HAS2 mRNA Research Articles

Skin Improvement Effects of Gardeniae fructus Extract in HaCaT Keratinocytes, B16F10 Melanocytes, and CCD-986sk Fibroblast Cells

The development of functional cosmetics with skin improvement effects from natural sources is necessary. In this study, the antioxidant, antiwrinkling, moisturizing, and whitening effects of Gardeniae fructus extract (GF) were investigated in keratinocytes, melanocytes, and fibroblast cells. Antioxidant activity was determined by a DPPH free radical scavenging assay. MMP-1, MMP-9, HAS1, and filaggrin mRNA levels were measured by RT-PCR in keratinocytes and fibroblast cells. MITF and tyrosinase protein levels were evaluated by blotting analysis in melanocytes. DPPH free radical activity was investigated to determine whether GF showed dose-dependent inhibitory activity. GF induced the upregulation of HAS1 and filaggrin mRNA expression in keratinocytes and fibroblast cells. GF led to the downregulation of MMP mRNA levels in keratinocytes and fibroblast cells. Western blotting was performed to confirm the whitening-related protein (MITF and tyrosinase) levels induced by GF in melanocytes, and the inhibitory activity was superior to that of the α-MSH used for the comparison test. GF showed marked antioxidant, antiwrinkling, skin moisturizing, and whitening activity in keratinocytes, melanocytes, and fibroblast cells. Through the results of these experiments, the applicability of GF as a natural and functional cosmetic material was verified.

The use of pituitary adenylate cyclase-activating polypeptide in the pre-maturation system improves in vitro developmental competence from small follicles of porcine oocytes

ObjectiveWe investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles.MethodsWe divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (Pre-SF[−]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF).ResultsCleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(−)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(−)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05).ConclusionThe PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

SAT-192 Chemokine Receptor CCR2 as a Novel Mediator of LH Signaling

Shortly before ovulation the LH surge induces processes critical for fertility, including cumulus-oocyte expansion (C-OE) and resumption of meiosis. While some of the paracrine-acting factors important for these events have been identified, the molecular mechanisms responsible for initiating such complex processes are not fully understood. Recent work from our laboratory showed a direct effect of the monocyte chemoattractant protein 1 (MCP1)/ C-C motif chemokine receptor 2 (CCR2) system, by increasing the mRNA levels of key genes involved in the ovulatory cascade in vitro. The aim of the present study was to determine whether inhibition of CCR2 signaling in the COC interfere with the expression of periovulatory genes and/or oocyte maturation. To test this, ovaries were surgically removed from adult female domestic cats (Felis catus, n=33) at unknown stages of the estrous cycle during the breeding season. Then, COCs were isolated from antral follicles and cultured for 3 hr (time when periovulatory genes peak in our culture system) or 28 hr [in vitro maturation (IVM)] with known inducers of C-OE and/or oocyte maturation [Gonadotropins (GNTs), amphiregulin (AREG) and PGE2)] in the presence or absence of a highly selective CCR2 antagonist (1 µM; RS 504393). At the end of culture (3 hr), total RNA from each COC (n=40) was individually extracted and the mRNA expression of periovulatory genes (HAS2, AREG, TSG6, PTX3) was analyzed by quantitative real-time PCR using specific TaqMan probes. In a second experiment, after IVM (28 hr) oocytes (n=161) were denuded and fixed to subsequent assess their nuclear oocyte maturation by immunofluorescence, due to the dark appearance of the feline oocytes. Expression results showed that RS 504393 was able to prevent or interfere (p< 0.05) with the stimulation of genes induced by either GNTs, AREG or PGE2. When combined with GNTs, RS 504393 decreased the expression of AREG, HAS2 and TSG6 mRNA within the COC (p<0.05). In contrast, the addition of RS 504393 together with the GNTs increased PTX3 mRNA levels within the COC (p<0.05). Also, RS 504393 significantly decreased the mRNA levels of AREG and TSG6 induced by AREG and PGE2 treatments. However, RS 504393 was not able to inhibit the stimulation of PTX3 mRNA levels induced by AREG (p> 0.05). Regarding oocyte maturation, RS 504393 did not provoke a significant reduction of the proportion of MII oocytes stimulated by GNT or AREG. No significant effect was observed in the presence of PGE2 under our IVM culture conditions. In conclusion, our results demonstrated that the GNT stimulation of AREG, HAS2 and TSG6 mRNA levels occurs, at least in part, through the CCR2/MCP1 pathway, proposing CCR2 receptor as a novel mediator of the ovulatory LH signaling. This study was supported by PRESTAMO BID PICT 2014 Nº 666 and by the Fogarty International Center, of the National Institutes of Health under Award Number R01TW009163.

