Phosphorylation of GATA4 serine 105 but not serine 261 is required for testosterone production in the male mouse.

BACKGROUND: GATA4 is a transcription factor essential for male sex determination, testicular differentiation during fetal development, and male fertility in the adult. GATA4 exerts part of its function by regulating multiple genes in the steroidogenic enzyme pathway. In spite of these crucial roles, how the activity of this factor is regulated remains unclear. OBJECTIVES: Studies in gonadal cell lines have shown that GATA4 is phosphorylated on at least two serine residues-serine 105 (S105) and serine 261 (S261)-and that this phosphorylation is important for GATA4 activity. The objective of the present study is to characterize the endogenous role of GATA4 S105 and S261 phosphorylation in the mouse testis. MATERIALS AND METHODS: We examined both previously described GATA4 S105A mice and a novel GATA4 S261A knock-in mouse that we generated by CRISPR/Cas9 gene editing. The male phenotype of the mutants was characterized by assessing androgen-dependent organ weights, hormonal profiles, and expression of multiple testicular target genes using standard biochemical and molecular biology techniques. RESULTS: The fecundity of crosses between GATA4 S105A mice was reduced but without a change in sex ratio. The weight of androgen-dependent organs was smaller when compared to wild-type controls. Plasma testosterone levels showed a 70% decrease in adult GATA4 S105A males. This decrease was associated with a reduction in Cyp11a1, Cyp17a1, and Hsd17b3 expression. GATA4 S261A mice were viable and testis morphology appeared normal. Testosterone production and steroidogenic enzyme expression were not altered in GATA4 S261A males. DISCUSSION AND CONCLUSION: Our analysis showed that blocking GATA4 S105 phosphorylation is associated with decreased androgen production in males. In contrast, S261 phosphorylation by itself is dispensable for GATA4 function. These results confirm that endogenous GATA4 action is essential for normal steroid production in males and that this activity requires phosphorylation on at least one serine residue.

MEF2 and NR2F2 cooperate to regulate Akr1c14 gene expression in mouse MA-10 Leydig cells.

Leydig cells are essential for male reproductive development and health throughout life. Production of androgens [testosterone, dihydrotestosterone (DHT)] as well as intermediate steroids [progesterone, dihydroprogesterone (DHP)] is tightly regulated. In the mouse, the 3α-hydroxysteroid dehydrogenase enzyme (3α-HSD, AKR1C14) catalyses the interconversion of DHP and DHT into less potent steroids. Despite its importance, nothing is currently known regarding the regulation of Akr1c14 expression in Leydig cells. Recently, the transcription factors MEF2 and NR2F2 were identified in the mouse testis including in Leydig cells where they were found to regulate expression of genes involved in steroidogenesis. Analyses of transcriptomic data from MEF2- or NR2F2-deficient MA-10 Leydig cells revealed a significant decrease in Akr1c14 mRNA levels. Using qPCR, we confirmed that Akr1c14 mRNA levels were decreased in MEF2- and in NR2F2-deficient conditions. Conversely, overexpression of MEF2A or/and NR2F2 in MA-10 Leydig cells led to an increase in endogenous Akr1c14 mRNA levels. Recruitment of MEF2 and NR2F2 to the Akr1c14 promoter was confirmed by ChIP while DNA precipitation assays revealed direct binding of MEF2 but not NR2F2 to this region. In functional promoter studies, NR2F2 was found to activate the Akr1c14 promoter while MEF2A on its own had no effect. Combination of both NR2F2 and MEF2A led to a cooperative activation of the Akr1c14 promoter and this required intact MEF2 and NR2F2 elements. Finally, co-immunoprecipitation experiments showed that MEF2 and NR2F2 are present in the same protein complex. In conclusion, our results identify a novel cooperation between MEF2 factors and NR2F2 in the expression of the Akr1c14 gene involved in the regulation of DHP/DHT levels.

Effects of laryngoscope handle light source on the light intensity from disposable laryngoscope blades.

