Diurnal rhythm in expression and release of yolk protein in the testis of Spodoptera littoralis (Lepidoptera: Noctuidae)

Circadian clocks (oscillators) regulate multiple life functions in insects. The circadian system located in the male reproductive tract of Lepidoptera is one of the best characterized peripheral oscillators in insects. Our previous research on the cotton leafworm, Spodoptera littoralis, demonstrated that this oscillator controls the rhythm of sperm release from the testis and coordinates sperm maturation in the upper vas deferens (UVD). We demonstrated previously that a protein that functions as yolk protein in females is also produced in cyst cells surrounding sperm bundles in the testis, and is released into the UVD. Here, we investigated the temporal expression of the yolk protein 2 (yp2) gene at the mRNA and protein level in the testis of S. littoralis, and inquired whether their expression is regulated by PER-based molecular oscillator. We describe a circadian rhythm of YP2 accumulation in the UVD seminal fluid, where this protein interacts with sperm in a circadian fashion. However, we also demonstrate that yp2 mRNA and YP2 protein levels within cyst cells show only a diurnal rhythm in light/dark (LD) cycles. These rhythms do not persist in constant darkness (DD), suggesting that they are non-circadian. Interestingly, the per gene mRNA and protein levels in cyst cells are rhythmic in LD but not in DD. Nevertheless, per appears to be involved in the diurnal timing of YP2 protein accumulation in cyst cells.     

Effects of period RNAi on V-ATPase expression and rhythmic pH changes in the vas deferens of Spodoptera littoralis (Lepidoptera: Noctuidae)

Circadian clocks (oscillators) regulate multiple aspects of insect behaviour and physiology. The circadian system located in the male reproductive tract of Lepidoptera orchestrates rhythmic sperm release from testis and sperm maturation in the upper vas deferens (UVD). Our previous research on the cotton leafworm, Spodoptera littoralis, suggested rhythmic changes in the V-ATPase levels in the UVD epithelium, which correlated with rhythmic pH fluctuations in the UVD lumen. However, it was not known whether UVD cells contain clock mechanism that generates these daily fluctuations. In the current paper, we show circadian rhythm in the expression of clock gene period at the mRNA and protein level in the UVD epithelium. To determine the role of PER in V-ATPase and pH regulation, testes–UVD complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment, which transiently lowered per mRNA and protein in the UVD, altered expression of V-ATPase c subunit. In addition, per RNAi caused a significant delay in the UVD lumen acidification. These data demonstrate that the UVD molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic V-ATPase activity and periodic acidification of the UVD lumen.     

Changes in protein patterns in sperm and vas deferens during the daily rhythm of sperm release in the gypsy moth

In the gypsy moth, Lymantria dispar, the release of sperm bundles from the testis into the upper vas deferens (UVD) is precisely timed within each 24 h period by a circadian mechanism located in the reproductive system. In males kept under light:dark cycles of 16:8, release of sperm bundles is limited to the 3 h period that starts before lights off. Sperm released from the testis remains in the UVD for about 12 h and then moves into the seminal vesicles, so that the UVD stays empty until the next cycle of sperm release begins. The rhythm of release appears to play a role in the terminal stages of sperm maturation and is essential for the fertility of males. Sperm bundles undergo substantial morphological changes during the release from the testis and while they are retained in the UVD. In this study, using gel electrophoresis, we compared protein patterns in sperm and in the UVD during the daily cycle of sperm release and maturation. Several protein bands evident in the sperm bundles contained in the testis were missing from the sperm bundles that had passed from the testis into the UVD. Furthermore, a number of new proteins appeared in the sperm bundles as they remained in the UVD. Some of these proteins appeared to be secreted from the UVD epithelium into the UVD lumen before being incorporated into sperm bundles. Correlations between changes in protein patterns and ultrastructural changes in sperm during the cycle of sperm release and maturation are discussed. © 1994 Wiley-Liss, Inc.     

Ontogeny of the circadian system controlling release of sperm from the insect testis.

In the gypsy moth, the release of sperm bundles from the testis into the vas deferens is rhythmic and is controlled by a circadian pacemaker located in the reproductive system. However, in males kept since pupation in constant darkness (DD) and temperature, the release of sperm was arrhythmic. The release of sperm became rhythmic when males were transferred from a light-dark cycle (LD 16:8) to DD 6-7 days after pupation. To further investigate the development of the circadian system during the pupal stage, we exposed DD pupae to a single 8-hr pulse of light or 8-hr pulse of a 4 degrees C temperature increase on different days after pupation. The pattern of sperm release was determined 5-6 days after the pulse. Males that were exposed to light or temperature pulses 5 days after pupation subsequently showed nonrhythmic sperm release. However, about half of the pupae that received the pulse on day 6 and most of the pupae that received it on day 7 subsequently showed synchronized sperm release. These results suggested that the clock underlying rhythmic release of sperm becomes operational at approximately 6 days after pupation--that is, 2 days prior to initiation of rhythmic sperm release from the testis.     

