In the Drosophila testis, hub cells form a specialized niche that sustains adjacent stem cells. The RNA-binding protein Lin28 is expressed in hub cells and is essential for their maintenance and niche function; however, the underlying mechanisms remain poorly understood. Lin28 is well-characterized as a suppressor of let-7 biogenesis, raising the possibility that Lin28 regulates let-7 and additional microRNAs to preserve hub cell identity. We performed genome-wide microRNA expression profiling and identified a set of microRNAs upregulated in lin28 mutant testes, including let-7, demonstrating that Lin28 represses let-7 in hub cells to protect let-7-targeted hub cell factors. Here, we report genetic analysis of miR-304, a microRNA identified from the profiling. miR-304 contains a seed site complementary to the 3' UTR of the IGF-II mRNA-binding protein (IMP), a critical hub-specific factor. We find that miR-304 mutant testes exhibit elevated levels of IMP specifically in hub cells, suggesting that miR-304 targets imp mRNAs and reduces imp expression in hub cells. Thus, in wild-type hub cells, Lin28 represses miR-304 to maintain IMP expression. We propose that Lin28 preserves niche function by restraining multiple microRNAs, thereby protecting essential hub cell factors from microRNA-mediated silencing.
Inflammation adversely affects reproductive function and early embryonic development; however, whether inflammatory conditions are associated with alterations in ganglioside expression during early embryogenesis remains unclear. The present study aimed to investigate whether lipopolysaccharide (LPS)-induced chronic inflammation is associated with alterations in ovarian function and early embryonic developmental competence in Institute of Cancer Research (ICR) mice, and whether subsequent melatonin intervention is accompanied by a partial recovery trend. Embryonic developmental competence was evaluated by superovulation and in vitro fertilization (IVF), and GM3 levels along with ST3GAL5 expression in ovarian tissues and blastocyst-stage embryos were assessed using biochemical analysis and immunofluorescence staining. LPS-induced inflammation was associated with reduced oocyte recovery, decreased blastocyst formation rates, and lower GM3 band intensities and ST3GAL5 fluorescence signals in ovarian tissues and embryos. Melatonin treatment partially improved reproductive outcomes and was accompanied by a partial recovery trend in GM3 levels and ST3GAL5 expression. These findings suggest that melatonin may partially alleviate LPS-induced reproductive impairment, and that recovery of ST3GAL5/GM3-related expression may be associated with this partial mitigating trend. However, the present study does not establish a direct causal relationship between these molecular changes and improved embryonic development.
Baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) (survivin) is known to play an important role in cell survival and tumor progression; however, its involvement in cancer stem cell (CSC) properties and epithelial-mesenchymal transition (EMT) in breast cancer remain unclear. In this study, we investigated the role of BIRC5 in regulating CSC-like characteristics and EMT in human breast cancer cells. Analysis of public datasets revealed that BIRC5 expression is significantly elevated in breast cancer tissues and is associated with poor patient prognosis. Treatment with CSC-inducing conditioned medium (CSC-CM) increased the expression of CSC markers, including CD44, ALDH1A1, and ALDH1A3. Silencing of BIRC5 using siRNA reduced the expression of CSC-associated markers as well as pluripotency-related transcription factors (Sox2, Oct4, and Nanog). In addition, BIRC5 knockdown significantly decreased sphere-forming ability, indicating reduced self-renewal capacity. Furthermore, suppression of BIRC5 altered EMT-related protein expression, as evidenced by increased E-cadherin and decreased N-cadherin, Vimentin, and EMT-associated transcription factors (Snail, Slug, Twist, and Zeb1). In addition, BIRC5 knockdown also significantly reduced cell migration and invasion. These results suggest that BIRC5 is involved in the regulation of CSC properties and EMT in breast cancer cells and may contribute to tumor progression.
