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PubMed2026

Fetal hypoxia causes oocyte oxidative stress damage via the Sirt3/Sod2 pathway and can be alleviated by nicotinamide mononucleotide.

Environmental hypoxia exerts detrimental effects on the reproductive capabilities of both humans and animals. A fetal hypoxia model was established in which fetal mice were kept in a high-plateau hypoxic setting from embryonic day (E) 0 to 16.5. In our previous research, we found that fetal hypoxia exposure perturbs the methylation of imprinted genes in adult sperm and causes intergenerational placental impairments in male offspring. However, the specific impacts of fetal hypoxia on the female reproductive system, particularly regarding oocyte maturation, remain poorly understood. First, we found that fetal hypoxia mice exhibited a significant reduction in the average number of pups per litter. We conducted a comprehensive analysis of the transcriptome in oocytes from the hypoxic group and investigated the metabolic alterations within the follicular microenvironment. Fetal hypoxic stress contributed to cleavage and blastocyst rate reduction and induced early apoptosis and DNA damage triggered by mitochondrial dysfunction, oxidative stress aggravation and Sirt3/Sod2 downregulation. Additionally, administration of nicotinamide mononucleotide (NMN) has been shown to prevent oocytes from mitochondrial dysfunction and developmental impairment by increasing the expression of Sirt3/Sod2 and autophagy. The number of pups per litter in fetal hypoxia mice was reduced by 57.7% compared to the control group, while NMN intervention could restore it to 73.1% of the control group. These results indicate that fetal hypoxia exposure exerts multiple potential damages to adult female reproduction, while highlighting the clinical potential of NMN supplementation as a targeted intervention to alleviate such hypoxia-associated female reproductive impairment.

Animals↗
PubMed1979

Oocyte maturation: aberrant post-fusion responses of the rabbit primary oocyte to penetrating spermatozoa.

Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated previously with spermatozoa, recovered 2--5 h later and examined by light and electron microscopy. At least 4 aspects of GV oocyte/sperm interaction were abnormal. Although the vestments and oolemma seem normally receptive to spermatozoa, fusion with the oolemma of the primary oocyte did not elicit exocytosis of cortical granules, and consequently multiple entry of spermatozoa into the ooplasm was common. Secondly, the GV oocyte cortex failed to achieve a normal englufment of the anterior part of the sperm head. It sank into the ooplasm capped by only a small rostral vesicle or left the stable inner acrosomal membrane as a patch in the oolemma. Only rarely then was there significant dispersion of the sperm chromatin, and this remained surrounded by nuclear envelope. The persistence of this envelope constitutes a further aberrant feature, for it disappears immediately in secondary oocytes and was absent in primary oocytes in which germinal vesicle breakdown had occurred. The results are discussed with particular reference to current ideas about male pronucleus formation.

Animals↗
PubMed1976

[Variability of the ova of mulberry silkworm (Bombyx mori L.) with regard to capacity for thermal parthenogenesis and heat resistance. 4. Relationship between the capacity of oocytes from different females for thermal parthenogenesis and heat resistance of ova at the early stage of development and heat resistance of the muscles of these females].

The reliable positive correlation was established between the variability of unfertilized eggs by their ability of thermal parthenogenesis and the heat resistance of fertilized eggs at the early stages of development. At the same time the ability of oocytes of parthenogenesis correlates negatively with the heat resistance of muscles. The data obtained suggest that the variability of oocytes by their ability of thermal parthenogenesis is partially due to the variability of heat resistance of their proteins.

Animals↗
PubMed1977

Hormonal and follicular factors affecting maturation of sheep oocytes in vitro and their subsequent developmental capacity.

