Comparison of Cryopreservation Media for Mesenchymal Stem Cell Spheroids.

Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.

Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Effect of severe acute respiratory syndrome coronavirus 2 infection during pregnancy in K18-hACE2 transgenic mice.

OBJECTIVE: This study aimed to examine the influence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on pregnancy in cytokeratin-18 (K18)-hACE2 transgenic mice. METHODS: To determine the expression of hACE2 mRNA in the female reproductive tract of K18-hACE2 mice, real-time polymerase chain reaction (RT-PCR) was performed using the ovary, oviduct, uterus, umbilical cord, and placenta. SARS-CoV-2 was inoculated intranasally (30 µL/mouse, 1×104 TCID50/mL) to plug-checked K18-hACE2 homozygous female mice at the pre-and post-implantation stages at 2.5 days post-coitum (dpc) and 15.5 dpc, respectively. The number of implantation sites was checked at 7.5 dpc, and the number of normally born pups was investigated at 20.5 dpc. Pregnancy outcomes, including implantation and childbirth, were confirmed by comparison with the non-infected group. Tissues of infected mice were collected at 7.5 dpc and 19.5 dpc to confirm the SARS-CoV-2 infection. The infection was identified by performing RT-PCR on the infected tissues and comparing them to the non-infected tissues. RESULTS: hACE2 mRNA expression was confirmed in the female reproductive tract of the K18-hACE2 mice. Compared to the non-infected group, no significant difference in the number of implantation sites or normally born pups was found in the infected group. SARS-CoV-2 infection was detected in the lungs but not in the female reproductive system of infected K18-hACE2 mice. CONCLUSION: In K18-hACE2 mice, intranasal infection with SARS-CoV-2 did not induce implantation failure, preterm labor, or miscarriage. Although the viral infection was not detected in the uterus, placenta, or fetus, the infection of the lungs could induce problems in the reproductive system. However, lung infections were not related to pregnancy outcomes.

Conventional versus modified nipple sparing mastectomy in immediate breast reconstruction: Complications, aesthetic, and patient-reported outcomes.

Background: Nipple-sparing mastectomy (NSM) followed by immediate breast reconstruction (IBR) is the optimal surgical treatment for breast cancer. However, investigations are ongoing to improve the surgical technique to achieve better results. This study aimed to evaluate the outcomes of modified NSM (m-NSM), which preserves the anterior lamellar fat layer, in patients who underwent IBR. Methods: All patients who underwent modified NSM (m-NSM) or conventional NSM (c-NSM) followed by IBR using autologous tissue or implants were retrospectively reviewed between January 2014 and January 2021. Two mastectomy types were compared in terms of postoperative complications and aesthetic outcomes using panel assessment scores by physicians and reported outcomes using Breast-Q. In addition, postoperative evaluations of the thickness of mastectomy flap was performed using CT scan images. Results: A total of 516 patients (580 breasts) with NSM (143 breasts with c-NSM and 437 breasts with m-NSM) followed by IBR were reviewed. The mean ± SD flap thickness was 8.48 ± 1.81 mm in patients who underwent m-NSM, while it was 6.32 ± 1.15 mm in the c-NSM cohort (p = 0.02). The overall major complications rate was lower in the m-NSM group (3.0% vs. 9.0%, p < 0.013). Ischemic complications of the mastectomy flap and nipple-areolar complex (NAC) were more in c-NSM, although the difference was not statistically significant. The mean panel assessment scores were higher in the m-NSM group (3.14 (good) and 2.38 (fair) in the m-NSM and c-NSM groups, respectively; p < 0.001). Moreover, m-NSM was associated with greater improvements in psychosocial (p < 0.001) and sexual (p = 0.007) well-being. Conclusion: Preserving the anterior lamellar fat in NSM was associated with thicker mastectomy flap, overall lower rates of complications, including ischemia of the mastectomy flap and nipple-areolar complex, and was associated with better aesthetic outcomes and improved quality of life.