Luteinizing hormone (LH) is a vital reproductive hormone produced by the anterior pituitary gland, essential for both fertility and sexual development. Interestingly, there is evidence indicates that rodent placenta is extrapituitary site of LH synthesis. On this basis, we performed immunohistochemistry (IHC) using specific antisera for LH subunits to investigate the tissue distribution of the LH subunits like molecules in mice placentae. Pregnant mice on gestation day 17 were sacrificed and the placentae were obtained Glycogen rich trophoblast cells (GlyT) in the junctional zone (JZ) were positive for CGα immunohistochemical staining, and the LH-β positive staining was found in all cell layers in both JZ and labyrinth zone (LZ). Immunoreactive LH-receptor (LH-R) staining was observed on the membrane of GlyT in JZ and partial syncytiotrophoblast cells layer in LZ. The present study demonstrated that immunoreactive LH subunits like substances were localized differentially in the microstructures of mice placentae. This finding that the substances exhibiting immune responses to these and their receptors exist in the same type of cells and/or adjacent cells, suggesting the possibility of local regulation through these substances. Further studies will be helpful for understanding the biochemical nature and the role(s) of local LH-like substances in sophisticated placental physiology.
The body color of crustaceans is an important factor influencing consumer preference and marketability, expressed through the accumulation of carotenoids within the body. However, crustaceans cannot synthesize carotenoids internally and rely entirely on dietary sourcess. Deficiency of this pigment can lead to poor body coloration. Haematococcus lacustris is a species of freshwater microalgae that accumulates astaxanthin at high concentrations, possessing significant industrial potential as a natural source of carotenoids. This study investigated the effects of adding H. lacustris, which is rich in astaxanthin, to feed on improving body color and health status in Macrobrachium rosenbergii post-larvae. The experimental results showed that body length exhibited similar growth trends across all groups, while body weight was significantly higher in the control (CON) group compared with the low concentration (LC) and high concentrations (HC) groups. Feed supplemented with H. lacustris exhibited a darkening effect on body color depending on concentration, and this effect persisted even after heating. Superoxide dismutase (SOD) expression levels in the hepatopancreas significantly increased at day 60 in the HC group. Crustin expression also significantly increased in the hepatopancreas on day 60, but no significant differences were observed between groups in the tail muscle. The addition of H. lacustris to the diet aided in the body color development of M. rosenbergii post-larvae, suggesting that this approach may also be applicable to the body color development of various crustaceans.
The Hippo signaling pathway is an evolutionarily conserved pathway from Drosophila to humans. Although key elements of the Hippo signaling pathway are well-defined, the factors that control the transcriptional outcome of Hippo have yet to be fully elucidated. Until now, mainly in mammals, the Hippo signaling pathway has been focused on serine 127 (S127) of yes-associated protein (YAP), a key target gene. Recently, it has been shown that nemo-like kinase (NLK) can crosstalk with the Hippo pathway by phosphorylating an unknown new site of YAP. NLK transfers YAP serine 128 (S128) to the nucleus through dissociation of the 14-3-3 binding with YAP, promoting transcriptional activity. However, this is worth investigating as it has not been studied in the mammalian reproductive system and the precise mechanisms of signaling pathway crosstalk in endometrial cells that are dynamically altered by steroid hormones remain unclear. In this study, we found that expression of NLK and YAP S128 changes the expression site or extent of expression in the endometrium during the estrous cycle. Furthermore, we demonstrated that regulation of its expression proceeds through estrogen and its receptors. We have shown that these responses are triggered and regulated in uterine epithelial cells, suggesting that their expression plays a role in uterine dynamics during the estrous cycle, as does the hippocampal signaling pathway.
Generation of primordial germ cell-like cells (PGCLCs) from pluripotent stem cells in vitro serve as key intermediates in the modeling of germline development. While previous studies have predominantly focused on the induction of PGCLCs from epiblast-like cells (EpiLCs), recent studies suggest that BMP4 signaling can also drive PGCLC specification from epiblast stem cells (EpiSCs). However, the efficiency of PGCLC induction from EpiSCs remains suboptimal and underexplored. We hypothesized that the dimensional structure of the culture environment significantly influences the differentiation efficiency. To evaluate PGCLC induction efficiency, we used Blimp1-mVenus×Stella-ECFP (BVSC) transgenic reporter embryonic stem cells. FACS analysis revealed that the proportion of Blimp1-mVenus+/Stella-ECFP+ double-positive PGCLCs was significantly higher in the 3D aggregate culture compared to the 2D monolayer system. Our findings demonstrate that a 3D culture environment enhances the efficiency of PGCLC induction from mouse EpiSCs compared to a 2D monolayer system. These results highlight the importance of culture dimensionality in optimizing germ cell differentiation protocols and provide a useful framework for further studies on germline development.
