Expression of the umami taste receptor T1R1/T1R3 in porcine testis of: Function in regulating testosterone synthesis and autophagy in Leydig cells.

Testosterone is a vital male hormone responsible for male sexual characteristics. The taste receptor family 1 subunit 3 (T1R3) regulates testosterone synthesis and autophagy in non-taste cells, and the links with the taste receptor family 1 subunit 1 (T1R1) for umami perception. However, little is known about these mechanisms. Thus, we aimed to determine the relationship between the umami taste receptor (T1R1/T1R3) and testosterone synthesis or autophagy in testicular Leydig cells of the Xiang pig. There was a certain proportion of spermatogenic tubular dysplasia in the Xiang pig at puberty, in which autophagy was enhanced, and the testosterone level was increased with a weak expression of T1R3. Silenced T1R3 decreased testosterone level and intracellular cyclic adenosine monophosphate (cAMP) content and inhibited the messenger RNA (mRNA) expression levels of testosterone synthesis enzyme genes [steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3ß-HSD1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and hydroxysteroid 17-beta dehydrogenase 3 (17ß-HSD3)]. In addition, T1R3 increased the number of acidic autophagy bubbles and upregulated the expression levels of autophagy markers [Microtubule-associated protein 1 A/1B-light chain 3 (LC3) and Beclin-1] in testicular Leydig cells of the Xiang pig. Using an umami tasting agonist (10 mM L-glutamate for 6 h), the activation of T1R1/T1R3 enhanced the testosterone synthesis ability by increasing the intracellular cAMP level and upregulated the expression levels of StAR, 3ß-HSD1, CYP17A1 and 17ß-HSD3 in Leydig cells. Furthermore, the number of acidic autophagy bubbles decreased in the T1R1/T1R3-activated group with the downregulation of the expression levels of the autophagy markers, including LC3 and Beclin-1. These data suggest that the function of T1R1/T1R3 expressed in testicular Leydig cells of the Xiang pig is related to testosterone synthesis and autophagy.

The metallic compound promotes primordial follicle activation and ameliorates fertility deficits in aged mice.

Background: Aged women and premature ovarian insufficiency (POI) patients have residual dormant primordial follicles that are hard to be activated through a physiological process. However, there are no effective and safe drugs to help them. Methods: We used the in vitro culture model of newborn mouse ovaries to identify the drugs that promote primordial follicle activation and study its mechanisms. It was verified by in vivo injection model of newborn mice and in vitro culture model of human ovarian tissue. In addition, we used the aged mice as a low infertility model to verify the effects of primordial follicle activation, and fertility by drugs. Results: Eleven metallic compounds activated mouse primordial follicles, and the five most effective compounds were selected for further study. Thapsigargin (TG), CrCl3, MnCl2, FeCl3 and ZnSO4 increased the levels of the glycolysis-related proteins (glucose transporter type 4, GLUT4; hexokinase 1, HK1; pyruvate kinase M2, PKM2; phosphofructokinase, liver type, PFKL), phosphorylated mammalian target of rapamycin (p-mTOR) in cultured mouse ovaries. The compound-promoted p-mTOR levels could be completely blocked by 2-DG (the inhibitor of glycolysis). The compounds also increased the levels of phosphorylated protein kinase B (p-Akt). TG-, CrCl3- and FeCl3-promoted p-Akt levels, but not MnCl2- and ZnSO4- promoted p-Akt levels, could be completely blocked by ISCK03 (the inhibitor of proto-oncogenic receptor tyrosine kinase, KIT). The injection of newborn mice with the compounds also activated primordial follicles and increased the levels of the glycolysis-related proteins, p-mTOR, and p-Akt. The oral administration of the compounds in adolescent and aged mice promoted primordial follicle activation, and had no obvious side effect. Importantly, ZnSO4 also increased ovulated oocytes, oocyte quality and offspring in aged mice. Furthermore, the compounds promoted human primordial follicle activation and increased the levels of the glycolysis-related proteins, p-mTOR, and p-Akt. Conclusion: The metallic compounds activate primordial follicles through the glycolysis-dependent mTOR pathway and/or the PI3K/Akt pathway, and the oral administration of ZnSO4 enhances fertility in aged mice. We suggest that these metallic compounds may be oral drugs to ameliorate fertility deficits in aged women and POI patients.

Sweet Taste Receptor T1R3 Expressed in Leydig Cells Is Closely Related to Homeostasis of the Steroid Hormone Metabolism Profile.