Tiliroside, a Constituent of Strawberry Seeds, Slightly Enhances Hyaluronan Production via Hyaluronan Synthase 2 Expression in Mouse Skin and Human Fibroblasts

Strawberry seeds are consumed with the sarcocarps, but their effects on the skin have not been well evaluated. We examined the effect of strawberry seed extract (SSE) and tiliroside, a major flavonol glycoside in SSE, on hyaluronan synthase (HAS) 2 expression and hyaluronan production in mouse skin and human fibroblasts. SSE and tiliroside were given orally to mice on a high-fat diet (HFD) or standard diet, and skin HAS2 mRNA expression and the skin hyaluronan content were evaluated. In addition, HAS2 protein expression and hyaluronan release were determined after fibroblasts were treated with SSE and tiliroside. Furthermore, binding of tiliroside to retinoic acid receptor (RAR) γ was examined. SSE (10 and 50 mg/kg) significantly increased HAS2 mRNA expression and the hyaluronan level in the skin of HFD-fed mice. Tiliroside (0.2 and 1 mg/kg) also increased skin HAS2 mRNA expression in HFD-fed mice. Moreover, SSE (10 mg/kg) increased HAS2 mRNA expression in mice on the standard diet. In human fibroblasts, both SSE (1 ug/mL) and tiliroside (0.3 ug/mL) significantly increased hyaluronan production, with HAS2 protein expression also being increased by SSE (10 ug/mL) and tiliroside (0.1 ug/mL). In the RARγ ligand assay, tiliroside (1 and 10 ug/mL) showed binding to RARγ. In conclusion, SSE and tiliroside increased hyaluronan synthase 2 expression in mouse skin and binding of tiliroside to RARγ was suggested to be involved.

Hyaluronic acid inhibition by 4-methylumbelliferone reduces the expression of cancer stem cells markers during hepatocarcinogenesis

Hyaluronic acid (HA) is a glycosaminoglycan of extracellular matrix related to cell surface which interacts with various cell types. To understand the role of HA during hepatocarcinogenesis, we assessed the effect of the inhibition of HA deposition and its association with heterogeneous hepatocellular carcinoma (HCC) cells. In this study, we used transgenic mice C57BL/6J-Tg(Alb1HBV)44Bri/J (HBV-TG) and normal C57BL/6 J (WT) for in vivo study, while HCC cells Huh7 and JHH6 as in vitro models. Both models were treated with an HA inhibitor 4-methylumbelliferone (4MU). We observed that 4MU treatments in animal model down-regulated the mRNA expressions of HA-related genes Has3 and Hyal2 only in HBV-TG but not in normal WT. As observed in vivo, in HCC cell lines, the HAS2 mRNA expression was down-regulated in Huh7 while HAS3 in JHH6, both with or without the presence of extrinsic HA. Interestingly, in both models, the expressions of various cancer stem cells (CD44, CD90, CD133, and EpCAM) were also decreased. Further, histological analysis showed that 4MU treatment with dose 25 mg/kg/day reduced fibrosis, inflammation, and steatosis in vivo, in addition to be pro-apoptotic. We concluded that the inhibition of HA reduced the expressions of HA-related genes and stem cells markers in both models, indicating a possible modulation of cells-to-cells and cells-to-matrix interaction.