Little is known about the regulation of gene expression related to the hypothalamus-pituitary (HP) axis around the onset of normal puberty. In the present study, we examined the expression profiles of genes in HP hormone circuit on every other day from postnatal day (PND) 29 to PND 43. Average vaginal opening (VO) date was PND 37 (66%), and the weight of reproductive organs increased significantly from PND 37. Serum steroid hormone levels significantly increased on PND 39. The appearance of a number of Graafian follicles and corpora lutea on PND 37. Generally, our polymerase chain reactions (PCR) results showed that most of the expression of hypothalamus and pituitary factors tended to increase after VO, and the patterns were rather unstable and no significant peak pattern such as LH surge shown in proestrus adults. The mRNA levels of gonadotropin-inhibitory hormone (GnIH)-GPR147 and neurokinin B(Tac)-TacR3 mostly reached a peak in the last period of the experimental schedule. In pituitary, mRNA level of gonadotropin subunits (Cgα, LH-β and FSH-β) also significantly increased on later experimental period. In conclusion, we could confirm the rapid growth and maturation of reproductive organs immediately after VO, and dynamic changes in gene expression of the HP axis factors. The gene expression patterns at peripubertal period were incomplete and unstable without showing the preovulatory LH surge-related gene expression pattern in adults. The present study on neuroendocrine control of peripubertal sexual maturation may offer a basis for understanding normo- and/or patho-physiological status of puberty.
Nitrogen-doped Carbon Quantum Dots (NCQDs) exhibit distinctive optical properties and potential bioactivity in mammalian systems. However, their effects on reproductive cells remain poorly understood. To clarify their role in gamete function and embryo development, the present study examined the influence of NCQDs on sperm activation, fertilization, and early embryo development in vitro using a mouse model. At a low concentration (10 µg/mL), NCQDs exposure markedly enhanced sperm motility, survival, and capacitation, as determined by computer-assisted sperm analysis (CASA). These functional improvements significantly increased fertilization rates and enhanced embryonic development up to the morula stage. In contrast, blastocyst formation was delayed, accompanied by reduced pluripotency (Oct4) and trophectoderm differentiation (Cdx2, Tead2). Elevated mRNA expression of endoplasmic reticulum (ER) stress markers (Atf6, Chop) in morula-stage embryos suggested that prolonged NCQD exposure induces cellular stress that may interfere with lineage specification. Collectively, these findings reveal a stage-specific, biphasic effect of NCQDs, promoting sperm activation and early cleavage while inhibiting later differentiation, highlighting the need for optimized dosing and exposure timing to safely harness their reproductive benefits.
Oxybenzone (Benzophenone-3; BP-3) is used as a component of sunscreens, and known to disrupt the endocrine system of marine organisms. This study evaluated BP-3 toxicity on Shimofuri goby (Tridentiger bifasciatus). We evaluated morphological changes during embryogenesis. In addition, hatching rate (HR) and embryo survival to hatching were assessed. Embryos were exposed to BP-3 in artificial seawater, and then they were randomly sampled every 12 hours for microscopic observation and cortisol analysis. 36 hours after exposure to BP-3 100 and 1,000 μg/L groups, the tail was not separated from the yolk sac. 72 hours after exposure, incompleted eye pigmentation was observed at 100 and 1,000 μg/L BP-3. After 48 and 60 hours of exposure, all individuals in the control group had elongated tails, whereas individuals in the all BP-3 treated group failed to elongate or showed signs of bent tails. Survival rate decreased dose-dependently (control: >90%) with LC50-96h=493 μg/L. HR significantly declined in all BP-3 groups in a dose-dependent manner. And, heartbeat was increased in response to BP-3 1,000 μg/L. High levels of cortisol were observed in the initial groups (0 hours) and decreased after 24 hours. The BP-3 100 and 1,000 μg/L showed significantly lower cortisol levels than the control at 96 hours of exposure. Overall, this study suggests that BP-3 can interfere with embryonic development, resulting in adverse effects on survival and HRs in T. bifasciatus embryos.