Taste receptor type 1 subunit 3 (T1R3) is initially expressed in mammal tongue for recognition and response of sweet/umami tastants and is critical to nutrient absorption, even endocrine. In this study, down-regulation of related steroidogenic enzymes such as StAR, 3ß-HSD, CYP17A1, and 17ß-HSD with the decrease of T1R3 expression was found in Leydig cells treated by a T1R3 inhibitor (lactisole). The abundances of progesterone, 17a-hydroxyprogesterone, androstenedione, testosterone, and deoxycorticosterone were down-regulated by 2.3, 3.5, 1.4, 1.6, and 2.2 times, respectively, after T1R3 inhibition. In addition, opposite results were found in saccharin sodium treatment. T1R3 activation contributed to intracellular cyclic adenosine monophosphate (cAMP) accumulation (14.41 ± 0.58 vs 20.21 ± 0.65) and increased testosterone (20.31 ± 3.49 vs 50.01 ± 7.44) and steroidogenic metabolite levels. Coadministration of human chorionic gonadotropin and saccharin sodium resulted in elevating the testosterone and cAMP levels and enhancing the expression levels of steroidogenic-related factors. Similarly, intratesticular injection of lactisole and saccharin sodium further confirmed that T1R3 inhibition/activation affected the expression of related steroidogenic enzymes and the testosterone levels in mice. The above findings suggest that T1R3 plays a role in testicular steroidogenesis.

RA-RAR signaling promotes mouse vaginal opening through increasing ß-catenin expression and vaginal epithelial cell apoptosis.

BACKGROUND: Retinoic acid (RA) plays important role in the maintenance and differentiation of the Müllerian ducts during the embryonic stage via RA receptors (RARs). However, the function and mechanism of RA-RAR signaling in the vaginal opening are unknown. METHOD: We used the Rarα knockout mouse model and the wild-type ovariectomized mouse models with subcutaneous injection of RA (2.5 mg/kg) or E2 (0.1 µg/kg) to study the role and mechanism of RA-RAR signaling on the vaginal opening. The effects of Rarα deletion on Ctnnb1 mRNA levels and cell apoptosis in the vaginas were analyzed by real-time PCR and immunofluorescence, respectively. The effects of RA on the expression of ß-catenin and apoptosis in the vaginas were analyzed by real-time PCR and western blotting. The effects of E2 on RA signaling molecules were analyzed by real-time PCR and western blotting. RESULTS: RA signaling molecules were expressed in vaginal epithelial cells, and the mRNA and/or protein levels of RALDH2, RALDH3, RARα and RARγ reached a peak at the time of vaginal opening. The deletion of Rarα resulted in 25.0% of females infertility due to vaginal closure, in which the mRNA (Ctnnb1, Bak and Bax) and protein (Cleaved Caspase-3) levels were significantly decreased, and Bcl2 mRNA levels were significantly increased in the vaginas. The percentage of vaginal epithelium with TUNEL- and Cleaved Caspase-3-positive signals were also significantly decreased in Rarα-/- females with vaginal closure. Furthermore, RA supplementation of ovariectomized wild-type (WT) females significantly increased the expression of ß-catenin, active ß-catenin, BAK and BAX, and significantly decreased BCL2 expression in the vaginas. Thus, the deletion of Rarα prevents vaginal opening by reducing the vaginal ß-catenin expression and epithelial cell apoptosis. The deletion of Rarα also resulted in significant decreases in serum estradiol (E2) and vagina Raldh2/3 mRNA levels. E2 supplementation of ovariectomized WT females significantly increased the expression of RA signaling molecules in the vaginas, suggesting that the up-regulation of RA signaling molecules in the vaginas is dependent on E2 stimulation. CONCLUSION: Taken together, we propose that RA-RAR signaling in the vaginas promotes vaginal opening through increasing ß-catenin expression and vaginal epithelial cell apoptosis.

Lithium treatment promotes the activation of primordial follicles through PI3K/Akt signaling†.

In mammals, dormant primordial follicles represent the ovarian reserve throughout reproductive life. In vitro activation of dormant primordial follicles has been used to treat patients with premature ovarian insufficiency (POI). However, there remains a lack of effective strategies to stimulate follicle activation in vivo. In this study, we used an in vitro ovarian culture system and intraperitoneal injection to study the effect of lithium treatment on primordial follicle activation. Lithium increased the number of growing follicles in cultured mouse ovaries and promoted pre-granulosa cell proliferation. Furthermore, lithium significantly increased the levels of phosphorylated protein kinase B (Akt) and the number of oocytes with forkhead Box O3a (FOXO3a) nuclear export. Inhibition of the phosphatidylinositol 3 kinase (PI3K)/Akt pathway by LY294002 reversed lithium-promoted mouse primordial follicle activation. These results suggest that lithium promotes mouse primordial follicle activation by the PI3K/Akt signaling. Lithium also promoted primordial follicle activation and increased the levels of p-Akt in mouse ovaries in vivo and in human ovarian tissue cultured in vitro. Taken together, lithium promotes primordial follicle activation in mice and humans by the PI3K/Akt signaling. Lithium might be a potential oral drug for treating infertility in POI patients with residual dormant primordial follicles.