Estrogen Regulates the Expression and Localization of YAP in the Uterus of Mice.

The dynamics of uterine endometrium is important for successful establishment and maintenance of embryonic implantation and development, along with extensive cell differentiation and proliferation. The tissue event is precisely and complicatedly regulated as several signaling pathways are involved including two main hormones, estrogen and progesterone signaling. We previously showed a novel signaling molecule, Serine/threonine protein kinase 3/4 (STK3/4), which is responded to hormone in the mouse uterine epithelium. However, the role and regulation of its target, YES-associated protein (YAP) remains unknown. In this study, we investigated the expression and regulation of YAP in mouse endometrium. We found that YAP was periodically expressed in the endometrium during the estrous cycle. Furthermore, periodic expression of YAP was shown to be related to the pathway under hormone treatment. Interestingly, estrogen was shown to positively modulate YAP via endometrial epithelial receptors. In addition, the knockdown of YAP showed that YAP regulated various target genes in endometrial cells. The knockdown of YAP down-regulated numerous targets including ADAMTS1, AMOT, AMOTL1, ANKRD1, CTNNA1, MCL1. On the other hand, the expressions of AREG and AXL were increased by its knockdown. These findings imply that YAP responds via Hippo signaling under various intrauterine signals and is considered to play a role in the expression of factors important for uterine endometrium dynamic regulation.

Epigenetic regulator Cfp1 safeguards male meiotic progression by regulating meiotic gene expression.

Meiosis occurs specifically in germ cells to produce sperm and oocytes that are competent for sexual reproduction. Multiple factors are required for successful meiotic entry, progression, and termination. Among them, trimethylation of histone H3 on lysine 4 (H3K4me3), a mark of active transcription, has been implicated in spermatogenesis by forming double-strand breaks (DSBs). However, the role of H3K4me in transcriptional regulation during meiosis remains poorly understood. Here, we reveal that mouse CXXC finger protein 1 (Cfp1), a component of the H3K4 methyltransferase Setd1a/b, is dynamically expressed in differentiating male germ cells and safeguards meiosis by controlling gene expression. Genetic ablation of mouse CFP1 in male germ cells caused complete infertility with failure in prophase I of the 1st meiosis. Mechanistically, CFP1 binds to genes essential for spermatogenesis, and its loss leads to a reduction in H3K4me3 levels and gene expression. Importantly, CFP1 is highly enriched within the promoter/TSS of target genes to elevate H3K4me3 levels and gene expression at the pachytene stage of meiotic prophase I. The most enriched genes were associated with meiosis and homologous recombination during the differentiation of spermatocytes to round spermatids. Therefore, our study establishes a mechanistic link between CFP1-mediated transcriptional control and meiotic progression and might provide an unprecedented genetic basis for understanding human sterility.

Generation of a B2M homozygous knockout human somatic cell nuclear transfer-derived embryonic stem cell line using the CRISPR/Cas9 system.

Beta2-microglobulin (B2M) is a subunit of human leukocyte antigen class-I (HLA-I) heterodimer that mediates immune rejection through activation of cytotoxic T cells. B2M binding to HLA-I proteins is essential for functional HLA-I on the cell surface. Here, we generated a B2M homozygous knockout somatic cell nuclear transfer-induced embryonic stem cell (SCNT-ESC) line using CRISPR/Cas9-mediated gene targeting. B2M KO cell line, which does not express HLA-I molecules on cell surface, has pluripotency and differentiation ability to three germ layers. This cell line provides a useful cell source for investigating immunogenicity of allogeneic ESCs and their derivatives for tissue regeneration.

FK866 Protects Human Dental Pulp Cells against Oxidative Stress-Induced Cellular Senescence.