Oocytes removed from, or retained within, non-atretic and atretic follicles of different sizes were cultured for 24 h in the presence of a variety of hormones in an attempt to identify the factors affecting oocyte maturation in vitro. Resumption of meiosis was assessed morphologically; the developmental capacity of oocytes after culture was determined by transfer to the oviducts of inseminated ewes. About 70% of oocytes cultured after removal from follicles of different sizes resumed meiosis in vitro, but they did not undergo normal development after transplantation. Oocytes cultured within the follicle in hormone-free medium remained at the germinal vesicle stage. In the presence of FSH and LH some oocytes reached the second meiotic metaphase: 19% in small (2-3 mm diam.) and 73% in larger (3-5 mm diam.) non-atretic follicles, and 54% in small and 45% in larger atretic follicles. Less than 5% of oocytes cultured in follicles developed into normal blastocysts after transplantation when either no hormone or only FSH and LH were added to the culture medium. The addition of oestradiol-17beta to medium containing FSH (2 mug/ml) and LH (1 mug/ml) resulted in the development to blastocysts of 26% of oocytes from small non-atretic follicles, 46% from large non-atretic follicles and 50% from atretic follicles. Blastocyst formation was greatly depressed and fragmentation rate signinificantly increased with concentrations of 10 mugFSH/ml and 2 mug LH/ml. Developmental capacity after culture was further demonstrated by the birth of lambs from 63% of blastocysts derived from oocytes matured in vitro; 52% of control blastocysts developed to lambs after transfer.

Animals↗
PubMed1977

Induction of oocytic maturation and differentiation: mode of progesterone action.

Full-grown amphibian oocytes, arrested in prophase I or meiosis, respond in vitro to progesterone and certain other steroids. They undergo an apparently normal sequence of nuclear maturation and cytoplasmic differentiation, which are necessary precedents for fertilization and embryogenesis. Individual oocytes or populations of these cells thus provide a model system for investigations concerning the nature and mechanism of hormone-cellular interactions. In this system, previous studies have shown that certain aspects of hormonal (progesterone, DOCA) induction of morphologic and biochemical differentiation in the nucleus can be induced in part, as a result of the formation of secondary cytoplasmic factors, some of which do not require the presence of the nucleus for their formation. In addition to initiating nuclear events, progesterone or DOCA alters the functional activity of the plasma membrane and establishes the conditions necessary for fertilization and activation. Uptake of radioactive vitellogenin, a yolk protein precursor, was inhibited by progesterone and DOCA. Maximum inhibition was dependent on induction of nuclear breakdown, the dose of steroid used, and was correlated with morphologic alterations at the oocytic surface. Estrone neither stimulated nor inhibited vitellogenin incorporation and had no effect on nuclear breakdown. Oocytic capacity to exhibit activation responses (vitelline membrane elevation) was dependent on oocytic exposure to progesterone or DOCA and development subsequent to the initiation of nuclear breakdown. Onset of the activation response after steroid treatment varied with the type of activation stimulus utilized (pricking or divalent ionophore A-23187). The results suggest that hormones cause ionic alterations in oocytes and that ions are directly involved in the activation response. To study steroid interaction with the cell surface, a method was developed for culturing oocytes that permits localized application of steroids to portions of the oocytic or follicular surface. The results obtained suggest that oocytes exposed to steroid over part of their surface do not respond, with regard to nuclear breakdown, in the same manner as do oocytes exposed over their entire surface to similar concentrations of steroid. Studies of isotopic distribution within the oocyte after local application of steroid indicate that hormone does not readily diffuse through the oocyte. Evidence for the role of cytoplasmic factors in the mediation of nuclear and cytoplasmic events is discussed.

Amphibians↗
PubMed1976

[Human oogenesis in organ culture].

The authors compared the extent of development of the germ cells of the human fetus ovaries developing in the organ culture and of control ovaries obtained of fetuses of "equivalent" age. The capacity to enter the meiosis prophase under conditions of the organ culture was expressed by the germ cells of fetuses of 8-9 week of gestation, and older; only individual oocytes reached the leptotene stage in the explants of the ovaries of 7-8 week fetuses. The oocytes at the leptotene, zygotene and pachitene stage appeared in the culture at the same periods as in the organism. However, the percentage of cells of these stages in the explant was as a rule lower than in control ovaries. In case of pulse administration of thymidine-3H into the culture labeled oocytes at the zygotene stage appeared in 4, and at the pachitene stage - in 14 days.