Different fixatives influence morphology, antigen preservation, and TUNEL staining in chicken (Gallus gallus) testis.

The optimized fixative for testis is still controversial. This study investigated the effects of Modified Davidson's Fluid (mDF), 4% Paraformaldehyde (4% PFA), and Bouin's Fluid (BF) fixatives on chicken testes in normal/cadmium (Cd) feeding groups using hematoxylin and eosin (HE), immunohistochemistry (IHC), and Terminal Transferase dUTP Nick End Labeling (TUNEL) staining. Compared to the mDF, we established that the testes fixed with 4% PFA and BF in the normal group had severe shrinkage in tubular and interstitial compartments. Moreover, compared with 4% PFA, the number of GATA4-positive Sertoli cells/mm2 reduced by 67.61% in mDF and 80.57% in BF for one seminiferous tubule. The TUNEL assay illustrated that more positive cells/mm2 in mDF group (28.47 ± 11.38) than in 4% PFA (10.49 ± 7.89). In Cd-treated testes, mDF showed more morphological details than 4% PFA and BF. In contrast, the number of GATA4-positive Sertoli cells/mm2 of 4% PFA was higher than that of mDF by 65.78% and BF by 64.80% in a seminiferous tubule. The number of TUNEL positive cells/mm2 in mDF (272.60 ± 34.41) were higher than in 4% PFA (175.91 ± 19.87). These results suggest that mDF fixative is suitable for normal and Cd-treated testis fixation for HE and TUNEL staining in chicken, whereas 4% PFA fixative is better for IHC examination.

Longitudinal Expression of Testicular TAS1R3 from Prepuberty to Sexual Maturity in Congjiang Xiang Pigs.

Testicular expression of taste receptor type 1 subunit 3 (T1R3), a sweet/umami taste receptor, has been implicated in spermatogenesis and steroidogenesis in mice. We explored the role of testicular T1R3 in porcine postnatal development using the Congjiang Xiang pig, a rare Chinese miniature pig breed. Based on testicular weights, morphology, and testosterone levels, four key developmental stages were identified in the pig at postnatal days 15-180 (prepuberty: 30 day; early puberty: 60 day; late puberty: 90 day; sexual maturity: 120 day). During development, testicular T1R3 exhibited stage-dependent and cell-specific expression patterns. In particular, T1R3 levels increased significantly from prepuberty to puberty (p < 0.05), and expression remained high until sexual maturity (p < 0.05), similar to results for phospholipase Cß2 (PLCß2). The strong expressions of T1R3/PLCß2 were observed at the cytoplasm of elongating/elongated spermatids and Leydig cells. In the eight-stage cycle of the seminiferous epithelium in pigs, T1R3/PLCß2 levels were higher in the spermatogenic epithelium at stages II-VI than at the other stages, and the strong expressions were detected in elongating/elongated spermatids and residual bodies. The message RNA (mRNA) levels of taste receptor type 1 subunit 1 (T1R1) in the testis showed a similar trend to levels of T1R3. These data indicate a possible role of T1R3 in the regulation of spermatid differentiation and Leydig cell function.

Postnatal differentiation and regional histological variations in the ductus epididymidis of the Congjiang Xiang pig.

The Congjiang Xiang pig is a rare Chinese miniature breed whose epididymal histologic features through the postnatal development are poorly understood. To clarify the histomorphological differences between each region of epididymis during postnatal development, 24 male Congjiang Xiang pigs aged from neonatal (15 d), peri-puberty (30 d), puberty (60 d) to adult (180 d) stages, were examined. Postnatal differentiation of the different regions (I-V) of the epididymis started from birth and continued until maturity that showed regional variations. Developmental progression was disto-proximal. At the neonatal stage, Wolffian duct differentiation starts in the distal region, then ascends to the middle region which forms regions V, IV and III, respectively. A simple lined cuboidal in the epidydimal epithelial, which gradually differentiated into a pseudostratified columnar with stereocilia from neonatal to post-pubertal. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility. After puberty cell rearrangement occurred in the epithelium, differentiation accelerated, and spermatozoon seen in the lumen, especially the lumen of region II. In region III, both halo and apical cells were frequently observed. At the post-pubertal stage, clear cells were frequently observed in Region IV-V, and the epididymal duct was markedly increased in size and fully packed with spermatozoa. The information presented in this study will be helpful for future evaluations of Congjiang Xiang pig fertility.

