Dim nighttime illumination alters photoperiodic responses of hamsters through the intergeniculate leaflet and other photic pathways.

In mammals, light entrains the central pacemaker within the suprachiasmatic nucleus (SCN) through both a direct neuronal projection from the retina and an indirect projection from the intergeniculate leaflet (IGL) of the thalamus. Although light comparable in intensity to moonlight is minimally effective at resetting the phase of the circadian clock, dimly lit and completely dark nights are nevertheless perceived differentially by the circadian system, even when nighttime illumination is below putative thresholds for phase resetting. Under a variety of experimental paradigms, dim nighttime illumination exerts effects that may be characterized as enhancing the plasticity of circadian entrainment. For example, relative to completely dark nights, dimly lit nights accelerate development of photoperiodic responses of Siberian hamsters transferred from summer to winter day lengths. Here we assess the neural pathways underlying this response by testing whether IGL lesions eliminate the effects of dim nighttime illumination under short day lengths. Consistent with previous work, dimly lit nights facilitated the expansion of activity duration under short day lengths. Ablation of the IGL, moreover, did not influence photoperiodic responses in animals held under completely dark nights. However, among animals that were provided dimly lit nights, IGL lesions prevented the short-day typical expansion of activity duration as well as the seasonally appropriate gonadal regression and reduction in body weight. Thus, the present data indicate that the IGL plays a central role in mediating the facilitative effects of dim nighttime illumination under short day lengths, but in the absence of the IGL, dim light at night influences photoperiodic responses through residual photic pathways.

Photic and nonphotic seasonal cues differentially engage hypothalamic kisspeptin and RFamide-related peptide mRNA expression in Siberian hamsters.

Seasonally breeding animals use a combination of photic (i.e. day length) and nonphotic (e.g. food availability, temperature) cues to regulate their reproduction. How these environmental cues are integrated is not understood. To assess the potential role of two candidate neuropeptides, kisspeptin and RFamide-related peptide-3 (RFRP), we monitored regional changes in their gene expression in a seasonally breeding mammal exposed to moderate changes in photoperiod and food availability. Adult male Siberian hamsters (Phodopus sungorus) were housed under a long (16 h light/day; 16 L) or intermediate (13.5 L) photoperiod and fed ad lib. or a progressive food restriction schedule (FR; reduced to 80% of ad lib.) for 11 weeks. Gonadal regression occurred only in FR hamsters housed under 13.5 L. Immunohistochemistry was used to identify diencephalic populations of kisspeptin- and RFRP-immunoreactive cells, and quantitative PCR was used to measure gene expression in adjacent coronal brain sections. Photoperiod, but not food availability, altered RFRP mRNA expression in the dorsomedial sections, whereas food availability but not photoperiod altered Kiss1 expression in the arcuate sections; intermediate photoperiods elevated RFRP expression, and food restriction suppressed Kiss1 expression. Regional- and neuropeptide-specific activity of RFamides may provide a mechanism for integration of multi-modal environmental information in the seasonal control of reproduction.

Different neural melatonin-target tissues are critical for encoding and retrieving day length information in Siberian hamsters.

Siberian hamsters exhibit several seasonal rhythms in physiology and behaviour, including reproduction, energy balance, body mass, and pelage colouration. Unambiguous long- and short day lengths stimulate and inhibit reproduction, respectively. Whether gonadal growth or regression occurs in an intermediate day length (e.g. 14 h L : 10 h D; 14L), depends on whether the antecedent day lengths were shorter (10L) or longer (16L). Variations in day length are encoded by the duration of nocturnal pineal melatonin secretion, which is decoded at several neural melatonin target tissues to control testicular structure and function. We assessed participation of three such structures in the acquisition and retrieval of day length information. Elimination of melatonin signalling to the nucleus reuniens (NRe), but not to the suprachiasmatic nucleus (SCN) or paraventricular thalamus (PVt), interfered with the acquisition of a long day reproductive response, whereas the obscuring of melatonin signals to the SCN and the NRe, but not to the PVt, interfered with the photoperiod history response. The SCN and NRe contribute in different ways to the melatonin-based system that mediates seasonal rhythms in male reproduction.

Perinatal influences of melatonin on testicular development and photoperiodic memory in Siberian hamsters.

We assessed the influence of perinatal melatonin on reproductive development and adult responsiveness to melatonin. Testicular growth in an intermediate day length (14 : 10 h light/dark cycle) was substantially reduced in Siberian hamsters gestated by pinealectomised compared to pineal-intact females; gonadal development was normalised in offspring of pinealectomised dams that were pinealectomised at 3-4 days of age. Hamsters deprived of melatonin only during gestation, or both pre- and postnatally, underwent testicular involution during treatment with melatonin in adulthood. Photoperiodic histories acquired prenatally did not endure as long as those acquired by adult hamsters. Hamsters first exposed to melatonin in adulthood were not more proficient in acquiring photoperiodic histories than were normal males. These findings indicate that pre- versus postnatal differences in melatonin signal duration determine rates of testicular development. Exposure to melatonin perinatally does not appear to organise the neuroendocrine substrate that mediates effects of day length and melatonin on the gonads of adult hamsters.

