Ultrastructural study of neuronal cells and localization of ghrelin-like peptide and its receptor in the ganglia of the golden apple snail (Pomacea canaliculata).

Pomacea canaliculata is an invasive snail species causing major problems in agriculture. The snail biology was then investigated. The main objective of the present study was to investigate the nervous system of the snail. The nervous system comprises pairs of cerebral, buccal, pedal, pleural, parietal ganglia and an unpaired visceral ganglion. Most neurons were concentrated at the periphery of the ganglia. The neurons were classified into four types: NR1, NR2, NR3, and NR4. The percentages of the NR3 and NR4 in the pleural and pedal ganglia were significantly higher than those of other ganglia. Ultrastructural study revealed that nuclei of all neuronal types exhibited mostly euchromatins. Many organelles including ribosomes and endoplasmic reticulum were found in their cytoplasm. However, various mitochondria were found in the NR2 and NR3. The immunohistochemistry revealed immunoreactivity of ghrelin-like peptide in the neurons of the cerebral, pleural and pedal ganglia. However, immunoreactivity of GHS-R1a-like peptide existed only in the neurons of the pleural and pedal ganglia. The present study is the first to demonstrate the existence of ghrelin-like peptide and its receptor in P. canaliculata nervous system.

The Efficiency of Neurospheres Derived from Human Wharton's Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats.

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and ß-tubulin 3 through the Wnt3A signaling pathway regulation markers (ß-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells.

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of ß-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-ß signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.

Antiadipogenesis of Oroxylum indicum (L.) Kurz Extract via PPARγ2 in 3T3-L1 Adipocytes.

Oroxylum indicum is regarded as a traditional food with medicinal properties and is used widely throughout Asia. It has previously been demonstrated that O. indicum extract (OIE) was able to suppress the differentiation of 3T3-L1 preadipocytes to adipocytes. However, the mechanism underlying the antiadipogenesis of this plant has not been fully investigated. The present study aimed to explore the impact of OIE at 50 to 200 µg mL-1 on the molecular mechanism involved in the antiadipogenic activity in 3T3-L1 cells at day 0 of their differentiation to adipocytes. The morphology and biochemistry of the cells on day 12 were investigated and compared to the relevant controls. Adiponectin was measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARγ2), sterol regulatory element-binding proteins 1c (SREBP-1c), fatty acid synthetase (FAS), glucose transporter (GLUT4), and leptin in adipocytes was determined by real-time PCR. The results demonstrated that the OIE at 200 µg mL-1 exhibited strongest suppression on intracellular lipid accumulation. The levels of adiponectin were dramatically increased in the untreated adipocytes, whereas significantly decreased in the 200 µg mL-1 OIE-treated adipocytes (P < 0.05). Expression of the mRNAs revealed that OIE-treated adipocytes at 200 µg mL-1 significantly inhibited the expression of PPARγ2 and SREBP-1c and lowered the level of expression of GLUT4, FAS, and leptin compared to the control (P < 0.05). These findings suggest that OIE inhibits adipocyte differentiation along with the downregulation of PPARγ2, SREBP-1c, and GLUT4, leading to the decrease in the expression of FAS and adipokine (leptin and adiponectin). Thus, OIE might be developed for hyperlipidemia and obesity prevention.

Enhanced Chondrogenic Differentiation of Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stem Cells by GSK-3 Inhibitors.

Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs) are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3) inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl) and SB216763 synergistically with TGF-ß3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs). hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and ß-catenin markers. Glycosaminoglycan (GAG) accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating ß-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration.

Localization of ghrelin-like peptide in the gastrointestinal tract of the golden apple snail (Pomacea canaliculata) and changing of its concentration during fasting.