Everestmab, a novel long-acting GLP-1/anti GLP-1R nanobody fusion protein, exerts potent anti-diabetic effects.

In the present study, a novel single domain antibody (sdAb) fusion protein, named everestmab, composing of a mutated GLP-1(A8G) fused to the tandem bispecific humanized GLP-1R-targeting and albumin-binding nanobodies was designed and characterized for the therapies for type 2 diabetes mellitus (T2DM). Surface plasmon resonance (SPR) measurements demonstrated everestmab associates with serum albumins of rat and monkey species with high affinity, and tends to be cross-reactive with rat and monkey species. In vitro GLP-1R binding and activation assays revealed that everestmab can specifically activate the GLP-1R, and the antagonist exendin-4 (9-39) did not inhibit the activation yet. In vivo multiple oral glucose tolerance tests (OGTTs) and hypoglycaemic efficacy tests proved that a single injection of everestmab reduced the blood glucose for at least 144 h in Goto-Kakizaki (GK) rats. The plasma half-lives of 4.1 and 7.8 days were observed after a single s.c. administration of everestmab in SD rats and cynomolgus monkeys, respectively. Chronic treatment of everestmab to GK and diet induced obese (DIO) rats achieved beneficial effects on weight reducing, HbA1c lowering, glucose tolerance, liver and pancreas islet function impairment. In summary, everestmab is a unique G-protein-coupled receptor-targeted nanobody fusion protein and exerts potential as a therapeutic treatment for T2DM.

Effects of 4% paraformaldehyde and modified Davidson's fluid on the morphology and immunohistochemistry of Xiang pig testes.

Modified Davidson's fluid (mDF) is a good fixative for morphological and antigen preservation. However, recent studies have shown that 4% paraformaldehyde (PFA) can better preserve the actin structure in rodent testes. It remains controversial which of these fixatives is best for testicular tissue. This study investigated the effects of both mDF and 4% PFA on the morphology and antigen preservation of Xiang pig testes using hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). The stronger testis penetration of mDF compared with that of 4% PFA was primarily manifested as testicular color change and decrease in tissue weight loss. Testes fixed with 4% PFA displayed a severe shrinkage of both the tubular and interstitial compartments and the seminiferous tubule area decreased by 12.02% compared with that in mDF-fixed tissues. In contrast, IHC results showed that 4% PFA fixation achieved better IHC-positive performance than mDF fixation for antigens specifically expressed in germ cells, Leydig cells and Sertoli cells. Due to this improved antigen preservation by 4% PFA fixation, the relative immunoreactions intensity significantly increased by 39.8%, 27.8%, and 76.4%, respectively, compared with that in mDF fixation. In summary, fixation of Xiang pig testes with mDF was suitable for HE staining, while fixation with 4% PFA was more suitable for IHC.

Periocular CO2 laser resurfacing: severe ocular complications from multiple unintentional laser impacts on the protective metal eye shields.

BACKGROUND AND OBJECTIVES: A 36-year-old woman underwent CO2 laser resurfacing for periocular rhytides using protective stainless steel Cox II ocular shields. Immediately after the treatment, corneal lesions were seen in both eyes. The left eye subsequent developed corneal ulceration and scarring, a deformed iris, cataract, and lower eye lashes showing signs of acute burns. The right cornea had a small inferior mid-peripheral superficial lesion and concomitant lower mid-peripheral burned eye lashes. Our objective was to determine the most likely cause of these ocular complications. STUDY: We estimated temperature-time combinations that could induce corneal injury and cataract. Heat conduction effects from a heated cornea to the lens and from a heated ring of periocular skin to the cornea were computed. The temperature response of a shield following CO2 laser irradiation was determined. RESULTS: We computed that cataract can develop when the corneal temperature reaches, for example, 80 °C for 14 seconds. A periocular ring of heated skin contributes little to the corneal temperature. After 7 pulses of consecutive CO2 laser bursts in 7.5 seconds, the total shield area already reached a homogeneous temperature of 63 °C. CONCLUSION: Despite uncertainties in procedural details and modeling of cataract temperatures, the eye injuries were caused beyond doubt by heating of tear-covered metal eye shields by at least 10 consecutive but unintentional laser impacts. Lasers Surg. Med. 50:980-986, 2018. © 2018 Wiley Periodicals, Inc.