The steroidogenic acute regulatory protein (StAR) governs the rate-limiting step of steroid hormone biosynthesis by facilitating cholesterol transfer from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM). This essential function initiates pregnenolone synthesis by P450 family 11 subfamily A member 1 (CYP11A1, cytochrome P450scc) within IMM. Beyond its biochemical role, StAR is a critical developmental protein, with spatiotemporally restricted expression during fetal adrenal and gonadal differentiation. Its activity is tightly regulated at multiple levels, including transcriptional control by transcription factors, GATA post-translational phosphorylation, mitochondrial targeting, and proteolytic degradation. Structurally, StAR functions through a dynamic molten globule-like conformation and a conserved StAR-related lipid transfer (START) domain that mediates cholesterol binding. StAR interacts with mitochondrial proteins such as nonselective voltage-gated ion channel VDAC (VDAC), translocator protein (TSPO), and ATPase family AAA domain-containing protein 3A (ATAD3A), forming part of the transduceosome complex that coordinates cholesterol transfer. Mutations in STAR, particularly within the START domain), cause lipoid congenital adrenal hyperplasia (CAH), a disorder marked by impaired steroidogenesis and disrupted endocrine organ development. This review integrates current knowledge on the molecular and developmental roles of STAR, emphasizing how its precise regulation is essential for embryonic steroidogenesis. Understanding StAR's function at the interface of lipid transport and organogenesis provides critical insight into congenital steroidogenic disorders and potential avenues for therapeutic intervention.
Neural progenitors of the ventral spinal cord differentiate into GABAergic Kolmer-Agduhr neurons (KA) under the control of Jagged2-meditated Notch signaling during late neurogenesis. Mib-mediated Notch signaling has also been demonstrated to regulate the number of KA neurons in the p3 domain. However, the relationship between Jagged2 and Mib during late neurogenesis remains unclear. Here we investigate how Mib is involved in the regulation of Jagged2 and the long-range Notch signaling. Ubiquitination of Jagged2 by Mib was found to promote its proteasome-dependent degradation in undifferentiated P19 cells, but not in differentiated P19 cells by retinoic acid. Co-IP assay revealed that Mib physically interacts with Jagged2, but not with the intracellular domain itself. Cell transplantation experiments showed that the formation of extracellular vesicles (EVs) containing Jagged2 was promoted by the co-expression of Mib. Our observations suggest that EVs containing Jagged2 and Mib may play a role in the Notch signaling in discrete compartments of the neural tube during the development of the vertebrate nervous system.
Germline cells are specified early in embryogenesis and are encapsulated by somatic cells to form the gonads (testis or ovary). This development requires genes with expression restricted to germline cells, such as the DEAD-box RNA helicase Vasa, an evolutionarily conserved protein exclusively expressed in the germline of the testis. However, the mechanisms underlying germline-specific expression remain poorly understood. To identify microRNAs that function in the somatic cells of the testis, we employed the binary Gal4/UAS expression system, which enables the expression of UAS-microRNA sponges in somatic cells driven by somatic Gal4 drivers. The screening identified the miR-932 sponge as a regulator. Testes with hub-specific Gal4 driven expression of the UAS-miR-932 sponge exhibit ectopic Vasa expression in the hub cells. Thus, our findings suggest that miR-932 in the somatic hub cells prevents Vasa expression in these cells.
The deleted in azoospermia like (DAZL) gene is a member of the DAZ gene family. It is firstly identified in male germ cells and recognized as a key molecule of their development, now it is extended to the female germ cells and the embryo. The DAZL gene is constructed with 11 exons, 10 introns, a 5' untranslated region (UTR), and a 3' UTR, and the enhancers at the upstream of the promoter in both human and mouse. It has been revealed that DAZL gene expression is not restricted to germ cells. The known mechanisms for expression regulation include the CpG methylation on the promoter region and post-transcriptional regulation by antagonistic proteins. DAZL protein has one RNA recognition motif (RRM) and one DAZ repeat. DAZL orchestrates the translation of numerous mRNAs essential for germ cell proliferation, differentiation, and survival. Several studies have unveiled DAZL's broader roles, including its involvement in stemness and tumorigenicity through post-transcriptional regulation via polyadenylation and potential functions in RNA stabilization. The alternatively spliced variants are also evaluated in different tissues. This review consolidates current knowledge on DAZL's molecular mechanisms, expression, and emerging research directions, and introduces DAZL gene anatomy.