Increased Hyaluronan and TSG-6 in Association with Neuropathologic Changes of Alzheimer's Disease.

Little is known about the extracellular matrix (ECM) during progression of AD pathology. Brain ECM is abundant in hyaluronan (HA), a non-sulfated glycosaminoglycan synthesized by HA synthases (HAS) 1-3 in a high molecular weight (MW) form that is degraded into lower MW fragments. We hypothesized that pathologic severity of AD is associated with increases in HA and HA-associated ECM molecules. To test this hypothesis, we assessed HA accumulation and size; HA synthases (HAS) 1-3; and the HA-stabilizing hyaladherin, TSG-6 in parietal cortex samples from autopsied research subjects with not AD (CERAD = 0, Braak = 0- II, n = 12-21), intermediate AD (CERAD = 2, Braak = III-IV, n = 13-18), and high AD (CERAD = 3, Braak = V-VI, n = 32-40) neuropathologic change. By histochemistry, HA was associated with deposits of amyloid and tau, and was also found diffusely in brain parenchyma, with overall HA quantity (measured by ELSA) significantly greater in brains with high AD neuropathology. Mean HA MW was similar among the samples. HAS2 and TSG-6 mRNA expression, and TSG-6 protein levels were significantly increased in high AD and both molecules were present in vasculature, NeuN-positive neurons, and Iba1-positive microglia. These results did not change when accounting for gender, advanced age (≥ 90 years versus <90 years), or the clinical diagnosis of dementia. Collectively, our results indicate a positive correlation between HA accumulation and AD neuropathology, and suggest a possible role for HA synthesis and metabolism in AD progression.

Expression of hyaluronan synthases upregulated by thyroid hormone is involved in intestinal stem cell development during Xenopus laevis metamorphosis.

During amphibian intestinal remodeling, thyroid hormone (TH) induces adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian one. We have previously shown that hyaluronan (HA) is newly synthesized and plays an essential role in the development of the stem cells via its major receptor CD44 in the Xenopus laevis intestine. We here focused on HA synthase (HAS) and examined how the expression of HAS family genes is regulated during natural and TH-induced metamorphosis. Our quantitative RT-PCR analysis indicated that the mRNA expression of HAS2 and HAS3, but not that of HAS1 and HAS-rs, a unique Xenopus HAS-related sequence, is upregulated concomitantly with the development of adult epithelial primordia consisting of the stem/progenitor cells during the metamorphic climax. In addition, our in situ hybridization analysis indicated that the HAS3 mRNA is specifically expressed in the adult epithelial primordia, whereas HAS2 mRNA is expressed in both the adult epithelial primordia and nearby connective tissue cells during this period. Furthermore, by treating X. laevis tadpoles with 4-methylumbelliferone, a HA synthesis inhibitor, we have experimentally shown that inhibition of HA synthesis leads to suppression of TH-upregulated expression of leucine-rich repeat-containing G protein-coupled 5 (LGR5), an intestinal stem cell marker, CD44, HAS2, HAS3, and gelatinase A in vivo. These findings suggest that HA newly synthesized by HAS2 and/or HAS3 is required for intestinal stem cell development through a positive feedback loop and is involved in the formation of the stem cell niche during metamorphosis.

Clinical significance of hyaluronan levels and its pro-osteogenic effect on mesenchymal stromal cells in myelodysplastic syndromes