[Ocular deviation as the cause of "psychiatric" problems]. / Onderliggende oogaandoening als oorzaak van psychiatrisch geduide klachten.

We describe two cases with complaints of atypical headache who were initially diagnosed as having psychiatric problems, but who, after careful evaluation, were found to have an underlying eye disorder. Both patients were known to have an ophthalmic history. Patient A, a 21-year-old female, presented with severe headache, which was so disabling that she was declared unfit for work. A diagnosis of tension headache was made and she received psychological treatment with neurofeedback, homeopathy and acupuncture, which had no effect. Patient B, a 26-year-old male, complained of extreme photophobia, nausea and dizziness. The patient was diagnosed as having a conversion disorder and was admitted to a psychiatric clinic and given psychotropic treatment. Careful ophthalmological examination revealed that an ocular deviation was causing the symptoms in both patients and could be treated appropriately with strabismus surgery. These cases emphasise the importance of a thorough general medical and ophthalmic history and proper examination at all times without putting a psychiatric stigma on the patient.

De novo characterization of the Anthurium transcriptome and analysis of its digital gene expression under cold stress.

BACKGROUND: Anthurium andraeanum is one of the most popular tropical flowers. In temperate and cold zones, a much greater risk of cold stress occurs in the supply of Anthurium plants. Unlike the freeze-tolerant model plants, Anthurium plants are particularly sensitive to low temperatures. Improvement of chilling tolerance in Anthurium may significantly increase its production and extend its shelf-life. To date, no previous genomic information has been reported in Anthurium plants. RESULTS: Using Illumina sequencing technology, we generated over two billion base of high-quality sequence in Anthurium, and demonstrated de novo assembly and annotation of genes without prior genome information. These reads were assembled into 44,382 unigenes (mean length = 560 bp). Based on similarity search with known protein in the non-redundant (nr) protein database, 27396 unigenes (62%) were functionally annotated with a cut-off E-value of 10-5. Further, DGE tags were mapped to the assembled transcriptome for gene expression analysis under cold stress. In total, 4363 differentially expressed genes were identified. Among these genes, 292, 805 and 708 genes were up-regulated after 1-h, 5-h and 24-h cold treatment, respectively. Then we mapped these cold-induced genes to the KEGG database. Specific enrichment was observed in photosynthesis pathway, metabolic pathways and oxidative phosphorylation pathway in 1-h cold-treated plants. After a 5-h cold treatment, the metabolic pathways and oxidative phosphorylation pathway were significantly identified as the top two pathways. After 24-h cold treatment, mRNA surveillance pathway, RNA transport pathway and plant-pathogen interaction pathway were significantly enriched. Together, a total of 39 cold-inducible transcription factors were identified, including subsets of AP2/ERF, Zinc figure, NAC, MYB and bZIP family members. CONCLUSION: Our study is the first to provide the transcriptome sequence resource for Anthurium plants, and demonstrate its digital gene expression profiling under cold conditions using the assembled transcriptome data for reference. These data provides a valuable resource for genetic and genomic studies under abiotic conditions for Anthurium plants.

Molecular characterization of cultivated bromeliad accessions with Inter-Simple Sequence Repeat (ISSR) Markers.

Bromeliads are of great economic importance in flower production; however little information is available with respect to genetic characterization of cultivated bromeliads thus far. In the present study, a selection of cultivated bromeliads was characterized via inter-simple sequence repeat (ISSR) markers with an emphasis on genetic diversity and population structure. Twelve ISSR primers produced 342 bands, of which 287 (~84%) were polymorphic, with polymorphic bands per primer ranging from 17 to 34. The Jaccard's similarity ranged from 0.08 to 0.89 and averaged ~0.30 for the investigated bromeliads. The Bayesian-based approach, together with the un-weighted paired group method with arithmetic average (UPGMA)-based clustering and the principal coordinate analysis (PCoA), distinctly grouped the bromeliads from Neoregelia, Guzmania, and Vriesea into three separately clusters, well corresponding with their botanical classifications; whereas the bromeliads of Aechmea other than the recently selected hybrids were not well assigned to a cluster. Additionally, ISSR marker was proven efficient for the identification of hybrids and bud sports of cultivated bromeliads. The findings achieved herein will further our knowledge about the genetic variability within cultivated bromeliads and therefore facilitate breeding for new varieties of cultivated bromeliads in future as well.