Effects of light and temperature on the circadian system controlling sperm release in moth Spodoptera littoralis

Reproductive physiology of male moths is regulated by a peripheral circadian system, which controls the timing of sperm release from the testis into the upper vas deferens (UVD) and timing of sperm transfer from the UVD to the seminal vesicles. We investigated various effects of light and temperature on sperm release and transfer rhythms in the moth Spodoptera littoralis. We report that both rhythms persist for up to 1 week in constant darkness without significant dampening and are also temperature compensated in the range from 20°C to 30°C. However, the duration of sperm retention in the UVD is temperature-dependent; consequently, temperature exerts a masking effect on the rhythm of sperm transfer. Experimental manipulations of light and temperature regime demonstrated that light dominates over temperature in entraining the timing of sperm release and transfer. Nevertheless, temperature plays a critical role in the absence of light Zeitgeber. Sperm release and transfer are arrhythmic in constant light (LL); however, both rhythms are restored by temperature cycles.     

Daily rhythm in myogenic contractions of vas deferens associated with sperm release cycle in a moth

In the gypsy moth, Lymantria dispar, release of sperm bundles from the testis into the upper vas deferens (UVD) and subsequent transfer of sperm bundles into the seminal vesicles (SV) occurs in a daily rhythm. The UVD undergoes different types of contractions despite the fact that its musculature appears to receive no innervation. Patterns of the UVD movements were recorded throughout the daily sperm release and transfer cycle. In males kept in light-dark cycles, transfer of sperm from the UVD to the SV was accompanied by a characteristic pattern of UVD contractions of high frequency and amplitude. In males kept in constant light, which fail to transfer sperm, this contraction pattern was absent. It is concluded that the vas deferens muscles undergo daily changes in contraction pattern in phase with the light-dark cycle. The increased muscular contractions appear to be a causal factor in the gated sperm transfer from the UVD to the SV.Abbreviations LD light-dark - LL constant light - SV seminal vesicle - UVD upper vas deferens     

The circadian rhythm of sperm release in the codling moth, Cydia pomonella

The release of sperm bundles from testes to the vas deferens is controlled by a circadian clock in several moth species. We investigated the pattern of sperm release in the codling moth, Cydia pomonnella L. (Lepidoptera: Tortricidae). Sperm release in the codling moth follows a two-step rhythm in light-dark cycles: sperm is released from the testis before lights-off, remains in the vas deferens during the dark phase and is transferred to the seminal vesicles after lights-on. This rhythm continues in constant darkness indicating that it has a circadian nature. The release of sperm is asynchronous in moths held in constant light. In contrast to previously investigated moths, constant light has no adverse effects on the male reproductive capacity in the codling moth.     

Spatial and circadian regulation of cry in Drosophila

In Drosophila, cryptochrome (cry) encodes a blue-light photoreceptor that mediates light input to circadian oscillators and sustains oscillator function in peripheral tissues. The levels of cry mRNA cycle with a peak at approximately ZT5, which is similar to the phase of Clock (Clk) mRNA cycling in Drosophila. To understand how cry spatial and circadian expression is regulated, a series of cry-Gal4 trans-genes containing different portions of cry upstream and intron 1 sequences were tested for spatial and circadian expression. In fly heads, cry upstream sequences drive constitutive expression in brain oscillator neurons, a novel group of nonoscillator cells in the optic lobe, and peripheral oscillator cells in eyes and antennae. In contrast, cry intron 1 drives rhythmic expression in eyes and antennae, but not brain oscillator neurons. These results demonstrate that intron 1 is sufficient for high-amplitude cry mRNA cycling, show that cry upstream sequences are sufficient for expression in brain oscillator neurons, and suggest that cry spatial and circadian expression are regulated by different elements.     

A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.     

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.     

Movement of spermatozoa in the reproductive tract of adult male Spodoptera litura: daily rhythm of sperm descent and the effect of light regime on male reproduction

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12 h:12 h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30 h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.     

