Serotonin/5-HT7 receptor provides an adaptive signal to enhance pigmentation response to environmental stressors through cAMP-PKA-MAPK, Rab27a/RhoA, and PI3K/AKT signaling pathways.

Serotonin (5-HT), a neurotransmitter, is essential for normal and pathological pigmentation processing, and its receptors may be therapeutical targets. The effect and behavior of the 5-HT7 receptor (5-HT7R) in melanogenesis in high vertebrates remain unknown. Herein, we examine the role and molecular mechanism of 5-HT7R in the pigmentation of human skin cells, human tissue, mice, and zebrafish models. Firstly, 5-HT7R protein expression decreased significantly in stress-induced depigmentation skin and vitiligo epidermis. Stressed mice received transdermal serotonin 5-HT7R selective agonists (LP-12, 0.01%) for 12 or 60 days. Mice might recover from persistent stress-induced depigmentation. The downregulation of tyrosinase (Tyr), microphthalmia-associated transcription factor (Mitf) expression, and 5-HT7R was consistently restored in stressed skin. High-throughput RNA sequencing showed that structural organization (dendrite growth and migration) and associated pathways were activated in the dorsal skin of LP-12-treated animals. 5-HT7R selective agonist, LP-12, had been demonstrated to enhance melanin production, dendrite growth, and chemotactic motility in B16F10 cells, normal human melanocytes (NHMCs), and zebrafish. Mechanistically, the melanogenic, dendritic, and migratory functions of 5-HT7R were dependent on the downstream signaling of cAMP-PKA-ERK1/2, JNK MAPK, RhoA/Rab27a, and PI3K/AKT pathway activation. Importantly, pharmacological inhibition and genetic siRNA of 5-HT7R by antagonist SB269970 partially/completely abolished these functional properties and the related activated pathways in both NHMCs and B16F10 cells. Consistently, htr7a/7b genetic knockdown in zebrafish could blockade melanogenic effects and abrogate 5-HT-induced melanin accumulation. Collectively, we have first identified that 5-HT7R regulates melanogenesis, which may be a targeted therapy for pigmentation disorders, especially those worsened by stress.

[Effects of 5 ml straws cryopreservation on the quality and ultrastructure of boar semen].

OBJECTIVE: To investigate damages to the quality of boar sperm frozen in 5 ml straws, pellet and 0.25 ml straws as well as the ultrastructural changes of frozen boar sperm in 5 ml straws. METHODS: We compared 3 different freezing packages of 5 ml straws, pellet and 0.25 ml straws to determine their effects on frozen boar semen, and observed the morphological and ultrastructural changes of the boar sperm in the 5 ml straws using scanning electron microscopy. RESULTS: In the 5 ml straws, the vitality and motility of the boar sperm after freezing were not significantly different from those in the other two formulations, the normal apical ridge (NAR) was 52.65%, higher than in the pellet but not significantly different from that in the 0.25 ml straws, and the sperm membranes were mostly bubbly, some locally broken, which indicated the damage induced by freezing and thawing. CONCLUSION: At the present time, boar semen frozen in 5 ml straws were not significantly different from those frozen in 0.25 ml straws. The existing freezing-thawing method may cause certain damage to the quality and ultrastructure of boar sperm, and therefore needs to be further improved.

Effects of six compounds with different chemical structures on melanogenesis.

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D3, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 µmol·L-1in vivo and 100 µmol·L-1in vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.

5-HT1A/1B receptors as targets for optimizing pigmentary responses in C57BL/6 mouse skin to stress.

Stress has been reported to induce alterations of skin pigmentary response. Acute stress is associated with increased turnover of serotonin (5-hydroxytryptamine; 5-HT) whereas chronic stress causes a decrease. 5-HT receptors have been detected in pigment cells, indicating their role in skin pigmentation. To ascertain the precise role of 5-HT in stress-induced pigmentary responses, C57BL/6 mice were subjected to chronic restraint stress and chronic unpredictable mild stress (CRS and CUMS, two models of chronic stress) for 21 days, finally resulting in abnormal pigmentary responses. Subsequently, stressed mice were characterized by the absence of a black pigment in dorsal coat. The down-regulation of tyrosinase (TYR) and tyrosinase-related proteins (TRP1 and TRP2) expression in stressed skin was accompanied by reduced levels of 5-HT and decreased expression of 5-HT receptor (5-HTR) system. In both murine B16F10 melanoma cells and normal human melanocytes (NHMCs), 5-HT had a stimulatory effect on melanin production, dendricity and migration. When treated with 5-HT in cultured hair follicles (HFs), the increased expression of melanogenesis-related genes and the activation of 5-HT1A, 1B and 7 receptors also occurred. The serum obtained from stressed mice showed significantly decreased tyrosinase activity in NHMCs compared to that from nonstressed mice. The decrease in tyrosinase activity was further augmented in the presence of 5-HTR1A, 1B and 7 antagonists, WAY100635, SB216641 and SB269970. In vivo, stressed mice received 5-HT precursor 5-hydroxy-l-tryptophan (5-HTP), a member of the class of selective serotonin reuptake inhibitors (fluoxetine; FX) and 5-HTR1A/1B agonists (8-OH-DPAT/CP94253), finally contributing to the normalization of pigmentary responses. Taken together, these data strongly suggest that the serotoninergic system plays an important role in the regulation of stress-induced depigmentation, which can be mediated by 5-HT1A/1B receptors. 5-HT and 5-HTR1A/1B may constitute novel targets for therapy of skin hypopigmentation disorders, especially those worsened with stress.

[Application of spindle-view in the enucleation porcine of oocytes].

The Spindle-view, a specialized instrument for observing spindle image, was applied to observe the meiotic spindles of vitro matured porcine oocytes at 36, 42, 44, 48h, and enucleation from porcine, comparing to the previously methods (McGrath-Solter's method and two-step-squeezing method) in the enucleated. The results showed that: (1) there was no noticeable differences at vicinity of spindle images and 1st polar body among in vitro matured porcine oocytes at 40-48 h under the instrument; (2) Spindle-view is suitable for the observation of meiotic spindles of matured oocytes and enucleation from porcine; the modified Spindle-view method for enucleation is significantly better than McGrath-Solter' s method and two-step-squeezing method in the enucleated rates (95.5%, 42.1%, 74.2%, P < 0.0l) of absolutely removing nuclei matter; (3) the spindle images could be used to monitor the oocyte qualities.

[Study on vitrification of porcine embryos by open pulled straw method].

291 embryos (Blastocyst/Morula) from 20 donor sows were vitrified by two step method with OPS (open pulled straw) in solution I (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3min, and solution II (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4mol/L SUC) for 1min, stored in liquid nitrogen for 3 months, and transferred into 8 recipient sows after warming, one recipient sow was pregnant and 8 alive piglets were born. This is the first paper to report getting alive piglets by vitrification in China.