Proteasome gene expression is controlled by coordinated functions of multiple transcription factors.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).

Therapeutic potential of clinically proven natural products in the management of dementia.

Dementia is a common neurodegenerative disorder connected to damage to nerve cells in the brain. Although some conventional drugs are available for dementia treatments and are still sanctified for dementia patients, their short- and long-term side effects and other limitations make treating patients more challenging. The authors aimed to explain novel options for treating dementia with natural products and unravel some clinically proven natural products. This article systematically reviewed recent studies that have investigated the role of natural products and their bioactive compounds for dementia. PubMed Central, Scopus, and Google Scholar databases of articles were collected, and abstracts were reviewed for relevance to the subject matter.In this review, we provide mechanistic insights of clinically validated natural products, including like- Yokukansan, Souvenaid, BDW, Hupergene, Bacopa monnier, Omega-3, Tramiprostate and Palmitoylethanolamide with which have therapeutic efficacy against dementia in the management of dementia. As shown by studies, certain natural ingredients could be used to treat and prevent dementia. We strongly believe that the medicinal plants and phytoconstituents alone or in combination with other compounds would be effective treatments against dementia with lesser side effects as compared to currently available treatments. Moreover, these products should be studied further in order to develop novel dementia medications.

FGF2, LIF, and IGF-1 supplementation improves mouse oocyte in vitro maturation via increased glucose metabolism†.

Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.

Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk.

Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. One limitation of our first protocol for human blastoid generation was relatively low efficiency. We now report an optimized protocol for the efficient generation of large quantities of high-fidelity human blastoids from naive pluripotent stem cells. This enabled proteomics analysis that identified phosphosite-specific signatures potentially involved in the derivation and/or maintenance of the signaling states in human blastoids. Additionally, we uncovered endometrial stromal effects in promoting trophoblast cell survival, proliferation, and syncytialization during co-culture with blastoids and blastocysts. Side-by-side single-cell RNA sequencing revealed similarities and differences in transcriptome profiles between pre-implantation blastoids and blastocysts, as well as post-implantation cultures, and uncovered a population resembling early migratory trophoblasts during co-culture with endometrial stromal cells. Our optimized protocol will facilitate broader use of human blastoids as an accessible, perturbable, scalable, and tractable model for human blastocysts.

FGF2, LIF, and IGF1 (FLI) supplementation during human in vitro maturation enhances markers of gamete competence.

