Search Results (46)

Newborn mice (up to 6 d after birth) are suitable for genetic manipulations, such as facial vein-mediated injection, owing to their hairless and thin skin. Their small body volumes also facilitate the rapid dissemination of injected solutions, supporting gene engineering-related experiments. However, anesthesia in newborns is challenging because of the potential risks associated with anesthetic agents. Isoflurane inhalation anesthesia is an option, although its effects on brain development remain under investigation. In this study, we established a reproducible protocol for delivering nucleic acids to juvenile mouse testes using a simple isoflurane-based anesthetic system prepared from common laboratory equipment. Using this system, nucleic acids were successfully delivered to juvenile mouse testes via intra-testicular injection, followed by in vivo electroporation. The present isoflurane-based method achieved >90% postoperative survival with normal maternal nursing observations. Gene delivery resulted in limited transfection of seminiferous tubules but efficient interstitial Leydig cell transfection. Thus, gene engineering in somatic and germ cells in neonatal mice will be facilitated using the anesthetic protocol established in this study. Full article

Background: Primordial germ cells (PGC) are the progenitor cells of sperm and eggs during the embryonic stage. The maternal gene vasa has been widely studied for its role in PGC origin, and other genes like dead end (dnd) have also been identified. Objectives: Spotted sea bass is an important economic marine fish, and the study of its germ cell characteristics provides important basic data for future population breeding and protection. Methods: In this study, we cloned the full-length sequences of Lmvasa (2384 bp, encoding 1905 aa) and Lmdnd (1523 bp, encoding 386 aa) using RACE. Temporal and spatial expression patterns of Lmvasa and Lmdnd in embryos and gonads were analyzed by PCR, immunohistochemistry, and in situ hybridization. We also used microinjections of chimeric RNA containing GFP and Lmvasa 3′ UTR to visualize PGCs. Results: Our results showed that Lmvasa and Lmdnd are expressed primarily in early embryonic development (pre-blastula stage) and were expressed only in the gonads. Immunohistochemistry revealed abundant expression of Lmvasa and Lmdnd proteins in spermatogonia, weak expression in spermatocytes, and no expression in spermatozoa. In ovaries, both genes were expressed throughout oogenesis. Furthermore, PGCs in spotted sea bass belonged to an early localization pattern. Microinjection experiments demonstrated that Lmvasa 3′ UTR effectively labeled PGCs in embryos of spotted sea bass, zebrafish, and medaka. Conclusions: These findings may contribute to understanding PGC development in spotted sea bass and other Percidae. Full article

Piwi-interacting RNAs (piRNAs) play a crucial role in silencing transposable elements (TEs) in the germ cells of Metazoa by acting as sequence-specific guides. Originating from distinct genomic loci, called piRNA clusters, piRNA can trigger an epigenetic conversion of TE insertions into piRNA clusters by means of a paramutation-like process. However, the variability in piRNA clusters’ capacity to induce such conversions remains poorly understood. Here, we investigated two Drosophila virilis strains with differing capacities to produce piRNAs from the subtelomeric RhoGEF3 and Adar gene loci. We found that active piRNA generation correlates with high levels of the heterochromatic mark histone 3 lysine 9 trimethylation (H3K9me3) over genomic regions that give rise to piRNAs. Importantly, the maternal transmission of piRNAs drives their production in the progeny, even from homologous loci previously inactive in piRNA biogenesis. The RhoGEF3 locus, once epigenetically converted, maintained enhanced piRNA production in subsequent generations lacking the original allele carrying the active piRNA cluster. In contrast, piRNA expression from the converted Adar locus was lost in offspring lacking the inducer allele. The present findings suggest that the paramutation-like behavior of piRNA clusters may be influenced not only by piRNAs but also by structural features and the chromatin environment in the proximity to telomeres, providing new insights into the epigenetic regulation of the Drosophila genome. Full article