Abdominally entrained periodicities of testis and vas deferens activity in the Mediterranean flour moth

In a light-dark (LD) regimen, sperm, first apyrene and then eupyrene, start moving out of the fused testes of the Mediterranean flour moth, Anagasta kuehniella, toward the beginning of the scotophase. At 27° ± 2°C, the sperm mass remains in the proximal part of the vasa deferentia for 10 to 12 hr and then passes rapidly into the seminal vesicles, remains in these organs for about 5 hr, and is then transported to the ductus ejaculatoris duplex where it becomes available for ejaculation. The phases of sperm movement appear to be closely related to sperm development, and the reproductive activity of the moths. In isolated abdomens there is a significant reduction in the amount of sperm released from the testes, but normal periodicity of sperm release and movement continues in either LD or continuous dark (DD) regimens, and rapid phase shifting occurs when a LD regimen is reversed. All stages of sperm movement are disrupted in continuous light (LL), but normal periodicity is usually resumed when isolated abdomens of the LL moths are placed in LD or DD regimens. Normal periodicity also occurs in moths paralyzed with tetrodotoxin or procaine. Removal of any one of the four abdominal ganglia from LL moths does not prevent increased sperm release when the moths are placed in LD, though with each ganglion there is some disruption of the normal pattern of movement down the vasa deferentia. It is thought that the testes and vasa deferentia down to at least the seminal vesicles represent a semiautonomous complex in which periodicity is maintained by endogenous circadian activity in cells of the testes (and possibly the vasa deferentia) or more probably in a peripheral control center.     

Analysis of period mRNA cycling in Drosophila head and body tissues indicates that body oscillators behave differently from head oscillators.

The period (per) gene is thought to be part of the Drosophila circadian pacemaker. The circadian fluctuations in per RNA and protein that constitute the per feedback loop appear to be required for pacemaker function, and have been measured in head neuronal tissues that are necessary for locomotor activity and eclosion rhythms. The per gene is also expressed in a number of neuronal and nonneuronal body tissues for which no known circadian phenomena have been described. To determine whether per might affect some circadian function in these body tissues, per RNA cycling was examined. These studies show that per RNA cycles in the same phase and amplitude in head and body tissues during light-dark cycles. One exception to this is the lack of per RNA cycling in the ovary, which also appears to be the only tissue in which PER protein is primarily cytoplasmic. In constant darkness, however, the amplitude of per RNA cycling dampens much more quickly in bodies than in heads. Taken together, these results indicate that circadian oscillators are present in head and body tissues in which PER protein is nuclear and that these oscillators behave differently.     

The luteinizing hormone surge regulates circadian clock gene expression in the chicken ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors' previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary.     

CLOCK is required for maintaining the circadian rhythms of Opsin mRNA expression in photoreceptor cells

In zebrafish, the expression of long-wavelength cone (LC) opsin mRNA fluctuated rhythmically between the day and night. In a 24-h period, expression was high in the afternoon and low in the early morning. This pattern of fluctuation persisted in zebrafish that were kept in constant darkness, suggesting an involvement of circadian clocks. Functional expression of Clock, a circadian clock gene that contributes to the central circadian pacemaker, was found to play an important role in maintaining the circadian rhythms of LC opsin mRNA expression. In zebrafish embryos, in which the translation of Clock was inhibited by anti-Clock morpholinos, the circadian rhythms of LC opsin mRNA expression diminished. CLOCK may regulate the circadian rhythms of LC opsin mRNA expression via cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In control retinas, the concentration of cAMP was high in the early morning and low in the remainder of the day and night. Inhibition of Clock translation abolished the fluctuation in the concentration of cAMP, thereby diminishing the circadian rhythms of opsin mRNA expression. Transient increase of cAMP concentrations in the early morning (i.e. by treating the embryos with 8-bromo-cAMP) restored the circadian rhythms of LC opsin mRNA expression in morpholino-treated embryos. Together, the data suggest that Clock plays important roles in regulating the circadian rhythms in photoreceptor cells.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)     

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

In all living organisms, behavior, metabolism and physiology are under the regulation of a circadian clock. The molecular machinery of this clock has been conserved throughout the animal kingdom. Besides regulating the circadian timing of a variety of processes through a central oscillating mechanism in the brain, these circadian clock genes were found to have a function in peripheral tissues in different insects. Here, we provide evidence that the circadian clock genes period (per) and timeless (tim) have a role in the male locust reproduction. A knockdown of either of the two genes has no effect on male sexual maturation or behavior, but progeny output in their untreated female copulation partners is affected. Indeed, the fertilization rates of the eggs are lower for females with a per or tim RNAi copulation partner as compared to the eggs deposited by females that mated with a control male. As the sperm content of the seminal vesicles is higher in per or tim knockdown males, we suggest that this phenotype could be caused by a disturbance of the circadian regulated sperm transfer in the male reproductive organs, or an insufficient maturation of the sperm after release from the testes.