Transcriptional coactivator with PDZ-binding motif (TAZ) functions as a transcriptional coactivator, which shuttles between the cytoplasm and the nucleus under the Hippo signaling. It is known to be involved in promoting cell proliferation, organ overgrowth, survival to stress, and dedifferentiation by interacting with TEAD transcription factors (TEADs). However, the regulation of TAZ by intrauterine hormones has not yet been investigated. In this study, we investigated TAZ expression during the estrous cycle in the normal mouse uterus and the effect of estrogen and progesterone on TAZ expression in the ovariectomized (OVX) mouse uterus. TAZ expression levels did not show a statistically significant change in the uterus during the estrous cycle. However, immunofluorescence revealed that TAZ nuclear localization significantly increased at the estrus stage. In the OVX mouse uterus, the expression levels of TAZ mRNA and protein dramatically increased in a time-dependent manner after estrogen treatment. Also, immunofluorescence showed that the nuclear TAZ expression increased at 6 h and 12 h after estrogen treatment compared to the oil treated OVX mouse uterus (0 h). Finally, pretreatment of an estrogen receptor (ER) antagonist ICI 182,780 efficiently reduced estrogen-induced TAZ expression. However, progesterone did not significantly affect the expression of TAZ in both mRNA and protein levels. In conclusion, TAZ expression is regulated and activated by estrogen through nuclear estrogen receptors, ERα, and ERβ in the uterine environment.
Previously, we developed a short-term incubation method of rat adrenal and demonstrated that nonylphenol (NP) exposure could induce changes in secretions of adrenal hormones. In the present study, the effects of NP on changes in hormonal secretion from hypothalamus were investigated. The catecholamine levels were measured using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) and the levels of gonadotropin-releasing hormone (GnRH) and kisspeptin were measured using enzyme-linked immunosorbent assay (ELISA). The norepinephrine (NE) levels from NP-treated male and female hypothalamus were not significantly changed across the entire treatment concentration (from 1 nM to 1 μM), except male 10 nM-treated group which were significantly lower than control (p<0.05). The epinephrine (E) levels from NP-treated female hypothalamus were significantly increased in 100 pM- and 1 nM-trated group (p<0.05). However, the E levels from NP-treated male hypothalamus were significantly decreased in 100 pM- and 10 nM-treated group (p<0.05). The GnRH levels from NP-treated hypothalamus showed an increasing trend, especially significant in male 10 nM- and 100 nM-treated groups (p<0.001) and female 1 nM-, 100 nM- and 1 μM-trated groups (p<0.05, p<0.01 and p<0.05, respectively). Also, the kisspeptin levels in incubated media of both sexes showed a strong increasing trend, especially significant in male 1 nM- and 10 nM-treated groups (p<0.05) and all of female groups except 10 nM-treated. In conclusion, our incubation method could be quite suitable for rapidly and effectively measuring endocrine disrupting chemicals (EDC) activity in hypothalamus. NP treatment shown stimulatory effects on both GnRH and kisspeptin secretions from hypothalamus of both sexes, suggesting possible relationship between NP exposure and reproductive phenomena and related disorders.
