Objective:To explore the effect of chronic incomplete sleep deprivation (SD) on ovarian reserve function in mice and its potential mechanisms.Methods:Sixteen 7-8 week old female C57BL/6 SPF mice were randomly divided into control and SD groups ( n=8 per group) after one week of acclimatization. The mice in the SD group were treated with SD from 7:00 to 10:00 using a deprivation rod for a total of 40 d. Before the first day and the last day of the experiment, vaginal smears were collected daily for 9 d to record and evaluate the estrous cycle. On the last day of the experiment, serum levels of estradiol, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and melatonin (MT) were measured by enzyme-linked immunosorbent assay (ELISA). The ovaries were dissected to calculate the ovarian index and count the number of primordial follicles by hematoxylin-eosin (HE) staining. Cardiac perfusion was performed to get the brains and the expression of c-Fos protein was observed in the suprachiasmatic nucleus (SCN) by immunohistochemistry. Results:Compared with control group, SD group had a tendency of estrous cycle disorders. Hormone levels of estradiol [(20.19±3.67) ng/L], progesterone [(2.88±0.53) μg/L], and FSH [(13.42±2.36) U/L] in the SD group were significantly lower than those in control group [(24.66±2.15) ng/L, P=0.010; (3.43±0.49) μg/L, P=0.049; (17.01±1.49) U/L, P=0.003]. Hormone levels of LH and MT in the SD group were lower than those in control group, but without statistical significances (all P>0.05). There was no significant change in the number of primordial follicles between the two groups ( P>0.05). However, the oocyte morphology was poor, the granulosa cells were disorderedly arranged, the number of atretic follicles was decreased, and ovarian fibrosis was obvious in the SD group. Immunohistochemical staining showed an upregulation of c-Fos protein expression in the SCN of the SD group. Conclusion:Continuous 3 h SD for 40 d impairs ovarian reserve function in mice, possibly related to excessive activation of the SCN.

Objective:To explore the effect of chronic incomplete sleep deprivation (SD) on ovarian reserve function in mice and its potential mechanisms.Methods:Sixteen 7-8 week old female C57BL/6 SPF mice were randomly divided into control and SD groups ( n=8 per group) after one week of acclimatization. The mice in the SD group were treated with SD from 7:00 to 10:00 using a deprivation rod for a total of 40 d. Before the first day and the last day of the experiment, vaginal smears were collected daily for 9 d to record and evaluate the estrous cycle. On the last day of the experiment, serum levels of estradiol, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and melatonin (MT) were measured by enzyme-linked immunosorbent assay (ELISA). The ovaries were dissected to calculate the ovarian index and count the number of primordial follicles by hematoxylin-eosin (HE) staining. Cardiac perfusion was performed to get the brains and the expression of c-Fos protein was observed in the suprachiasmatic nucleus (SCN) by immunohistochemistry. Results:Compared with control group, SD group had a tendency of estrous cycle disorders. Hormone levels of estradiol [(20.19±3.67) ng/L], progesterone [(2.88±0.53) μg/L], and FSH [(13.42±2.36) U/L] in the SD group were significantly lower than those in control group [(24.66±2.15) ng/L, P=0.010; (3.43±0.49) μg/L, P=0.049; (17.01±1.49) U/L, P=0.003]. Hormone levels of LH and MT in the SD group were lower than those in control group, but without statistical significances (all P>0.05). There was no significant change in the number of primordial follicles between the two groups ( P>0.05). However, the oocyte morphology was poor, the granulosa cells were disorderedly arranged, the number of atretic follicles was decreased, and ovarian fibrosis was obvious in the SD group. Immunohistochemical staining showed an upregulation of c-Fos protein expression in the SCN of the SD group. Conclusion:Continuous 3 h SD for 40 d impairs ovarian reserve function in mice, possibly related to excessive activation of the SCN.

Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAalpha1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAalpha1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAalpha1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2-4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and tested their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAalpha1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAalpha1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.
Animals ; Base Sequence ; Chiroptera ; genetics ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Plasminogen Activators ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis, DNA