Development of a High-Throughput Serum Neutralization Test Using Recombinant Pestiviruses Possessing a Small Reporter Tag

A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections

Early Postnatal Exposure to a Low Dose of Decabromodiphenyl Ether Affects Expression of Androgen and Thyroid Hormone Receptor-Alpha and Its Splicing Variants in Mouse Sertoli Cells

<div><p>Decabromodiphenyl ether (decaBDE) adversely affects reproduction and development. Our previous study showed that postnatal exposure to a low dose of decaBDE (0.025 mg/kg body weight/day) by subcutaneous injection on postnatal days (PNDs) 1 through 5 leads to reductions in testicular size and number of Sertoli cells and sperm, while higher dose of decaBDE (2.5 mg/kg body weight/day) had no significant differences about these. In the present study, we examined the molecular mechanism of these effects on mouse testes following postnatal exposure to a low decaBDE dose. We hypothesized that postnatal exposure to decaBDE may alter levels of serum thyroid hormones (THs) and testosterone, or the level of TH receptor alpha (<i>Thra</i>) transcripts and its splicing variants and androgen receptor (<i>Ar</i>) in Sertoli cells, adversely affecting spermatogenesis. To test this hypothesis, we examined serum TH and testosterone levels and the levels of transcripts of the <i>Ar, Thra</i> and its splicing variants, and <i>Thra</i> splicing factors (<i>Hnrnpa1</i>, <i>Srsf1</i>, and <i>Hnrnph1</i>) with qPCR in isolated mouse Sertoli cells exposed postnatally to decaBDE (0.025, 0.25, and 2.5 mg/kg). Levels of serum testosterone and transcripts encoding <i>Ar</i>, <i>Thra</i>, and its variant, <i>Thra1,</i> declined significantly in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg. No significant differences in serum TH level or <i>Thra2, Hnrnph1</i>, or <i>Srsf1</i> transcript levels were observed between control and decaBDE-exposed mice. However, the <i>Thra1</i>:<i>Thra2</i> and <i>Hnrnpa1</i>:<i>Srsf1</i> ratios were altered in Sertoli cells of mice exposed to 0.025 mg decaBDE/kg but not in cells exposed to 0.25 or 2.5 mg decaBDE/kg. These results indicate that postnatal exposure to a low dose of decaBDE on PNDs 1 through 5 lowers the testosterone level and the levels of <i>Ar</i> and <i>Thra</i> transcripts in Sertoli cells, accompanied by an imbalance in the ratios of <i>Thra</i> splicing variants, resulting in smaller testicular size and impaired spermatogenesis.</p></div

3,4-Dihydroxybenzaldehyde Derived from Prunus mume Seed Inhibits Oxidative Stress and Enhances Estradiol Secretion in Human Ovarian Granulosa Tumor Cells

Granulosa cells form ovarian follicles and play important roles in the growth and maturation of oocytes. The protection of granulosa cells from cellular injury caused by oxidative stress is an effective therapy for female infertility. We here investigated an effective bioactive compound derived from Prunus mume seed extract that protects granulosa cells from hydrogen peroxide (H2O2)-induced apoptosis. We detected the bioactive compound, 3,4-dihydroxybenzaldehyde (3,4-DHBA), via bioactivity-guided isolation and found that it inhibited the H2O2-induced apoptosis of granulosa cells. We also showed that 3,4-DHBA promoted estradiol secretion in granulosa cells and enhanced the mRNA expression levels of steroidogenic factor 1, a promoter of key steroidogenic enzymes. These results suggest that P. mume seed extract may have clinical potential for the prevention and treatment of female infertility

Levels of <i>Thra</i> and <i>Ar</i> transcripts in isolated Sertoli cells.

<p>Levels of transcripts of <i>Thra</i> (A), <i>Thra1</i> (B), <i>Thra2</i> (C), and <i>Ar</i> (D) were measured using qPCR in control and decaBDE-exposed isolated Sertoli cells cultured for 1 week. Transcript expression was normalized to the level of <i>Actb</i> transcript expression and is shown as the ratio relative to <i>Actb</i> compared with the control (set as a value of 1.0). Each value is the mean ± SD of 6-9 samples per group. *<i>P</i><0.05 compared with the control.</p

Serum testosterone levels in control and decaBDE-exposed mice.

<p>Testosterone levels in the serum of control and decaBDE-exposed mice were measured by ELISA. Each value is the mean ± SD of 6 samples per group. *<i>P</i><0.05 compared with the control.</p

Possible mechanism following early postnatal exposure to a low dose of decaBDE (0.025 mg/kg) in mouse testis.

<p>Early postnatal exposure to decaBDE from PND 1 to 5 causes to reduce serum testosterone level and <i>Ar</i> transcript levels in Sertoli cells, resulting in smaller testes size, reduced sperm count and a lower number of Sertoli cells, as well as decreased transcript levels of <i>Thra</i> and its splicing variant, <i>Thra1,</i> and an imbalance in the expression ratios of <i>Thra1</i>:<i>Thra2</i> and <i>Hnrnpa1</i>:<i>Srsf1</i> (gray box). It remains unknown if these latter changes are related to smaller testes size, reduced sperm count and lower number of Sertoli cells (dotted line).</p