BackgroundHyaluronan (HA), a major component of the extracellular matrix, has been proven to play a crucial role in tumor progression. However, it remains unknown whether HA exerts any effects in myelodysplastic syndromes (MDS).MethodsA total of 82 patients with MDS and 28 healthy donors were investigated in this study. We firstly examined the bone marrow (BM) serum levels of HA in MDS by radioimmunoassay. Then we determined HA production and hyaluronan synthase (HAS) gene expression in BM mesenchymal stromal cells (MSC) and mononuclear cells derived from MDS patients. Finally, we investigated the effects of HA on osteogenic differentiation of MSC.ResultsThe BM serum levels of HA was increased in higher-risk MDS patients compared to normal controls. Meanwhile, patients with high BM serum HA levels had significantly shorter median survival than those with low HA levels. Moreover, the HA levels secreted by MSC was elevated in MDS, especially in higher-risk MDS. In addition, HAS-2 mRNA expression was also up-regulated in higher-risk MDS-MSC. Furthermore, we found that MSC derived from MDS patients with high BM serum HA levels had better osteogenic differentiation potential. Moreover, MSC cultured in HA-coated surface presented enhanced osteogenic differentiation ability.ConclusionsOur results show that elevated levels of BM serum HA are related to adverse clinical outcome in MDS. Better osteogenic differentiation of MSC induced by HA may be implicated in the pathogenesis of MDS.

Depot-specific characteristics of adipose tissue-derived stromal cells in thyroid-associated orbitopathy

BackgroundThyroid-associated orbitopathy (TAO) causes inflammatory fibroproliferation of periocular connective tissues. We compared adipose tissue-derived stem/stromal cells (ADSCs) from three adipose depots of each patient with TAO on mesenchymal, myofibrogenic, adipogenic...

The Hyaluronic Acid System is Intact in Menstrual Endometrial Cells in Women With and Without Endometriosis.

To characterize the production and degradation of hyaluronic acid (HA) in menstrual endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) in women with and without endometriosis. To identify the presence of CD44, the primary receptor of HA, in menstrual EECs and ESCs in women with and without endometriosis. In vitro study. Academic center. Deidentified patient samples from women with and without endometriosis. EECs and ESCs were isolated from menstrual endometrial biopsies performed on women with (N = 9) and without (N = 11) endometriosis confirmed by laparoscopy. Real-time polymerase chain reaction, Western blot, and immunohistochemistry were used to assess hyaluronic acid synthase (HAS) isoforms 1, 2, and 3; hyaluronidase (HYAL) isoforms 1 and 2; and standard CD44. Student t test was used to analyze the results. There was no significant difference in messenger RNA (mRNA) or protein expression of HAS2, HAS3, HYAL1, or HYAL2 in EECs or ESCs from women with or without endometriosis. HAS1 mRNA was variably detected, whereas HAS1 protein was similarly expressed in EECs and ESCs from women with and without endometriosis. Standard CD44 was expressed in both cell types, and expression did not differ in cells from women with or without endometriosis. The HA system is expressed in eutopic menstrual ESCs and EECs from women with and without endometriosis. There are no differences in expression in HA production or degradation enzymes in EECs or ESCs from women with and without endometriosis. Standard CD44 expression does not differ in eutopic menstrual endometrial cells from women with and without endometriosis.

Changes in Hepatic Expression of Hyaluronan Synthases (HAS2 and HAS3) After Treatment with Steptozotocin in Channel Catfish

Streptozotocin (STZ) is commonly used experimentally to induce hyperglycemia in rodents. As an alternative model organism for investigating human obesity, channel catfish show a genetic predisposition toward an accelerated growth rate resulting in a phenotype similar to obese humans. In our previous studies, STZ treatment resulted in hypoglycemia and abnormal liver morphology in channel catfish, indicating that STZ treatment possibly induces liver damage. The mechanisms involved in the STZ‐induced hypoglycemic and tissue damage have never been examined. Another noted marker for liver damage stems from hyaluronan synthases (HAS), which have been associated with tissue damage in both humans and rodents. We hypothesized the expression of HAS mRNA would change in response to liver damage induced by STZ in channel catfish. Juvenile channel catfish were treated with 0, 3.6, 36, 180, or 360 mg STZ/kg body weight (n=4 fish/treatment) on day 0. Liver samples were collected on day 7. Hepatic expression of HAS2 and HAS3 mRNA was examined, using real‐time PCR. Expression of HAS2 and HAS3 mRNA decreased in response to an increased dose of STZ (p &lt; 0.05 and p &lt; 0.10, respectively). These results suggest STZ induces severe liver damage that down regulates the expression of HAS2 and HAS3 mRNA. However, the effects of STZ on gene expression appear to be global, and the tissue damage induced by STZ is not reversible by changes in the amount or activity of HAS.Support or Funding InformationThis research was funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS‐1238831). It was also supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyaluronan content and distribution in the rat ventral prostate after castration.

