Noncircadian regulation and function of clock genes period and timeless in oogenesis of Drosophila melanogaster.

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period (per) and timeless (tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock (Clk) and cycle (cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.

Disruption of sperm release from insect testes by cytochalasin and beta-actin mRNA mediated interference.

Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at aspecific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of F-actin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release.

Circadian rhythm of glycoprotein secretion in the vas deferens of the moth, Spodoptera littoralis.

BACKGROUND: Reproductive systems of male moths contain circadian clocks, which time the release of sperm bundles from the testis to the upper vas deferens (UVD) and their subsequent transfer from the UVD to the seminal vesicles. Sperm bundles are released from the testis in the evening and are retained in the vas deferens lumen overnight before being transferred to the seminal vesicles. The biological significance of periodic sperm retention in the UVD lumen is not understood. In this study we asked whether there are circadian rhythms in the UVD that are correlated with sperm retention. RESULTS: We investigated the carbohydrate-rich material present in the UVD wall and lumen during the daily cycle of sperm release using the periodic acid-Shiff reaction (PAS). Males raised in 16:8 light-dark cycles (LD) showed a clear rhythm in the levels of PAS-positive granules in the apical portion of the UVD epithelium. The peak of granule accumulation occurred in the middle of the night and coincided with the maximum presence of sperm bundles in the UVD lumen. These rhythms persisted in constant darkness (DD), indicating that they have circadian nature. They were abolished, however, in constant light (LL) resulting in random patterns of PAS-positive material in the UVD wall. Gel-separation of the UVD homogenates from LD moths followed by detection of carbohydrates on blots revealed daily rhythms in the abundance of specific glycoproteins in the wall and lumen of the UVD. CONCLUSION: Secretory activity of the vas deferens epithelium is regulated by the circadian clock. Daily rhythms in accumulation and secretion of several glycoproteins are co-ordinated with periodic retention of sperm in the vas deferens lumen.

Loss of circadian clock function decreases reproductive fitness in males of Drosophila melanogaster.

Circadian coordination of life functions is believed to contribute to an organism's fitness; however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. Here we examined the consequences of loss of clock function on reproductive fitness in Drosophila melanogaster with mutated period (per(0)), timeless (tim(0)), cycle (cyc(0)), and Clock (Clk(Jrk)) genes. Single mating among couples with clock-deficient phenotypes resulted in approximately 40% fewer progeny compared with wild-type flies, because of a decreased number of eggs laid and a greater rate of unfertilized eggs. Male contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per(0) and tim(0) males mated with wild-type females. The important role of clock genes for reproductive fitness was confirmed by reversal of the low-fertility phenotype in flies with rescued per or tim function. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles, and these tissues displayed rhythmic and autonomous expression of clock genes. By combining molecular and physiological approaches, we identified a circadian clock in the reproductive system and defined its role in the sperm release that promotes reproductive fitness in D. melanogaster.

Temporal and spatial expression of the period gene in the reproductive system of the codling moth.

The authors examined patterns of spatial and temporal expression of Drosophila per gene homologue in the codling moth, Cydia pomonella. Since sperm release in moths is regulated in a circadian manner by an autonomous clock that is independent from the brain, the authors investigated per expression in male reproductive system along with its expression in moth heads. per mRNA is rhythmically expressed with the same phase and amplitude in both tissues under light-dark (LD) conditions. The levels of per mRNA are low during the day, start to increase before lights-off, reach the peak in dark, and decrease after lights-on. In constant darkness (DD), cycling of per mRNA continued in heads with severely blunted amplitude. No cycling of per mRNA was detected in testis in DD. In situ hybridization and immunocytochemistry revealed distinct spatial patterns of per expression in the moth reproductive system. There is no expression of per in cells forming the wall of testes or in sperm bundles. However, per mRNA and protein are rhythmically expressed in the epithelial cells forming the wall of the upper vas deferens (UVD) and in the cells of the terminal epithelium, which are involved in the circadian gating of sperm release. Increase in per mRNA in the UVD coincides with sperm accumulation in this part of the insect reproductive system.

Two alternatively spliced transcripts from the Drosophila period gene rescue rhythms having different molecular and behavioral characteristics.

The period (per) and timeless (tim) genes encode key components of the circadian oscillator in Drosophila melanogaster. The per gene is thought to encode three transcripts via differential splicing (types A, B, and C) that give rise to three proteins. Since the three per mRNA types were based on the analysis of cDNA clones, we tested whether these mRNA types were present in vivo by RNase protection assays and reverse transcriptase-mediated PCR. The results show that per generates two transcript types that differ only by the presence (type A) or absence (type B') of an alternative intron in the 3' untranslated region. Transgenic flies containing transgenes that produce only type B' transcripts (perB'), type A transcripts (perA), or both transcripts (perG) rescue locomotor activity rhythms with average periods of 24.7, 25.4, and 24.4 h, respectively. Although no appreciable differences in type A and type B' mRNA cycling were observed, a slower accumulation of PER in flies making only type A transcripts suggests that the intron affects the translation of per mRNA.

Vaccinia virus G4L gene encodes a second glutaredoxin.

Vaccinia virus (VV) was previously shown to encode a functional glutaredoxin, the product of the o2l gene, which is synthesized late in infection, after the onset of DNA replication. Here we report that an open reading frame in the VV genome designated as g4l encodes a protein that has sequence similarity to glutaredoxins and possesses thioltransferase and dehydroascorbate reductase activities. G4L protein in infected cells can be detected as early as 4 hr after infection and is constitutively expressed up to 24 hr postinfection. A protein homologous to G4L and retaining the predicted glutaredoxin active center is encoded by the recently sequenced Molluscum Contagiosum virus (MCV), whereas O2L protein is not conserved, suggesting that the glutaredoxin activity of G4L may be involved in replication of all poxviruses.

Identification of a second functional glutaredoxin encoded by the bacteriophage T4 genome.

Thioredoxins and glutaredoxins are small ubiquitous redox proteins that were discovered as hydrogen donors for ribonucleotide reductase, the key enzyme for deoxyribonucleotide biosynthesis. Some organisms encode more than one redox protein. In this study, we demonstrate that an open reading frame in the bacteriophage T4 genome, reported earlier and designated as Y55.7 (Tomaschewski, J., and Rüger, W. (1987) Nucleic Acids Res. 15, 3632-3633), encodes a second functional redox protein. Gene y55.7 was cloned and expressed in Escherichia coli. Purified Y55.7 protein had glutathione-dependent thioltransferase and dehydroascorbate reductase activities indicative of a functional glutaredoxin. The protein is expressed at all stages of the T4 infection cycle and can serve as a hydrogen donor for the phage ribonucleotide reductase in in vitro experiments.