FK866 possesses various functional properties, such as anti-angiogenic, anti-cancer, and anti-inflammatory activities. We previously demonstrated that premature senescence of human dental pulp cells (hDPCs) was induced by hydrogen peroxide (H2O2). The present study aimed to investigate whether H2O2-induced premature senescence of hDPCs is affected by treatment with FK866. We found that FK866 markedly inhibited the senescent characteristics of hDPCs after exposure to H2O2, as revealed by an increase in the number of senescence-associated ß-galactosidase (SA-ß-gal)-positive hDPCs and the upregulation of the p21 and p53 proteins, which acts as molecular indicators of cellular senescence. Moreover, the stimulatory effects of H2O2 on cellular senescence are associated with oxidative stress induction, such as excessive ROS production and NADPH consumption, telomere DNA damage induction, and upregulation of senescence-associated secretory phenotype factors (IL-1ß, IL-6, IL-8, COX-2, and TNF-α) as well as NF-κB activation, which were all blocked by FK866. Thus, FK866 might antagonize H2O2-induced premature senescence of hDPCs, acting as a potential therapeutic antioxidant by attenuating oxidative stress-induced pathologies in dental pulp, including inflammation and cellular senescence.

TRITC-Loaded PLGA Nanoparticles as Drug Delivery Carriers in Mouse Oocytes and Embryos.

The structural layers around oocytes make it difficult to deliver drugs aimed at treating infertility. In this study, we sought to identify nanoparticles (NPs) that could easily pass through zona pellucida (ZP), a special layer around oocytes, for use as a drug delivery carrier. Three types of NPs were tested: quantum dot NPs, PE-polyethylene glycol (PEG)-loaded poly(lactic-co-glycolic acid) (PLGA) NPs (PEG/PL), and tetramethylrhodamine-loaded PLGA NPs (TRNPs). When mouse oocytes were treated with NPs, only TRNPs could fully pass through the ZP and cell membrane. To assess the effects of TRNPs on fertility and potential nanotoxicity, we performed mRNA sequencing analysis to confirm their genetic safety. We established a system to successfully internalize TRNPs into oocytes. The genetic stability and normal development of TRNP-treated oocytes and embryos were confirmed. These results imply that TRNPs can be used as a drug delivery carrier applicable to germ cells.

Sirtuin 6 deficiency induces endothelial cell senescence via downregulation of forkhead box M1 expression.

Cellular senescence of endothelial cells causes vascular dysfunction, promotes atherosclerosis, and contributes to the development of age-related vascular diseases. Sirtuin 6 (SIRT6), a conserved NAD+-dependent protein deacetylase, has beneficial effects against aging, despite the fact that its functional mechanisms are largely uncharacterized. Here, we show that SIRT6 protects endothelial cells from senescence. SIRT6 expression is progressively decreased during both oxidative stress-induced senescence and replicative senescence. SIRT6 deficiency leads to endothelial dysfunction, growth arrest, and premature senescence. Using genetically engineered endothelial cell-specific SIRT6 knockout mice, we also show that down-regulation of SIRT6 expression in endothelial cells exacerbates vascular aging. Expression microarray analysis demonstrated that SIRT6 modulates the expression of multiple genes involved in cell cycle regulation. Specifically, SIRT6 appears to regulate the expression of forkhead box M1 (FOXM1), a critical transcription factor for cell cycle progression and senescence. Overexpression of FOXM1 ameliorates SIRT6 deficiency-induced endothelial cell senescence. In this work, we demonstrate the role of SIRT6 as an anti-aging factor in the vasculature. These data may provide the basis for future novel therapeutic approaches against age-related vascular disorders.

BIRC5 Expression is Regulated in Uterine Epithelium During the Estrous Cycle.

Baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), also known as survivin, is a member of the inhibitor of apoptosis (IAP) family of proteins and regulates the size of tissues through cell division control. The uterus is the most dynamically sized organ among tissues during the estrous cycle. Although Birc5 is expressed in some terminally differentiated cells, the regulation of its expression in the uterus remains unknown. We investigated the regulation of Birc5 expression in the mouse uterus. RT-PCR analysis showed that Birc5 was expressed in various tissues, including the uterus; the expression level of Birc5 was significantly higher at the diestrus stage. Immunohistochemistry and Western blotting analysis revealed that Birc5 was more active in luminal and glandular epithelium than in endometrial stroma. In ovariectomized mice, Birc5 expression in the uterus was gradually increased by estrogen treatment; however, progesterone injection decreased its expression. Estrogen-induced Birc5 expression was blocked by treatment with estrogen receptor antagonist, ICI 182, 780 and progesterone-reduced Birc5 expression was inhibited by the progesterone receptor antagonist RU486. These results suggest that Birc5 expression is dynamically regulated by a combination of estrogen and progesterone via their receptor-mediated signaling.

Epigenetic priming by Dot1l in lymphatic endothelial progenitors ensures normal lymphatic development and function.

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood-lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.

Decrease of peripheral blood lymphocyte count predicts response to neoadjuvant chemotherapy in breast cancer patients.

Purpose: Pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) holds promise as a significant prognostic factor to predict NAC response in breast cancer patients. The absolute peripheral blood lymphocyte (PBL) count has been suggested as an independent predictor of response to NAC. The current study evaluated the relationship between pCR and the change of PBL count in patients treated with NAC. Methods: A total of 61 patients with histologically confirmed breast cancer treated with NAC followed by mastectomy between January 2010 and December 2019 were analyzed retrospectively. Correlational analyses confirmed a statistically significant relationship between PBL count and pCR. Following conformational correlational analyses, patients were divided into two groups according to cutoff values using the receiver operating characteristics curve and a logistic regression was conducted to determine the optimal conditions for achieving pCR. Results: A total of 14 patients (22.9%) achieved pCR. Most PBL counts decreased after NAC relevant to pCR. Logistic regression analysis revealed that a small decrease of PBL was associated with pCR (P=0.028). The cutoff value of PBL decrease was 755×106/L, which was used to divide patients into high and low reduction groups. The pCR rate was 11.43% and 38.46% for the high and low reduction group, respectively (area under the curve, 0.707; 95% confidence interval, 0.556-0.858; P=0.020). The high reduction group was found to have more difficulty achieving pCR. Conclusion: The decrease of PBL is significantly associated with pCR. Our data support that the decrease of PBL after NAC may be useful factors in predicting the response to NAC in breast cancer patients.

STK3/4 Expression Is Regulated in Uterine Endometrial Cells during the Estrous Cycle.

The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle.

Differential Regulation of TLE3 in Sertoli Cells of the Testes during Postnatal Development.

Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.

Synergistic protective effects of a statin and an angiotensin receptor blocker for initiation and progression of atherosclerosis.

AIM: Although the atheroprotective effects of statins and angiotensin II receptor blockers (ARBs) are well-established, little is known about their additive effects, especially during the early period of atherosclerosis. The aim of this study was to investigate whether combination of a statin and an ARB exerts synergistic anti-atherosclerotic effects, and to elucidate the mechanisms of combined effects. METHODS: Atherosclerotic plaques were developed in arteries of 23 rabbits using a high-cholesterol diet (HCD) and intra-arterial balloon inflation. Rabbits received one of five different treatment strategies for 4 weeks: positive control [n = 5, HCD]; negative control [n = 3, regular chow diet]; statin [n = 5, HCD and rosuvastatin 10 mg]; ARB [n = 5, HCD and olmesartan 20 mg]; and combination [n = 5, HCD and statin+ARB]. RESULTS: Histological analysis demonstrated that development of atherosclerotic plaques was inhibited more in combination group than in statin group (P = 0.001). Although macrophage infiltration identified by RAM11 staining was not significantly different between combination and individual treatment groups (31.76±4.84% [combination] vs. 38.11±6.53% [statin; P = 0.35] or 35.14±2.87% [ARB; P = 0.62]), the relative proportion of pro-inflammatory M1-macrophages was significantly lower in combination group than in ARB group (3.20±0.47% vs. 5.20±0.78%, P = 0.02). Furthermore, M2-macrophage polarization was higher in combination group than in statin group (17.70±3.04% vs. 7.86±0.68%, P = 0.001). CONCLUSION: Combination treatment with a statin and an ARB produced synergistic protective effects for atherosclerosis initiation and progression, which may be attributed to modulation of macrophage characteristics in the early period of atherosclerosis.