Hyaluronan (HA) has been implicated in tissue remodeling, healing, and tumor growth. This study investigated the variation in hyaluronan content, distribution, and metabolism in the rat ventral prostate (VP) in response to androgen deprivation after castration. The mRNA abundance of hyaluronan synthases (Has1-3) and hyaluronidases (Hyal 1-3) were assessed by reverse transcription (RT)-PCR and immunohistochemistry, respectively. The results demonstrated an increased concentration, but an overall reduction in HA content. HA was located in both epithelium and stroma of the prostate of both the noncastrated and castrated animals. Quantitative RT-PCR (qRT-PCR) showed that Has1 and Has2 are major synthases, and that Hyal 1 was the predominant hydrolase expressed in the VP. qRT-PCR also showed that Has1 and Has2 mRNA increased transiently after castration, whereas Has3 mRNA declined markedly. While Hyal 1 mRNA increased slowly up to day 21 after castration, Hyal 2 and Hyal 3 mRNA dropped significantly. CD44 was found in the epithelial cells and in some stromal cells in both hormonal conditions. In conclusion, castration results in increased abundance of Has1 and Has2 mRNA, but is associated with a decrease in the total content of HA, with an increased concentration, and a predominance of short-chain HA molecules.

Stromal-derived factor 1 directly promotes genes expressed within the ovulatory cascade in feline cumulus oocyte complexes.

We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24h. MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.

4-Methylumbelliferone inhibits enhanced hyaluronan synthesis and cell migration in pancreatic cancer cells in response to tumor-stromal interactions.

Hyaluronic acid (HA) in tumor stroma promotes tumor invasion and progression. 4-Methylumbelliferone (4-MU) is a potent HA synthesis inhibitor. In the present study, the effects of 4-MU on enhanced HA synthesis and cell migration in pancreatic ductal adenocarcinoma (PDAC) cells, in response to co-culture with stromal fibroblasts, was investigated. The HA concentration was determined using ELISA and a Transwell migration assay was used to analyze cell migratory capability. The mRNA expression levels of hyaluronan synthases (HAS1, HAS2 and HAS3) were determined using the quantitative polymerase chain reaction. Co-culture between Panc-1 cells and stromal fibroblasts markedly increased cell migration in association with increasing HA production, which was markedly associated with an increase in HAS3 mRNA expression. Treatment with 4-MU markedly decreased the HA production and cell migration of Panc-1 cells in the co-culture system. The results of the present study suggested that interactions between PDAC cells and stromal fibroblasts increased HA production, resulting in a marked increase in migration of PDAC cells, and 4-MU may be used as a chemotherapeutic agent to inhibit the enhanced migration of PDAC cells in response to tumor-stromal interactions.

Expression Pattern of Hyaluronan Synthase2 in Inflammatory Breast Cancer Cells

Breast cancer (BC) Is the most widely recognized threatening malignant disease around the whole world. It Is the second most incessant reason for malignant tumor growth related to death among woman at different ages. Inflammatory Breast Cancer (IBC) Is the most lethal form of BC with low survival rate. Because of lacking molecular markers, identification of new unique markers for IBC Is warranted. Hyaluronic acid synthases (HASs), particularly HAS2, have shown a critical role in the metastasis of non-inflammatory breast cancer (non-IBC). In this study, we assessed HAS2 mRNA expression by qPCR in the human triple negative non-IBC MDA-MB-231 and IBC SUM-149 cell lines. Our data demonstrated that HAS2 mRNA expression was significantly upregulated in IBC vs. non-IBC breast cancer cell lines. In conclusion, the high expression of HAS2 may represents a marker for IBC and could have a role in IBC pathogenesis.