Female↗
PubMed2024

Integrative Omics Reveals the Metabolic Patterns During Oocyte Growth.

Well-controlled metabolism is associated with high-quality oocytes and optimal development of a healthy embryo. However, the metabolic framework that controls mammalian oocyte growth remains unknown. In the present study, we comprehensively depict the temporal metabolic dynamics of mouse oocytes during in vivo growth through the integrated analysis of metabolomics and proteomics. Many novel metabolic features are discovered during this process. Of note, glycolysis is enhanced, and oxidative phosphorylation capacity is reduced in the growing oocytes, presenting a Warburg-like metabolic program. For nucleotide biosynthesis, the salvage pathway is markedly activated during oocyte growth, whereas the de novo pathway is evidently suppressed. Fatty acid synthesis and channeling into phosphoinositides are specifically elevated in oocytes accompanying primordial follicle activation; nevertheless, fatty acid oxidation is reduced in these oocytes simultaneously. Our data establish the metabolic landscape during in vivo oocyte growth and serve as a broad resource for probing mammalian oocyte metabolism.

Animals↗
PubMed2026

Generation of eight-cell embryo-like cells from human pluripotent stem cells.

Mammalian embryonic development is a highly orchestrated process initiated by the fusion of the oocyte with sperm to generate the zygote. In humans, the zygote remains transcriptionally quiescent until the major wave of zygotic genome activation (ZGA) occurs around the eight-cell (8C) stage (day 3 after fertilization). These cells and the derived morula cells are totipotent: they have the capacity to form a whole individual. Our understanding of human totipotency is very limited because of ethical concerns using embryos and the scarcity of material available for research. Recently, we established a controllable transgene-free methodology to generate totipotent eight-cell embryo-like cells (8CLCs) from human pluripotent stem cells (PSCs) in vitro. These 8CLCs are produced using a novel medium, formulated by us, containing specific chemical compounds and cytokines. Here, we provide a detailed protocol for inducing, isolating and characterizing 8CLCs generated with this medium. The induction process can be done either in a stepwise manner (primed-naive-8CLC) that requires only 5 d starting from naive PSCs or directly from primed PSCs, which takes ~7 d. The resulting 8CLCs exhibit transcriptional and epigenetic features resembling those of human 8C embryo cells. On the basis of our experience, we expect that an individual with ~1 year of experience working with human PSC culture would be able to carry out this protocol. Our approach provides a valuable model for studying human early embryogenesis, particularly the molecular events surrounding ZGA.

Journal Article↗
PubMed2026

Research note: Efficient preservation of genome-edited chicken germplasm via N-methylacetamide-based semen cryopreservation.

The rapid development of genome-edited chicken lines for agricultural and biomedical use requires effective methods for long-term preservation. In birds, cryopreservation of oocytes and embryos is challenging due to the structure of the egg. This makes semen cryopreservation one of the most practical alternatives, however, this method has not been validated in genome-edited chickens. This study evaluated the feasibility of cryopreserving semen from genome-edited chickens using an N-methylacetamide (N-MA)-based protocol. Two genome-edited chicken lines were used in this study which are a DAZL-GFP germ cell reporter line and a RAG1 knockout line. Semen was stored for either one week or one month prior to assessing fertility and hatchability. The results demonstrated that cryopreserved semen from both genome-edited chicken lines maintained their fertilizing capacity, with no significant differences in fertility or hatchability compared to cryopreserved wild-type groups at either storage duration. Hatchability remained above 80% across all groups, indicating that semen cryopreservation did not significantly affect embryo development after successful fertilization. Furthermore, SYBR-14/PI staining revealed no significant differences in sperm viability between genome-edited and control groups, although both showed a significant decrease in viability compared to fresh semen. These findings demonstrate that N-MA-based semen cryopreservation provides a reliable and practical method for the preservation of genome-edited chicken lines.