Ghrelin is an endogenous hormone detected in the gastrointestinal tracts (GI) of various species. In the present study, ghrelin-like peptide (ghrelin-LP) was identified in the GI tract of the golden apple snail, Pomacea canaliculata. Using immunohistochemistry, the result revealed an immunoreactivity (-ir) of ghrelin-LP in regions of the GI tract. The ghrelin-LP-ir was observed in both opened-type and closed-type cells of the esophagus, stomach, and small and large intestines. The highest density of ghrelin-LP immunoreactive cells was found in the esophagus and the least density was detected in the stomach. The highest percentages of the opened-type and closed-type cells were present in the esophagus and small intestine, respectively. In immunoblotting, the molecular weight of ghrelin-LP was related to the human ghrelin peptide (∼13kDa). Moreover, the concentration of ghrelin-LP was significantly higher in snails that were fasted for 24h compared with fed snails. The concentration decreased after refeeding. The present study could be useful for understanding the physiological role of ghrelin-LP in mollusk species.

Formaldehyde exposure in gross anatomy laboratory of Suranaree University of Technology: a comparison of area and personal sampling.

Cadavers are usually preserved by embalming solution which is composed of formaldehyde (FA), phenol, and glycerol. Therefore, medical students and instructors have a higher risk of exposure to FA inhalation from cadavers during dissection. Therefore, the objective of this study was to evaluate the FA exposure in indoor air and breathing zone of medical students and instructors during dissection classes in order to investigate the relationship between them. The indoor air and personal air samples in breathing zone were collected three times during anatomy dissection classes (in January, August, and October of 2014) with sorbent tubes, which were analyzed by high-performance liquid chromatography (HPLC). The air cleaner machines were determined by weight measurement. Pulmonary function tests and irritation effects were also investigated. The mean of FA concentrations ranged from 0.117 to 0.415 ppm in the indoor air and from 0.126 to 1.176 ppm in the breathing zone of students and instructors. All the personal exposure data obtained exceeded the threshold limit of NIOSH and WHO agencies. The air cleaner machines were not significant difference. The pulmonary function of instructors showed a decrease during attention of classes and statistically significant decreasing in the instructors more than those of the students. Clinical symptoms that were observed in nose and eyes were irritations with general fatigue. We suggested that the modified exhaust ventilation and a locally ventilated dissection work table were considered for reducing FA levels in the gross anatomy dissection room.

Cytochalasin B efficiency in the cryopreservation of immature bovine oocytes by Cryotop and solid surface vitrification methods.

The present study was undertaken to compare the efficacies of Cryotop (CT), solid surface vitrification (SSV) methods and cytochalasin B (CB) treatment for the cryopreservation of immature bovine oocytes, in terms of survival, nuclear maturation, and in vitro development. Solution exposed oocytes were in vitro maturated and fertilized. No difference was found in the rates of survival, nuclear maturation and blastocyst among solution exposed groups and fresh control group, except blastocysts rates in oocytes exposed to CB, cryoprotectant (CPA) and fluorescein diacetate (FDA) group (CB-CPA-FDA) (23%) significantly lower than that of control group (32%). CB pretreated ((+)CB) or non-pretreated ((-)CB) COCs were vitrified either by SSV or CT. Among four vitrified groups the nuclear maturation rates (CT(-)CB: 58%, CT(+)CB: 57%, SSV(-)CB: 60%, SSV(+)CB: 63%), cleavage (CT(-)CB: 36%, CT(+)CB: 24%, SSV(-)CB: 34%, SSV(+)CB: 26%) and blastocysts rates (CT(-)CB: 6%, CT(+)CB: 7%, SSV(-)CB: 4%, SSV(+)CB: 6%) did not differ, but the rates of the four vitrified groups were significantly lower than those of non-vitrified group (81%, 71% and 26%, respectively). We thus conclude that CT and SSV perform equally in vitrification of bovine immature oocytes, and CB did not increase the viability, nuclear maturation, or in vitro development of vitrified oocytes.

Plasma leptin concentrations during the reproductive cycle in the native Thai chicken (Gallus domesticus).