STUDY QUESTION: Does a chemically defined maturation medium supplemented with FGF2, LIF, and IGF1 (FLI) improve in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) obtained from children, adolescents, and young adults undergoing ovarian tissue cryopreservation (OTC)? SUMMARY ANSWER: Although FLI supplementation did not increase the incidence of oocyte meiotic maturation during human IVM, it significantly improved quality outcomes, including increased cumulus cell expansion and mitogen-activated protein kinase (MAPK) expression as well as enhanced transzonal projection retraction. WHAT IS KNOWN ALREADY: During OTC, COCs, and denuded oocytes from small antral follicles are released into the processing media. Recovery and IVM of these COCs is emerging as a complementary technique to maximize the fertility preservation potential of the tissue. However, the success of IVM is low, especially in the pediatric population. Supplementation of IVM medium with FLI quadruples the efficiency of pig production through improved oocyte maturation, but whether a similar benefit occurs in humans has not been investigated. STUDY DESIGN, SIZE, DURATION: This study enrolled 75 participants between January 2018 and December 2021 undergoing clinical fertility preservation through the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago. Participants donated OTC media, accumulated during tissue processing, for research. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants who underwent OTC and include a pediatric population that encompassed children, adolescents, and young adults ≤22 years old. All participant COCs and denuded oocytes were recovered from media following ovarian tissue processing. IVM was then performed in either a standard medium (oocyte maturation medium) or one supplemented with FLI (FGF2; 40 ng/ml, LIF; 20 ng/ml, and IGF1; 20 ng/ml). IVM outcomes included meiotic progression, cumulus cell expansion, transzonal projection retraction, and detection of MAPK protein expression. MAIN RESULTS AND THE ROLE OF CHANCE: The median age of participants was 6.3 years, with 65% of them classified as prepubertal by Tanner staging. Approximately 60% of participants had been exposed to chemotherapy and/or radiation prior to OTC. On average 4.7 ± 1 COCs and/or denuded oocytes per participant were recovered from the OTC media. COCs (N = 41) and denuded oocytes (N = 29) were used for IVM (42 h) in a standard or FLI-supplemented maturation medium. The incidence of meiotic maturation was similar between cohorts (COCs: 25.0% vs 28.6% metaphase II arrested eggs in Control vs FLI; denuded oocytes: 0% vs 5.3% in Control vs FLI). However, cumulus cell expansion was 1.9-fold greater in COCs matured in FLI-containing medium relative to Controls and transzonal projection retraction was more pronounced (2.45 ± 0.50 vs 1.16 ± 0.78 projections in Control vs FLIat 16 h). Additionally, MAPK expression was significantly higher in cumulus cells obtained from COCs matured in FLI medium for 16-18 h (chemiluminescence corrected area 621,678 vs 2,019,575 a.u., P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Our samples are from human participants who exhibited heterogeneity with respect to age, diagnosis, and previous treatment history. Future studies with larger sample sizes, including adult participants, are warranted to determine the mechanism by which FLI induces MAPK expression and activation. Moreover, studies that evaluate the developmental competence of eggs derived from FLI treatment, including assessment of embryos as outcome measures, will be required prior to clinical translation. WIDER IMPLICATIONS OF THE FINDINGS: FLI supplementation may have a conserved beneficial effect on IVM for children, adolescents, and young adults spanning the agricultural setting to clinical fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Department of Obstetrics and Gynecology startup funds (F.E.D.), Department of Surgery Faculty Practice Plan Grant and the Fertility & Hormone Preservation & Restoration Program at the Ann & Robert H. Lurie Children's Hospital of Chicago (M.M.L. and E.E.R.). M.M.L. is a Gesualdo Foundation Research Scholar. Y.Y.'s research is supported by the internal research funds provided by Colorado Center of Reproductive Medicine. Y.Y., L.D.S., R.M.R., and R.S.P. have a patent pending for FLI. The remaining authors have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Proteasome gene expression is controlled by the coordinated functions of multiple transcription factors.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo , we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 d after denervation) it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PAL NRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PAL NRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g. type-2 diabetes, cancer).

Assembly checkpoint of the proteasome regulatory particle is activated by coordinated actions of proteasomal ATPase chaperones.

The proteasome holoenzyme regulates the cellular proteome via degrading most proteins. In its 19-subunit regulatory particle (RP), a heterohexameric ATPase enables protein degradation by injecting protein substrates into the core peptidase. RP assembly utilizes "checkpoints," where multiple dedicated chaperones bind to specific ATPase subunits and control the addition of other subunits. Here, we find that the RP assembly checkpoint relies on two common features of the chaperones. Individual chaperones can distinguish an RP, in which their cognate ATPase persists in the ATP-bound state. Chaperones then together modulate ATPase activity to facilitate RP subunit rearrangements for switching to an active, substrate-processing state in the resulting proteasome holoenzyme. Thus, chaperones may sense ATP binding and hydrolysis as a readout for the quality of the RP complex to generate a functional proteasome holoenzyme. Our findings provide a basis to potentially exploit the assembly checkpoints in situations with known deregulation of proteasomal ATPase chaperones.

Two alternative mechanisms regulate the onset of chaperone-mediated assembly of the proteasomal ATPases.

The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as 'Rpt ring', assembles through six ATPase subunits (Rpt1-Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2, and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early-stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism-degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully-formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone-mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation.