Background/Objectives: Vasa and PL10 belong to the DEAD-box protein family, which plays crucial roles in various cellular functions, such as DNA replication, DNA repair, and RNA processing. Additionally, DEAD-box family genes have also been identified as being related to gonadal development in many species. However, the function of vasa and PL10 in abalone is poorly understood on a molecular level. Methods: In the present study, we individually isolated and characterized the vasa and PL10 orthologs in Haliotis discus hannai (Hdh-vasa and Hdh-PL10). We also characterized the mRNA distributions of vasa and PL10 in various tissues from adult organisms and different embryonic developmental stages using real-time PCR (RT-qPCR) techniques. Furthermore, spatial and temporal expression of Hdh-vasa and Hdh-PL10 throughout embryonic and larval development was examined by whole-mount in situ hybridization (WMISH). Results: The two predicted amino acid sequences contained all of the conserved motifs characterized by the DEAD-box family. Homology and phylogenetic analyses indicate that they belong to the vasa and PL10 subfamilies. We found that vasa and PL10 mRNA were not solely restricted to gonads but were widely expressed in various tissues. WMISH showed that Hdh-vasa and Hdh-PL10 largely overlapped, with both being maternally expressed and specifically localized to the micromere lineage cells during early cleavage stages. By the gastrulation stage, Hdh-vasa were expressed strongly in two bilaterally symmetrical paraxial clusters, but Hdh-PL10 was dispersed in entire endodermal region. Our results suggest that Hdh-vasa-expressing cells are located as a subpopulation of undifferentiated multipotent cells that express Hdh-PL10. As such, we infer that primordial germ cells are specified from these vasa-expressing cells at some point during development, and inductive signals (epigenesis) play an important role in specifying primordial germ cells (PGCs) in H. discus hannai. Conclusions: This study provides valuable insights into the molecular characteristics and expression patterns of Hdh-vasa and Hdh-PL10, contributing to a better understanding of their roles in germ cell specification and early embryonic development in H. discus hannai. Full article

F8 gene inversion variants originate in male germ cells during spermatogenesis. Our recent study revealed that de novo variants (DNVs) caused F8 noninversion variants (NIVs) in sporadic hemophilia A (HA). Here, we conducted a direct clinical determination of sex differences in the origin of sporadic HA-NIV according to the data of two new HA-NIV families, as well as of the families demonstrated in the previous study. Of the 126 registered families with HA, 23 were eligible for inclusion. We conducted a linkage analysis with F8 gene markers and an amplification refractory mutation system–quantitative polymerase chain reaction to confirm the origin of the sporadic NIVs (~0% mutant cells) or the presence of a mosaic variant, requiring further confirmation of the origin in the parent. Two sporadic DNV events were determined. One event occurred in grandparents, consisting of five maternal grandmothers and seven maternal grandfathers, who were the origins; their respective daughters became carrier mothers who gave birth to probands. The other event included 11 mothers, who were the origins exclusively; their respective sons became probands. In this study, we found that sporadic HA-NIVs originate mostly from females (16 out of 23). Our study contributes to a better understanding of the genetic pathogenesis of HA. Full article

Maternal cadmium exposure during pregnancy has been demonstrated to have detrimental effects on offspring development. However, the impact of maternal cadmium exposure on offspring oocytes remains largely unknown, and the underlying mechanisms are not fully understood. In this study, we found that maternal cadmium exposure during pregnancy resulted in selective alteration in epigenetic modifications of mouse oocytes in offspring, including a decrease in H3K4me2 and H4K12ac, as well as an increase in DNA methylation of H19. Although ROS levels and mitochondrial activity remain at normal levels, the DNA damage marker γH2AX was significantly increased and the DNA repair marker DNA-PKcs was remarkably decreased in offspring oocytes from maternal cadmium exposure. These alterations are responsible for the decrease in the quality of mouse oocytes in offspring induced by maternal cadmium exposure. As a result, the meiotic maturation of oocytes and subsequent early embryonic development are influenced by maternal cadmium exposure. RNA-seq results showed that maternal cadmium exposure elicits modifications in the expression of genes associated with metabolism, signal transduction, and endocrine regulation in offspring ovaries, which also contribute to the disorders of oocyte maturation and failures in early embryonic development. Our research provides direct evidence of transgenerational epigenetic inheritance of cadmium reproductive toxicity in mouse germ cells. Full article

The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I–V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus. Full article

Maternal obesity and/or Western diet (WD) is associated with an increased risk of metabolic dysfunction-associated steatotic liver disease (MASLD) in offspring, driven, in part, by the dysregulation of the early life microbiome. Here, using a mouse model of WD-induced maternal obesity, we demonstrate that exposure to a disordered microbiome from WD-fed dams suppressed circulating levels of endogenous ligands of the aryl hydrocarbon receptor (AHR; indole, indole-3-acetate) and TMAO (a product of AHR-mediated transcription), as well as hepatic expression of Il10 (an AHR target), in offspring at 3 weeks of age. This signature was recapitulated by fecal microbial transfer from WD-fed pregnant dams to chow-fed germ-free (GF) lactating dams following parturition and was associated with a reduced abundance of Lactobacillus in GF offspring. Further, the expression of Il10 was downregulated in liver myeloid cells and in LPS-stimulated bone marrow-derived macrophages (BMDM) in adult offspring, suggestive of a hypo-responsive, or tolerant, innate immune response. BMDMs from adult mice lacking AHR in macrophages exhibited a similar tolerogenic response, including diminished expression of Il10. Overall, our study shows that exposure to maternal WD alters microbial metabolites in the offspring that affect AHR signaling, potentially contributing to innate immune hypo-responsiveness and progression of MASLD, highlighting the impact of early life gut dysbiosis on offspring metabolism. Further investigations are warranted to elucidate the complex interplay between maternal diet, gut microbial function, and the development of neonatal innate immune tolerance and potential therapeutic interventions targeting these pathways. Full article