Long-Term Experience of Sperm Cryopreservation in Cancer Patients in a Single Fertility Center.

PURPOSE: Sperm cryopreservation before cancer treatment is the most effective method to preserve the fertility of male patients. We present our 21 years experience with sperm cryopreservation for cancer patients, including an examination of semen quality, the current status of cryopreserved sperm, and the rate of sperm use for assisted reproductive technology (ART). MATERIALS AND METHODS: A total of 721 cancer patients at Fertility Center of CHA Gangnam Medical Center successfully performed sperm cryopreservation for fertility preservation from January 1996 to December 2016. Medical chart review was used to analyze patient age, marital status, cancer type, semen volume, sperm counts and motility, length of storage, and current banking status. RESULTS: The major cancers of the 721 patients were leukemia (28.4%), lymphoma (18.3%), testis cancer (10.0%). The mean age at cryopreservation was 27.0 years, and 111 patients (15.4%) performed sperm cryopreservation during or after cancer treatment. The mean sperm concentration was 66.7±66.3 ×106/mL and the mean sperm motility was 33.8%±16.3%. During median follow-up duration of 75 months (range, 1-226 months), 44 patients (6.1%) used their banked sperm at our fertility center for ART and 9 patients (1.2%) transferred their banked sperm to another center. The median duration from cryopreservation to use was 51 months (range, 1-158 months). CONCLUSIONS: Sperm cryopreservation before gonadotoxic treatment is the most reliable method to preserve the fertility of male cancer patients. Sperm cryopreservation should be offered as a standard of care for all men planning cancer therapy.

Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients.

Using Protein-Fragment Complementation Assays (PCA) and Peptide Arrays to Study Telomeric Protein-Protein Interactions.

Studying protein-protein interactions is critical to our understanding of signaling pathways. Telomere Interactome is assembled around telomeres and consists of proteins and factors from diverse pathways. Dissecting how this protein network contributes to telomere protection and length regulation requires the elucidation of the complex and dynamic interactions between the proteins within the interactome. Here, we focus on three assays, Bi-molecular Fluorescence Complementation (BiFC), Gaussia Luciferase Protein-fragment Complementation Assay (GLuc PCA), and OPAL peptide array, which have proven vital in our studies of telomere protein interaction networks.

Rapid expression of RASD1 is regulated by estrogen receptor-dependent intracellular signaling pathway in the mouse uterus.

Dexamethasone-induced RAS-related protein 1 (RASD1) is a signaling protein that is involved in various cellular processes. In a previous study, we found that RASD1 expression was down-regulated in the uterine endometrium of repeated implantation failure patients. The study aim was to determine whether RASD1 is expressed in the endometrium of mouse uterus and how it is regulated by steroid hormones during the estrous cycle. In this study, we investigated RASD1 expression and regulation in an ovariectomized female mouse model. Rasd1 mRNA was highly expressed in mouse reproductive tissues, including the uterus. Rasd1 expression was detected exclusively in the endometrial epithelium at the proestrus stage of the estrous cycle. Rasd1 expression in uteri increased with administration of estradiol, but not progesterone. Its expression was rapidly induced within 2 h after E2 treatment. Pretreatment with ICI 182,780, an estrogen receptor antagonist, reduced RASD1 protein expression. In addition, we identified that rapid expression of Rasd1 was mediated by the estrogen intracellular signaling including both p38-mitogen-activated protein kinase and the extracellular signal-regulated kinase. These findings suggest that RASD1 acts as a novel signaling molecule and plays an important role in regulating dynamic uterine remodeling during the estrous cycle in the uterus.