (Oct-4). They have a normal chromosome karyotype (40) and can be maintained in an undifferentiated state for extended periods of time. When NF-pES cells were injected into severe combined immunodeficient mice, teratomas with all three embryonic germ layers were obtained. The in vitro differentiation potential of NF-pES cells was analyzed by embryonic bodies (EB) formation. The expression of germ layer markers, such as nestin (ectoderm), desmin (mesoderm), and alpha-fetoprotein (endoderm) demonstrated that the NF-pES cells can differentiate into all three germ layers.

High-frequency generation of viable mice from engineered bi-maternal embryos

Mammalian development to adulthood typically requires both maternal and paternal genomes, because genomic imprinting places stringent limitations on mammalian development, strictly precluding parthenogenesis. Here we report the generation of bi-maternal embryos that develop at a high success rate equivalent to the rate obtained with in vitro fertilization of normal embryos. These bi-maternal mice developed into viable and fertile female adults. The bi-maternal embryos, distinct from parthenogenetic or gynogenetic conceptuses, were produced by the construction of oocytes from fully grown oocytes and nongrowing oocytes that contain double deletions in the H19 differentially methylated region (DMR) and the Dlk1-Dio3 intergenic germline-derived DMR. The results provide conclusive evidence that imprinted genes regulated by these two paternally methylated imprinting-control regions are the only paternal barrier that prevents the normal development of bi-maternal mouse fetuses to term.

Long-Term Effects of In Vitro Growth of Mouse Oocytes on Their Maturation and Development

Since very few oocytes grow completely in vivo, in vitro growth (IVG) of ovarian oocytes may provide a new source of functional oocytes. The long-term effects of in vitro maturation (IVM) of oocytes and in vitro culture of fertilized eggs have been reported; however, the effects of IVG of oocytes are unknown. Here in, we report the long-term effects of IVG of oocytes. Ovaries from 1-day-old mice containing non-growing oocytes were cultured for 10 days; the isolated follicles were then cultured for 11 days. Secondary follicles from 10-day-old mice were also cultured for 11 days. The nuclei of oocytes collected from the IVG and Graafiais follicles of adult mice were transferred to enucleated oocytes grown in vivo, respectively. Developmental competence was examined following IVM of the reconstituted oocytes. Chronologically, oocytes of 1-day-old, 10-day-old and adult mice were cultured for 22, 12 and 1 day(s). The result showed that the reconstituted eggs developed into pups at high rates after nuclear transfer and in vitro fertilization (IVF) in all the experimental groups (29-45%). However, the pups from reconstituted eggs containing the nuclei of 22-day cultured oocytes were heavier than the control pups (P<0.05). We concluded that long-term culture of oocytes did not affect their nuclear ability to develop to term; however, fetal growth was affected by the culture duration or culture conditions during the initial phase of follicular growth.

Aberrant DNA methylation of imprinted loci in superovulated oocytes

There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The sex-specific epigenetic modifications that are imposed during gametogenesis act as a primary imprint to distinguish maternal and paternal alleles. The most likely candidate for the gametic mark is DNA methylation. However, the timing of DNA methylation acquisition in adult oocytogenesis and the effects of superovulation are unknown. We examined the maternal methylation of PEG1(MEST), LIT1(KCNQ1OT1) and ZAC(PLAGL1) and the paternal methylation of H19 in adult growing oocytes of humans and mice and compared them with the methylation status of mouse neonatal growing oocytes by using bisulphite sequencing. Furthermore, we examined the effects of superovulation in the human and mouse. Maternal methylation of these genes has already been initiated to some extent in adult human and mouse non-growing oocytes but not in mouse neonates. In addition, the methylation dynamics during adult human and mouse oocyte development changed more gradually than those during neonatal oocyte development. Furthermore, we found the demethylation of PEG1 in growing oocytes from some ART-treated infertile women and a gain in the methylation of H19. We also detected methylation changes in superovulated mice. Our studies in the human and mouse suggest that superovulation can lead to the production of oocytes without their correct primary imprint and highlight the need for more research into ARTs.