Caffeine (1,3,7-trimethylxanthine) is a widely consumed bioactive substance worldwide. Our recent study showed that a reduction in both reproduction and yolk protein production (vitellogenesis) caused by caffeine intake were improved by vitamin B12 supplementation, which is an essential co-factor in methionine metabolism. In the current study, we investigated the role of methionine in the reproduction of caffeine-ingested animals (CIAs). We assessed the effect of methionine metabolism on CIAs and found that caffeine intake decreased both methionine levels and essential enzymes related to the methionine cycle. Furthermore, we found that the caffeine-induced impairment of methionine metabolism decreased vitellogenesis and increased germ cell apoptosis in an LIN-35/RB-dependent manner. Interestingly, the increased germ cell apoptosis was restored to normal levels by methionine supplementation in CIAs. These results indicate that methionine supplementation plays a beneficial role in germ cell health and offspring development by regulating vitellogenesis. Full article

Maternal exposures during pregnancy can impact the establishment of the ovarian reserve in offspring, the lifetime supply of germ cells that determine a woman’s reproductive lifespan. However, despite alcohol consumption being common in women of reproductive age, the impact of prenatal alcohol on ovarian development is rarely investigated. This study used an established rat model of periconceptional ethanol exposure (PCEtOH; 12.5% v/v ethanol) for 4 days prior to 4 days post-conception. Ovaries were collected from neonates (day 3 and day 10), and genes with protein products involved in regulating the ovarian reserve analyzed by qPCR. Adult offspring had estrous cycles monitored and breeding performance assessed. PCEtOH resulted in subtle changes in expression of genes regulating apoptosis at postnatal day (PN) 3, whilst those involved in regulating growth and recruitment of primordial follicles were dysregulated at PN10 in neonatal ovaries. Despite these gene expression changes, there were no significant impacts on breeding performance in adulthood, nor on F2-generation growth or survival. This contributes additional evidence to suggest that a moderate level of alcohol consumption exclusively around conception, when a woman is often unaware of her pregnancy, does not substantially impact the fertility of her female offspring. Full article

Sporadic hemophilia A (HA) enables the persistence of HA in the population. F8 gene inversion originates mainly in male germ cells during meiosis. To date, no studies have shown the origin and timing of HA sporadic noninversion variants (NIVs); herein, we assume that HA-sporadic NIVs are generated as a de novo variant. Of the 125 registered families with HA, 22 were eligible for inclusion. We conducted a linkage analysis using F8 gene markers and amplification refractory mutation system–quantitative polymerase chain reaction to confirm the origin of the sporadic NIVs (~0% mutant cells) or the presence of a mosaic variant, which requires further confirmation of the origin in the parent. Nine mothers, four maternal grandmothers, and six maternal grandfathers were confirmed to be the origin of sporadic NIVs, which most likely occurred in the zygote within the first few cell divisions and in single sperm cells, respectively. Three mothers had mosaic variants, which most likely occurred early in postzygotic embryogenesis. All maternal grandparents were free from sporadic NIV. In conclusion, F8 NIVs in sporadic HA were found to be caused primarily by de novo variants. Our studies are essential for understanding the genetic pathogenesis of HA and improving current genetic counseling. Full article

The mechanisms of immune tolerance of a mother against an antigenically foreign fetus without a concomitant loss of defense capabilities against pathogens are the factors underlying the success of a pregnancy. A significant role in human defense is played by killer immunoglobulin-like receptor (KIR) receptors, which regulate the function of the natural killer (NK) cells capable of destroying antigenically foreign cells, virus-infected cells, or tumor-lesioned cells. A special subpopulation of NK cells called uterine NK cells (uNK) is found in the uterus. Disruption of the tolerance process or overactivity of immune-competent cells can lead to immune infertility, a situation in which a woman’s immune system attacks her own reproductive cells, making it impossible to conceive or maintain a pregnancy. Since the prominent role of the inflammatory response in infertility, including KIR receptors and NK cells, has been postulated, the process of antigen presentation involving major histocompatibility complex (MHC) molecules (HLA) appears to be crucial for a successful pregnancy. Proper interactions between KIR receptors on female uNK cells and HLA class I molecules, with a predominant role for HLA-C, found on the surface of germ cells, are strategically important during embryo implantation. In addition, maintaining a functional balance between activating and inhibitory KIR receptors is essential for proper placenta formation and embryo implantation in the uterus. A disruption of this balance can lead to complications during pregnancy. The discovery of links between KIR and HLA-C has provided valuable information about the complexity of maternal–fetal immune interactions that determine the success of a pregnancy. The great diversity of maternal KIR and fetal HLA-C ligands is associated with the occurrence of KIR/HLA-C combinations that are more or less favorable for reproductive success. Full article