Reconsidering the roles of endogenous estrogens and xenoestrogens: the membrane estradiol receptor G protein-coupled receptor 30 (GPR30) mediates the effects of various estrogens.

Estrone (E1) and estriol (E3) are considered "weak" estrogens, which exert suppressive effects through estrogen receptors α and ß. However, recent studies have demonstrated that E1 and E3, as well as estradiol (E2), suppress gonadotropin-releasing hormone-induced luteinizing hormone secretion from bovine gonadotrophs via G-protein-coupled receptor 30, which is expressed in various reproductive organs. Currently, there is a lack of fundamental knowledge regarding E1 and E3, including their blood levels. In addition, xenoestrogens may remain in the body over long time periods because of enterohepatic circulation. Therefore, it is time to reconsider the roles of endogenous estrogens and xenoestrogens for reproduction.

Expression of macrophage migration inhibitory factor (MIF) in bovine oviducts is higher in the postovulatory phase than during the oestrus and luteal phase.

Whether macrophage migration inhibitory factor (MIF) in the bovine oviduct is important for early embryogenesis has not been well substantiated. The aim of the present study was to test the hypothesis that bovine oviduct expresses higher levels of MIF during the post-ovulation phase. Both ampullary and isthmic samples were collected from Japanese black heifers during oestrus (Day 0; n=5), postovulation (Day 3; n=6) and luteal phase (Days 9-12; n=5). MIF mRNA and protein were extracted from the ampullary and isthmic samples and their levels measured by real-time polymerase chain reaction and western blot analysis respectively. Fluorescent immunohistochemistry was performed on frozen ampullary and isthmic sections using antibodies against MIF. MIF mRNA and protein expression was higher in the postovulatory phase than during oestrus and the luteal phase (P<0.05). Fluorescent immunohistochemistry confirmed that in all phases of the oestrous cycle evaluated, the primary site of MIF expression in the ampulla and isthmus was the tunica mucosa. In conclusion, the bovine ampulla and isthmus have higher MIF expression during the postovulatory phase. Further studies are needed to clarify the role of MIF in bovine oviducts.

Positive correlations of age and parity with plasma concentration of macrophage migration inhibitory factor in Japanese black cows.

Plasma Macrophage migration inhibitory factor (MIF) concentration correlates positively with age, and negatively with self-rated health in women, and optimal MIF concentration may promote proper reproductive function. This study was conducted to evaluate the hypotheses that plasma MIF concentration changes with parturition or postpartum first ovulation, and that age in months and parity correlate with plasma MIF concentration in Japanese black cows. Western blotting utilizing an anti-MIF mouse monoclonal antibody of various tissues and plasma from females indicated that MIF expression was stronger in the anterior pituitary than in other tissues. We developed a competitive EIA utilizing the same anti-MIF mouse monoclonal antibody with sufficient sensitivity and reliable performance for measuring bovine plasma samples. We then measured MIF concentrations in bovine plasma collected from 4 weeks before parturition to 4 weeks after postpartum first ovulation. There was no significant difference in plasma MIF concentration pre- and post-parturition, or before and after the postpartum first ovulation. Plasma MIF concentrations were positively correlated (P < 0.01) with parity (r = 0.703), age in months on the day of parturition (r = 0.647), and age in months on the day of the postpartum first ovulation (r = 0.553) when we used almost all data, except for that from a third-parity cow with an abnormally high plasma MIF concentration. We therefore concluded that plasma MIF concentrations may increase with age in months and parity, but do not change either before and after parturition or before and after postpartum first ovulation in Japanese black cows.

Suppressed expression of macrophage migration inhibitory factor in the oviducts of lean and obese cows.