A bench-top study was performed to assess the effects of different laryngoscope handles on the light intensity delivered from disposable metal or plastic laryngoscope blades. The light intensity from both the handle light sources themselves and the combined handle and laryngoscope blade sets was measured using a custom-designed testing system and light meter. Five samples of each disposable blade type were tested and compared with a standard re-usable stainless steel blade using three different handle/light sources (Vital Signs LED, Heine 2.5 V Xenon and 3.5 V Xenon). The light intensity delivered by the disposable blades ranged from 790 to 3846 lux for the different handle types. Overall, the 3.5 V Heine handle delivered the highest light output (p < 0.007) in comparison with the other handles. For the disposable blades, the overall light output was significantly higher from the plastic than the metal blades (p < 0.001).

Effects of bacillus thuringiensis and destruxins (Metarhizium anisopliae mycotoxins) combinations on spruce budworm (Lepidoptera: tortricidae)

Laboratory bioassays were performed in order to assess the efficacy of a combination of a commercial preparation of Bacillus thuringiensis var. kurstaki (B.t.k.) (Thuricide) with destruxins (Metarhizium anisopliae mycotoxins) on the fifth instar of Choristoneura fumiferana Clemens. Lethal doses were determined for each microbial agent and used as a basis for the bioassays involving a combination of the two agents. Interaction or the lack of interaction was determined by comparing the observed and the theoretically expected mortality rates. Seven out of 10 different combinations demonstrated synergism between the two agents. The modelization applied on results allowed us to establish the following general equation: LDmixture = 1.259(LDDx) + 1.129(LDBt) - 0.016(LDDx)(LDBt) + 12.196. Such an equation explains the relationship which exists between the two lethal agents (R2 = 0.99). Our results suggest that the two agents contribute to the synergism in the system and that a combination of both could be an efficient means of controlling C. fumiferana populations and of reducing the dose of B.t.k. which is usually required for such a control. Copyright 1998 Academic Press.

Isoflurane-induced hypotension in orthognathic surgery.

The effect of isoflurane-induced hypotension on reduction of blood loss, improvement of surgical field, and postoperative edema was investigated in 52 patients undergoing combined maxillary and mandibular osteotomies. Anesthesia was maintained with fentanyl, N2O, O2, and isoflurane. Deliberate hypotension was induced by increasing isoflurane inspired concentration. Blood loss in the hypotensive group (MAP 55-65 mm Hg) was significantly less than that in the control group (MAP 75-85 mm Hg); 454.0 +/- 211.3 mL versus 755.3 +/- 334.6 mL (P less than 0.001). Fewer patients had to be transfused in the hypotensive group, 12.0% versus 44.4% (P less than 0.02). The surgical field was significantly improved by the hypotensive technique, but operative time was not shortened. Subjective and objective measurements of postoperative edema failed to show any effect of deliberate hypotension. Our data suggest that isoflurane-induced hypotension effectively reduces blood loss and the number of transfusions in orthognathic surgery.

Myalgia in outpatient surgery: comparison of atracurium and succinylcholine.

We studied 60 outpatients randomly divided into two groups. Anesthesia was induced with fentanyl 1.5 micrograms.kg-1 plus thiopentone 5-7 mg.kg-1. Patients in Group I were intubated with the aid of succinylcholine 1.5 mg.kg-1 after pre-treatment with d-tubocurarine 0.05 mg.kg-1. Group II received atracurium 350 micrograms.kg-1 three minutes after a priming dose of 50 micrograms.kg-1. Anesthesia was maintained with isoflurane 1-2 per cent in a mixture of nitrous oxide 60 per cent and oxygen 40 per cent. No supplemental doses of fentanyl or atracurium were given. Intubation conditions were satisfactory for all patients in both groups. There was no significant difference in intubation score between the two groups. The incidence of myalgia was 76 per cent in the succinylcholine group compared to 23 per cent in the atracurium group (p less than 0.005). Fifty per cent of the patients in the succinylcholine group had myalgia necessitating bed rest or analgesics compared to 23 per cent in the atracurium group (p less than 0.05). We conclude that atracurium is a suitable neuromuscular relaxant for outpatient surgery and that myalgia is a major morbidity factor in this population that can be reduced by the use of atracurium instead of succinylcholine.