During gestation, maternal (F0), embryonic (F1), and migrating primordial germ cell (F2) genomes can be simultaneously exposed to environmental influences. Accumulating evidence suggests that operating epi- or above the genetic DNA sequence, covalent DNA methylation (DNAme) can be recorded onto DNA in response to environmental insults, some sites which escape normal germline erasure. These appear to intrinsically regulate future disease propensity, even transgenerationally. Thus, an organism’s genome can undergo epigenetic adjustment based on environmental influences experienced by prior generations. During the earliest stages of mammalian development, the three-dimensional presentation of the genome is dramatically changed, and DNAme is removed genome wide. Why, then, do some pathological DNAme patterns appear to be heritable? Are these correctable? In the following sections, I review concepts of transgenerational epigenetics and recent work towards programming transgenerational DNAme. A framework for editing heritable DNAme and challenges are discussed, and ethics in human research is introduced. Full article

Emerging evidence has indicated the involvement of extrahypothalamic Kisspeptin and GnRHR in reproductive function. In this study, we evaluate if maternal exposure to the pesticide chlorpyrifos (CPF) and/or a high-fat diet (HFD) has an impact on the expression of Kisspeptin and GnRHR in the reproductive organs of rats’ offspring. A total of 16 pregnant rats are divided into four groups: a control group (n = 4), CPF group (4 rats exposed daily to 1/mg/kg/day), HFD group (4 rats randomly fed a 5.25 kcal/g HFD), and coexposed group (4 rats exposed to CPF and HDF). At postnatal development postnatal day (PND) 60, male and female offspring were sacrificed. The reproductive organs (ovary and testis) were removed, and histological and immunohistological analysis and in silico quantification (TissueGnostics software 6.0.1.102, TissueFAXS, HistoQuest) were applied to investigate the impact of different treatments on Kisspeptin and GnRHR expression in reproductive organs. The main outcomes of the study showed a significant decrease in rat offspring’s body weight in the CPF group from PND30 and PND60 (p < 0.05 and p < 0.01, respectively). Histological analysis showed a significant increase in the atretic follicle and abnormal testis structure with germ cell desquamation in the CPF-exposed group. The immunodetection quantification of protein shows a significant decrease in GnRHR and Kisspeptin in the HFD and CPF exposed groups, respectively, in testis rat offspring. Perinatal exposure to CPF and HFD exposure affect the reproduction function of rat offspring. Full article

Recent developments in stem cell technologies have made significant advancements in the field of in vitro gametogenesis. In vitro gametogenesis (IVG) is a promising technology where functional gametes (sperm or egg cells) can be generated from stem cells. Scientists have made continuous advancements in the field and successfully derived fully functional sperm from stem cells in mice. Two recent papers generated excitement in IVG by generating bi-maternal and bi-paternal mice from embryonic stem cells (ESCs) and pluripotent stem cells (PSCs). IVG is a promising technology with potential applications that include infertility treatment, fertility preservation, same-sex reproduction, bypassing oocyte depletion in women with advanced age, conservation biology, genetic disorder prevention, and research into human germ cell development. In vitro spermatogenesis (IVS) is the attempt to recreate the process of spermatogenesis in a culture system. Spermatogenesis is essential for male fertility and reproductive health, but it can be impaired by various factors such as genetic defects, environmental toxicants, infections, aging, or medical therapies. Spermatogenesis is a complex and highly regulated process involving multiple cell proliferation, differentiation, and maturation stages. The main challenges of IVS are to provide a suitable microenvironment that mimics the testis in vivo, to support the survival and development of all the cell types involved in spermatogenesis, and to achieve complete and functional spermatogenesis. Therefore, there is a great interest in developing methods to study spermatogenesis in vitro, both for basic research and clinical applications. This review covers recent developments in in vitro spermatogenesis in the past two years. Advances in tissue engineering and regenerative medicine have introduced techniques like ex vivo tissue culture and technologies such as bioreactors, microfluidic systems, and organoids. Bioreactors and microfluidic systems replicate physiological conditions for tissue and cell cultivation, while organoids model organ functionality. Meanwhile, scaffolds, made from various materials, provide essential structural support, guiding the growth and organization of cells into functional tissues. Full article