Oviducts synthesise macrophage migration inhibitory factor (MIF) to promote sperm capacitation and embryogenesis. This study aimed to test a hypothesis that the oviducts of obese cows may express MIF at a lower level than those of normal and lean cows. Ampullar and isthmic oviduct sections were collected from lean (n=5; body condition score (BCS) on a 5-point scale, 2.5), normal (n=6; BCS, 3.0) and obese (n=5; BCS, 4.0) Japanese Black cows. MIF mRNA and protein were extracted from ampullae and isthmuses and their levels measured by real-time polymerase chain reaction or western blot. Immunohistochemistry was performed on frozen sections of ampullae and isthmuses by using antibodies to MIF. MIF mRNA and protein expression were lower in the obese and lean groups than in the normal group (P<0.05). Immunohistochemistry revealed that the primary site of MIF expression in the ampulla and isthmus is the tunica mucosa. In conclusion, obese cows have suppressed MIF expression in the ampullae and isthmuses of their oviducts, as hypothesised, but, unexpectedly, MIF expression was also lower in lean cows.

Gonadotropin-releasing hormone (GnRH) receptors of cattle aggregate on the surface of gonadotrophs and are increased by elevated GnRH concentrations.

The presence of gonadotropin-releasing hormone (GnRH) receptors (GnRHRs) on gonadotrophs in the anterior pituitary (AP) is an important factor for reproduction control. However, little is known regarding GnRHR gene expression in gonadotrophs of cattle owing to the lack of an appropriate anti-GnRHR antibody. Therefore, an anti-GnRHR antibody for immunohistochemistry, flow cytometry, and immunocytochemistry assays was developed to characterize GnRHR gene expression in gonadotrophs. The anti-GnRHR antibody could suppress GnRH-induced LH secretion from cultured AP cells of cattle. The GnRHR, luteinizing hormone (LH), and follicle stimulating hormone (FSH) in the AP tissue was analyzed by fluorescence immunohistochemistry. The GnRHRs were aggregated on a limited area of the cell surface of gonadotrophs, possibly localized to lipid rafts. The LH secretion was stimulated with increasing amounts of GnRH; however, excessive concentrations (> 1 nM) resulted in a decrease in LH secretion. A novel method to purify gonadotrophs was developed using the anti-GnRHR antibody and fluorescence-activated cell sorting. Flow cytometric analysis using the anti-GnRHR antibody for cultured bovine AP cells, however, failed to support the hypothesis that GnRH induces GnRHR internalization and decreases GnRHR on the surface of GnRHR-positive AP cells. In contrast, immunocytochemistry using primary antibodies for cultured bovine AP cells showed that 10 nM (P < 0.05) and 100 nM (P < 0.01) GnRH, but not 0.01-1 nM GnRH, increased GnRHR in the cytoplasm of LH-positive cells. In conclusion, these data suggested that GnRHRs were aggregated on the surface of gonadotrophs and GnRHR inside gonadotrophs increased with elevated concentrations of GnRH.

Suppressed expression of granulocyte macrophage colony-stimulating factor in oviduct ampullae of obese cows.

Obese heifers have been found to produce fewer excellent-grade embryos than lean and normal heifers due to unknown mechanisms. Oviducts synthesize granulocyte macrophage colony-stimulating factor (GMCSF) to promote embryogenesis, and GMCSF expression may be down-regulated in the oviducts of obese cows. The present study evaluated the relationship between the degree of obesity and GMCSF expression in the ampullary or isthmic section of oviducts in lean [n=5; body condition score (BCS) on a 5-point scale, 2.5], normal (n=6; BCS, 3.0), and obese (n=5; BCS, 4.0) Japanese Black cows. GMCSF mRNA and protein expression in the ampulla, measured by real-time PCR and western blotting, respectively, were less (P<0.05) in the obese group than in the normal group. mRNA and GMCSF protein did not differ significantly in the isthmus among the three groups. The obese group had less GMCSF immuno-reactivity in the tunica mucosa, the primary site of GMCSF gene expression, of the ampulla than the normal and lean groups. In conclusion, unlike normal and lean cows, obese cows had suppressed GMCSF gene expression in the ampulla.