Gonadotropins, such as follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG), are widely used to induce ovarian hyperovulation during in vitro fertilization and embryo transfer (IVF-ET) for the treatment of infertility. However, the effects of repeated administration of these gonadotropins on immune function, particularly on T cell development in the thymus, remain poorly understood. This study investigated the effects of repeated administration of pregnant mare serum gonadotropin (PMSG) and hCG on thymic T cell development in mice. Histological analysis revealed structural changes in the thymus, including a blurred boundary between the medulla and cortex and reduced vascularization after repeated administration of PMSG and hCG. Quantitative real-time PCR showed increased expression of adipogenesis-related genes [phosphoenolpyruvate carboxykinase (PEPCK), adipocyte fatty acid-binding protein 2 (aP2), peroxisome proliferator-activated receptor gamma (PPARγ)] but no significant changes in thymic epithelial cell-related genes [autoimmune regulator (AIRE), epithelial V-like antigen (EVA), interleukin 7 (IL-7)]. Flow cytometry revealed a decrease in CD4+CD8+ T cells and an increase in CD4-CD8-T cells with altered CD25/CD44 subsets. In addition, CD4+ and CD8+ T cells in the spleen were significantly reduced. These findings suggest that repeated gonadotropin exposure may disrupt thymic T cell development and peripheral T cell populations, potentially impairing immune function. Further research is needed to elucidate the underlying mechanisms and broader immunologic consequences of gonadotropin use in infertility treatment.
The egg quality is a representative limiting factor in developing culture techniques for certain fish species. It is a known predictor of subsequent larval viability, quality, and stress resistance related to aquaculture productivity. Here we tracked egg quality in a second generation of broodstock small yellow croacker, Larimichthys polyactis. Cultured second generation broodstock (3 years old, 600 fishes) was reared in indoor tank (30 tons). We induced natural spawning with increasing water temperature (11.5°C22.0°C) and regulation of photoperiod (9L:15D). We used spawning events from spawning period and monitored basic morphometrics such as: egg viability, egg diameter (ED), oil droplet diameter (OD) and oil droplet volume. Natural spawning of the broodstock was maintained for 23 days. EDs and OD for L. polyactis decreased as the spawning season progressed and water temperature increased. We showed that smaller eggs lead to higher quality with viability, and that using eggs later in the spawning season would lead to better production. In addition, the volume of oil droplet was the strongest factors for prediction of egg viability for cultured second generation of small yellow croaker.
We previously reported that metformin, a widely prescribed antidiabetic drug, induces the accumulation of triglyceride (TG) together with the apoptotic death of H4IIE via AMP-activated protein kinase (AMPK) in hepatocellular carcinoma (HCC) cells. However, the effect of cytoplasmic fat accumulation on the growth of HCCs remains controversial. Herein, we investigated the effect of fatty acid synthase (FASN) inhibitors on the basal- or metformin-induced changes including the content of cytoplasmic TG and the viability of HCC cells. Cerulenin and C75, inhibitors of FASN, did not significantly affect the basal TG content but dose-dependently suppressed the metformin-induced increase in the cytoplasmic TG content. Metformin-induced apoptosis of H4IIE cells was also significantly reduced by cerulenin and C75. Metformin enhanced the generation of reactive oxygen species which was suppressed by adding cerulenin or T75. Cerulenin also stimulated cell migration, which was suppressed by metformin. However, the degree of suppressive effect of metformin on TG synthesis, apoptosis, and cell migration was much more prominent by the inhibition of AMPK by compound C than cerulenin. In conclusion, our study found that excess fat accumulation is responsible for the apoptosis of H4IIE HCC cells and is informative for designing anti-tumor reagents, especially in HCC.
Maintenance of neural progenitors requires Notch signaling in vertebrate development. Previous study has shown that Jagged2-mediated Notch signaling maintains proliferating neural progenitors in the ventral spinal cord. However, components for Jagged-mediated signaling remain poorly defined during late neurogenesis. Here we performed yeast-two hybrid screening by using the intracellular domain (ICD) of zebrafish Jagged2, and investigated a possible role of PEX1 as a component of Notch signaling for the cell-fate decision and the differentiation of neural precursors in p3 domain. Western blotting showed that zebrafish PEX1 might interacts with the ICD of zebrafish Jagged2 physically. PEX1 morpholino-injected embryos showed the increased number of GABAergic KA" neurons as well as the ectopic expression of secondary motor neurons in the p3 domain. The increased number of KA" neurons was also observed in the zebrafish embryos with PEX1 mutation induced by CRISPR/Cas9. These phenotypes resemble with that of Jagged2 morphant. Our observations imply that a critical role of PEX1 in the cell-fate decision of proliferating neural precursors in the p3 domain during the continuing growth and development of the vertebrate nervous system.