Chicken↗
PubMed1979

The effect of LH on the fertilizability and developmental capacity of rat oocytes matured in vitro.

The effect of adding LH (10 microgram NIH-LH-B8/ml) to the medium in which oocytes were undergoing maturation in vitro was studied. The fertilizability of the oocytes was evaluated in the sterile oviduct of a unilaterally ovariectomized, mated recipient. Freshly ovulated oocytes, used as a control of the method, were fertilized at a rate of 72%. Only 14% of oocytes matured in culture (without LH) were penetrated by spermatozoa, and 11% were fertilized normally. Addition of LH to the medium increased these proportions to 43 and 33% respectively. Oocytes matured in the presence of LH were able to develop into apparently normal rats. It is concluded that, although oocytes can mature in vitro spontaneously, and that these matured oocytes can be fertilized, addition of LH increases the numbers 3-fold. LH therefore has a direct maturation-promoting action on the rat oocyte-cumulus complex in vitro.

Animals↗
PubMed1977

[Tumors in BALB C mice after transplacental exposure to N-nitrosoethylurea in progenesis].

In the experiments with transplacental exposure of mice to N-nitrosoethyl urea (NEU) an enhanced carcinogenesis was noted not only in the first but also in the second generation. This effect in mice of the second generation was manifested in earlier development, as compared with control animals, of different neoplasms as well as in somewhat increased frequency of their detection. Postnatal exposure to NEU would enhance carcinogenesis in mice of both experimental and control groups, whereas no enhancement of carcinogenesis was observed in animals exposed to NEU in progenesis, compared with control mice. Postnatal x-ray irradiation also failed to produce enhanced carcinogenesis, compared with control animals i.e. those exposed to NEU in progenesis. While in female mice of the same group, contrary to the control, ovarian tumors under the effect of irradiation did not arise. It is suggested that there is a pathogenic connection between the inheritance of carcinogenic effect by oocytes and the loss by the ovary its capacity to produce neoplasms in response to irradiation.

Adenocarcinoma↗
PubMed2026

Loss of maternal PADI6 disrupts DNA methylation and genomic imprinting maintenance in late preimplantation mouse embryos.

BACKGROUND: The maternal-effect protein PADI6, which is part of the subcortical maternal complex, is involved in proper spindle assembly, organelle distribution, ribosome storage, and cytoplasmic lattice organization in mouse oocytes. In humans, variants of PADI6 are associated with female infertility and multilocus imprinting disturbance in offspring. Recently, it was demonstrated that PADI6 plays a role in the storage and cytoplasmic localization of epigenetic factors, including UHRF1 and DNMT1. Moreover, maternal PADI6 depletion leads to defective epigenetic reprogramming and zygotic genome activation but not to an imprinting defect in two-cell mouse embryos. These findings raise the possibility that imprinting disturbances arise later in development. RESULTS: By employing combined single-blastocyst RNA-seq/BS-seq and immunostaining validation in the embryos derived from Padi6P620A-mutant oocytes, we investigated the role of Padi6 in late preimplantation development. We demonstrated that embryos that overcame the two-cell stage block had a dramatic reduction in UHRF1 and DNMT1 protein levels, a decrease in H3K9me3, and whole-genome hypomethylation, including most imprinted loci and repetitive elements, at the blastocyst stage. Furthermore, these maternal mutant embryos showed deregulation of inner cell mass markers and defective blastocyst implantation, but no effect on trophoblast differentiation. CONCLUSION: Our results demonstrate that maternal PADI6 is a key regulator of the stability of epigenetic factors required to maintain repressive marks in late preimplantation mouse embryos. Its deficiency results in genomic imprinting defects that closely resemble those found in human patients and provide a mechanistic explanation for MLID caused by maternal PADI6 variants. Furthermore, the impairment of blastocyst implantation capacity, likely due to dysregulation of inner cell mass differentiation, provides new mechanistic insights into the control of female fertility and embryo development exerted by PADI6.

DNA Methylation↗
PubMed1979

The production of progesterone, androgens, and estrogens by granulosa cells, thecal tissue, and stromal tissue from human ovaries in vitro.