Plasma leptin concentrations were investigated during the reproductive cycle in the native Thai chicken. The plasma leptin concentration was high during non-laying (0.69±0.15ng/ml), lowered to a minimum concentration during egg laying (0.07±0.02ng/ml), and gradually increased during egg incubation and rearing of the chicks (0.53±0.22 and 0.74±0.29ng/ml, respectively). However, the differences were not significant. Incubating chickens that were deprived of their nests for 3 weeks showed a significant decrease in plasma leptin concentrations (0.29±0.04ng/ml, P<0.05) compared to those of their corresponding incubating controls (0.77±0.08ng/ml). Similarly, plasma leptin concentration of chickens that were deprived of their chicks for 4 weeks was significantly lower (0.09±0.11ng/ml, P<0.05), when compared to those of chickens that rearing their chicks (0.71±0.18ng/ml). These findings taken together with the results that the low plasma leptin concentrations were observed in chickens having relatively greater ovary and oviduct weights led to the suggestion that circulating leptin concentrations are associated with the reproductive states of the birds, especially the ovarian activity (i.e. ovarian steroid hormone concentrations) in the native Thai chicken, a tropical and continuous breeding species.

Role of vitelline envelope during fertilization in the black tiger shrimp, Penaeus monodon.

Animal eggs possess investments through which sperm must penetrate. The aim of the present study was to investigate the role of the egg coating, the vitelline envelope, during sperm-egg interactions in the black tiger shrimp, Penaeus monodon. The site(s) of primary binding between sperm and egg and the possible binding molecule(s) for sperm were identified. In vitro adsorption of the vitelline envelope protein onto the sperm surface showed that primary binding occurred between the sperm anterior spike of acrosome intact sperm and the vitelline envelope. Results from streptavidin blotting revealed that the component of the vitelline envelope that interacts with the sperm integral membrane protein is a 370kDa protein. In addition, it was shown that the vitelline envelope protein had no ability to induce acrosome reaction. These results suggest that the function of the vitelline envelope is as a primary binding site for sperm in shrimp, but not a sole trigger for the acrosome reaction.

The identification and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii.

In the present study, we demonstrated the existence of GnRH-like peptides in the central nervous system (CNS) and ovary of the giant freshwater prawn, Macrobrachium rosenbergii using immunocytochemistry. The immunoreactivity (ir) of lamprey (l) GnRH-III was detected in the soma of medium-sized neurons located in neuronal cluster number 11 in the middle part of supraesophageal ganglion (deutocerebrum), whereas ir-octopus (oct) GnRH was observed in the soma of both medium-sized and large-sized neurons in thoracic ganglia, as well as in the fibers innervating the other medium-sized and large-sized neuronal cell bodies in the thoracic ganglia. In addition, ir-lGnRH-I was observed in the cytoplasm of late previtellogenic oocyte and early vitellogenic oocyte. These data suggest that M. rosenbergii contain at least three isoforms of GnRH: two GnRH isoforms closely related to lGnRH-III and octGnRH in the CNS, whereas another isoform, closely related to lGnRH-I, was localized in the ovary. This finding provides supporting data that ir-GnRH-like peptide(s) may exist in this decapod crustacean.

The presence and distribution of gonadotropin-releasing hormone-liked factor in the central nervous system of the black tiger shrimp, Penaeus monodon.

The distribution and presence of gonadotropin-releasing hormone (GnRH) in the central nervous system (CNS) of Penaeus monodon were examined by immunocytochemistry, high performance liquid chromatography (HPLC), and radioimmunoassay (RIA). We demonstrated the existence of octopus (oct)GnRH-liked immunoreactivity (ir-octGnRH) and lamprey (l)GnRH-III-liked immunoreactivity (ir-lGnRH-III) in cell bodies of medium-sized neurons of the anterior part (protocerebrum) of the supraesophageal ganglion (brain). In addition, only the ir-octGnRH was detected in the nerve fibers located in the brain and segmental ganglia (subesophageal, thoracic, and abdominal ganglia). Moreover, some branches of these fibers also innervated the neurons in the middle (deutrocerebrum), posterior (tritocerebrum) brain and segmental ganglia. There was no ir-lGnRH-I and ir-salmon (s)GnRH detected in the shrimp CNS. The results from HPLC and RIA showed ir-GnRH in the CNS using anti-lGnRH-III, but not with anti-mammalian (m)GnRH. The data from immunocytochemistry, HPLC and RIA suggest that ir-GnRH in shrimp may be more similar to octGnRH and lGnRH-III than the other forms. These findings support the hypothesis that GnRH-liked factor(s) may be an ancient peptide that also exists in this decapod crustacean.

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm.

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.