Glucocorticoids Improve Myogenic Differentiation In Vitro by Suppressing the Synthesis of Versican, a Transitional Matrix Protein Overexpressed in Dystrophic Skeletal Muscles.

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.

The effect of vasopressin on the expression of genes of key enzymes of the interstitial hyaluronan turnover and concentration ability in WAG rat kidneys

In mammals arginine-vasopressin (AVP) is a major hormone involved in the regulation of renal water reabsorption, acting via the increase of the osmotic permeability of the epithelium of the collecting duct. The AVP-induced intracellular events include, as a core step, the trafficking of the vesicles containing the water channels, aquaporin-2, to the apical plasma membrane of the collecting duct’s principal cells. The interstitium of the renal inner medulla contains abundant linear negatively charged glycosaminoglycan, hyaluronan (HA), which affects the water flow between structures of the concentrating mechanism, depending on its polymeric state. Using real-time RT-PCR, we tested the assumption that the renal hyaluronan may be involved in the long-term vasopressin effect on water reabsorption. The expression of the genes encoding hyaluronan synthase-2 (Has2), hyaluronidase-1, and hyaluronidase-2 (Hyal1 and Hyal2) in the kidneys of Wistar Albino Glaxo (WAG) was studied. The Has2 mRNA content was the highest in the papilla of the kidneys of the hydrated rats. The V2 receptor-selective vasopressin analog dDAVP (100 μg/kg of body weight, twice a day intraperitoneally for 2 days) induced a considerable decrease in the Has2 mRNA content in the papilla with less pronounced changes in the cortex. In contrast to Has2, the dDAVP treatment caused a significant increase in the Hyal1 and Hyal2 mRNA content in the renal papilla. There was a good fit between the Hyal1 and Hyal2 transcriptional levels and changes in hyaluronidase activity in the renal tissue. It was suggested that vasopressin is able to inhibit the synthesis of hyaluronan and concomitantly promotes its degradation in the renal papilla interstitium, thereby facilitating water flow between the elements of the renal countercurrent system. The implications for this effect are discussed in the context of the data in the literature.

Effect of MT3 on Retinal and Choroidal TGF-β2 and HAS2 Expressions in Form Deprivation Myopia of Guinea Pig

Purpose To confirm its dose-dependent effect on form deprivation myopia and evaluate the effect of MT3 at different tissue concentrations on changes in mRNA and protein expression for TGF-β2 and HAS2. Methods MT3 was intravitreally injected into deprived eyes at two-day intervals. Refraction was measured by streak retinoscopy after cycloplegia. The axial dimensions were measured by A-scan ultrasound. The quantitative RT-PCR and Western blot were used to detect the changes of TGF-β2 and HAS2 expressions in the retina and choroid of guinea pigs. Results MT3 treatment produced a significant dose-dependent reduction in relative myopia compared to FD group (both p < 0.001). There were statistically significant increases in retinal and choroidal mRNA levels for both TGF-β2 and HAS2 after injections of 10 μM of MT3, when compared to the FD group. There were no significant differences in retinal and choroidal TGF-β2 protein expression levels between the MT3 treatment groups and FD group (all p > 0.05). The injections of 10 μM of MT3 caused a marked decrease in retinal HAS2 protein expression level, when compared to the FD group (p = 0.001). Conclusion MT3 can inhibit form deprivation myopia, and MT3 treatment can result in changes of retinal and choroidal TGF-β2 and HAS2 mRNA and protein expressions.

Effects of exosome-like vesicles on cumulus expansion in pigs in vitro.