The concentrations of steroids in antral fluid, the number of granulosa cells, the status of the oocyte, and the diameter of each follicle were determined in human ovaries so that follicles at each stage of the menstrual cycle could be classified as large (greater than or equal to 8 mm diameter) or small (less than 8 mm diameter) and healthy or atretic. The granulosa cells and thecal-enriched tissue from each follicle and the stromal tissue from each ovary were cultured for 6 days in vitro. The amounts of progesterone (P), androstenedione (delta 4), testosterone, dihydrotestosterone, estrone, and estradiol (E2) generated by the different tissues were measured on days 0, 2, 4, and 6 of culture. It was found that granulosa cells, thecal tissue, and stromal tissue all have the biosynthetic capacity to produce P, delta 4, testosterone, dihydrotestosterone, estrone, and E2. No individual steroid-secreting compartment of the ovaries studied, whether part of the follicle or of the stroma, had the exclusive capability of producing any of the above-named steroids at any stage of the menstrual cycle or at any stage of antral follicle growth or atresia. Although the steroids produced by the human follicle appear not to be unique to any one cell type, the patterns of steroidogenesis by the granulosa and thecal compartments differ from one another and from the stroma throughout follicular maturation and atresia. During follicular development, granulosa cells produce large amounts of E2 and small amounts of delta 4. During the preovulatory phase, cells from large follicles (greater than or equal to 8 mm diameter) differentiate from an estrogen-secreting state into a P- and, to a lesser extent, an delta 4-secreting one. By contrast, during follicular atresia, granulosa cells continue to synthesize delta 4, but their capacity to synthesize estrogen is substantially reduced. Furthermore, granulosa cells from atretic follicles are incapable of transforming from an androgen-secreting state into a P-secreting one in tissue culture. During follicular growth, thecal tissue secretes about 2--3 times more delta 4 than E2. By contrast, during follicular atresia, thecal tissue retains its capacity to synthesize delta 4 but loses much of its capacity to synthesize E2. The in vitro capacity of thecal tissue to produce steroids exceeds that of the stroma (on a per weight basis) from 2- to 500-fold. Thecal tissue from healthy but not from atretic follicles is capable of differentiating from an androgen- and estrogen-secreting state to a predominantly P-secreting one in tissue culture. It is postulated that although steroid synthesis may not be rigidly compartmentalized during follicular development, appreciable amounts of the steroids secreted by the granulosa and theca may enter different compartments before leaving the ovary...

Adult↗
PubMed1979

Detection of nondisjunction in mammals.

Methods have been developed in the past to assess spontaneous and induced chromosomal aneuploidy in germ cells and in early pre- and postimplantation mammalian embryos. Some of these methods yield still more information when combined with chromosome banding techniques. Various chemicals and x-rays have been tested in mammalian oogenesis and x-rays in spermatogenesis. The inference may be drawn from these studies that spontaneous nondisjunction is considered to occur only rarely in mouse and hamster oogenesis and spermatogenesis. X-rays induce nondisjunction during male and femlae meiosis, thus giving rise to significantly more aneuploid oocytes and F1 embryos. The alkylating agents trenimone and cyclophosphamide induce chromosomal missegregation in oocytes; the incidence depends on the dose injected. Hormones used as oral contraceptives did cause aneuploidy in oocytes, but only after daily treatment with high doses. Hormones used for stimulated ovulation did not interfere with chromosome segregation in the mouse and Chinese and Syrian hamsters. The following problems may be considered in futre studies: the problem of a species-specificity for induced nondisjunction; the question of a stage sensitivity (transplacental treatment); what happens after chronic exposure, also at low doses; the presence of a threshold; the existence of a dose-effect relation; the nature of cellular target(s) responsible for induced nondisjunction (spindle, regulatory proteins for polymerization of microtubules and ther depolymerization, centrioles, centromeres, RNA, or gene expression); whether DNA is involved and whether repair capacity plays a role.

Alkylating Agents↗