Cell-secreted vesicles, such as exosomes, have recently been recognized as mediators of cell communication. A recent study in cattle showed the involvement of exosome-like vesicles in the control of cumulus expansion, a prerequisite process for normal ovulation; however, whether this is the case in other mammalian species is not known. Therefore, this study aimed to examine the presence of exosome-like vesicles in ovarian follicles and their effects on cumulus expansion in vitro in pigs. The presence of exosome-like vesicles in porcine follicular fluid (pFF) was confirmed by transmission electron microscopic observation, the detection of marker proteins, and RNA profiles specific to exosomes. Fluorescently labeled exosome-like vesicles isolated from pFF were incorporated into both cumulus and mural granulosa cells in vitro. Exosome-like vesicles were not capable of inducing cumulus expansion to a degree comparable to that induced by follicle-stimulating hormone (FSH). Moreover, exosome-like vesicles had no significant effects on the expression levels of transcripts required for the normal expansion process (HAS2, TNFAIP6, and PTGS2). Interestingly, FSH-induced expression of HAS2 and TNFAIP6 mRNA, but not of PTGS2 mRNA, was significantly increased by the presence of exosome-like vesicles; however, the degree of FSH-induced expansion was not affected. In addition, porcine exosome-like vesicles had no significant effects on the expansion of mouse cumulus-oocyte complexes. Collectively, the present results suggest that exosome-like vesicles are present in pFF, but they are not efficient in inducing cumulus expansion in pigs.

170 SUPPLEMENT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE DURING IN VITRO MATURATION ACTIVATES SMAD2 AND cAMP RESPONSIVE ELEMENT BINDING PROTEIN SIGNALING

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated reactive oxygen species (ROS) levels, specific gene transcription levels in oocytes and cumulus cells (CC) after IVM, and specific protein expression and activation levels in matured CC by Western blotting. Each concentration (0, 1, 10, and 100 ng mL−1) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analysed by ANOVA followed by Duncan using SPSS (IBM Corp., Armonk, NY). Data are presented as the mean (replicate numbers) and differences were considered significant at P &lt; 0.05. After 44 h of IVM, oocytes are mechanically denuded from CC with 0.1% hyaluronidase and only metaphase II stage oocytes are counted as nuclear matured oocytes. Each group of matured oocytes are stained by 2′,7′-dichlorodihydrofluorescein diacetate and the fluorescence was captured as graphic files under an epifluorescence microscope. The fluorescence intensities of oocytes were measured using Image J software (National Institutes for Health, Bethesda, MD). The groups treated with 10 and 100 ng mL−1 of GDF8 showed significantly more than 10% decrease in intracellular ROS levels compared with other groups (5 times). To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, real-time PCR was performed. In matured oocytes, the developmental competence marker POU5F1, antioxidant enzymes regulator NRF2, and antiapoptosis gene BCL-2 mRNA transcription levels were significantly increased in the 10 ng mL−1 treatment group compared with control (4 times). In CC, the 10 ng mL−1 treatment groups showed significantly higher PCNA and NRF2 mRNA levels, and the 1 and 10 ng mL−1 treatment groups observed significantly increased cumulus expansion-related genes HAS2, PTX3, and TNFAIP6 mRNA expression levels after IVM (4 times). To determine effect of GDF8 treatment during IVM, GDF8 downstream effector and oocyte ovulation-related protein expression and activation levels were analysed in CC after IVM by Western blotting. The 1 and 10 ng mL−1 treatment groups showed significantly increased phosphorylated (P)-SMAD2 (1.25 and 1.23 times increased compared with the control) and cyclic adenosine monophosphate responsive element binding protein (CREB; 1.31 and 1.32 times increased compared with the control) activation levels (4 times). In conclusion, supplementation of 10 ng mL−1 of GDF8 during IVM effectively increased the oocytes cytoplasmic maturation by reducing of intracellular ROS, and it seems correlated with significantly increased P-SMAD2, which is possibly related with induction of the cumulus cell expansion related genes expression and P-CREB while process of IVM. This work was supported by a grant from the Cooperative Research Program for Agriculture Science &amp; Technology Development (Project No. PJ011288), Rural Development Administration, Republic of Korea.