Termination of neuroendocrine refractoriness to melatonin in Siberian hamsters (Phodopus sungorus).

Siberian hamsters maintained from birth in a short day length (DL), unlike their long-day counterparts, fail to undergo reproductive development by 5 weeks of age. Instead, reproductive maturation of short-day males is delayed for approximately 20 weeks, at which point neuroendocrine refractoriness to the inhibitory effects of short DLs develops, resulting in growth of the gonads. To terminate refractoriness and re-establish responsiveness to short photoperiods, 10-15 weeks of long-day exposure is required. We assessed whether continuous exposure to long days is necessary to terminate refractoriness or whether the first few weeks of long days initiate a process that culminates several months later in the breaking of refractoriness. Male hamsters refractory to short DLs were transferred to a long-day photoperiod, pinealectomized (PINx) after 0, 3, 6 or 15 weeks, and subsequently infused for 6 weeks with a short-day melatonin signal. This melatonin treatment induces gonadal regression in photosensitive but not in photorefractory hamsters. Six percent of males PINx at week 0 and 88% of those PINx at week 15 underwent gonadal atrophy by the end of the melatonin infusion treatment initiated on week 15. Among hamsters PINx on week 6, 17% versus 76% underwent testicular involution in response to melatonin infusions initiated on week 6 and week 15, respectively. This finding indicates that a fraction of the long days that hamsters experience during spring and summer are sufficient to trigger the processes that restore responsiveness to short DLs. Additional groups of pineal-intact photorefractory animals were given 3, 6 or 15 weeks of long-day exposure and then returned to a short DL for several months; only those treated for 15 weeks terminated refractoriness. The breaking of refractoriness, once triggered by long-day melatonin signals, proceeds to completion only in the absence of short-day melatonin signals.

Testicular and somatic growth in Siberian hamsters depend on the melatonin-free interval between twice daily melatonin signals.

In Siberian hamsters, day length is encoded by the duration of the nocturnal melatonin signal; short and long melatonin signals over the course of several weeks stimulate and inhibit somatic and gonadal development, respectively, in prepubertal males. We sought to determine whether juvenile male Siberian hamsters respond to multiple melatonin signals each day and the manner in which the sequence of melatonin signals and the duration of the melatonin-free interval between signals affects development. Twenty-one day old male Siberian hamsters, gestated and maintained in a short-day photoperiod of 10 h light/day (10 L), were transferred to constant light to suppress endogenous melatonin secretion and received s.c. infusions of melatonin or saline for 12 days. Hamsters infused with saline retained small testes, whereas one short melatonin infusion each day resulted in significant testicular growth. Other hamsters were provided with two melatonin signals each day, one long (9 h) and one short (4 or 5 h); the order in which these signals was administered and the duration of the melatonin-free interval after each signal varied between groups. In asymmetrical melatonin infusions, the first and second daily infusions were followed by 3-h and 7-h melatonin-free intervals, respectively, whereas in symmetrical infusions, each melatonin signal was followed by a 5-h melatonin-free interval. In the asymmetrical sequence, the melatonin signal that immediately preceded the longer melatonin-free interval determined the rate gonadal growth. Equal melatonin-free intervals after each of the long and short daily melatonin infusions produced intermediate increases in gonadal and somatic development. The hypothalamic-pituitary-gonadal axis of Siberian hamsters can respond to multiple melatonin signals each day, with the rate of testicular growth determined primarily by the duration of the melatonin-free interval following each infusion.

Photoperiodic control of oestrous cycles in Syrian hamsters: mediation by the mediobasal hypothalamus.

To assess whether the mediobasal hypothalamus (MBH) is necessary for photoperiodic control of oestrous cycles and prolactin secretion, we tested intact female Syrian hamsters (controls) and those that had sustained unilateral or bilateral lesions of the MBH. All hamsters displayed 4-day oestrous cycles postoperatively in the long-day photoperiod (14 h light/day); control females and those with unilateral MBH damage ceased to undergo oestrous cycles approximately 8 weeks after transfer to a short-day photocycle (10 h light/day), whereas 12 of 15 females with bilateral MBH lesions continued to generate 4-day oestrous cycles throughout 22 weeks in short days. Serum prolactin concentrations were either undetectable or low in all hamsters 8 or 14 weeks after the transfer to short-day lengths, but increased above long-day baseline values by week 22. We conclude that melatonin-binding sites in the MBH mediate suppression of oestrous cycles but not prolactin secretion by short-day lengths; recovery of prolactin secretion in females during prolonged exposure to short-day lengths reflects development of refractoriness to melatonin in a substrate distinct from the MBH. These findings suggest that separate neural pathways mediate photoperiodic control of gonadotropin and prolactin secretion in female hamsters.

Refractoriness to melatonin occurs independently at multiple brain sites in Siberian hamsters.

The mid-winter development of refractoriness to melatonin (Mel) triggers recrudescence of the atrophied reproductive apparatus of rodents. As a consequence, over-wintering animals become reproductively competent just before the onset of spring conditions favorable for breeding. The neural target tissues that cease to respond to winter Mel signals have not been identified. We now report that the suprachiasmatic nucleus of the hypothalamus, which contains the principal circadian clock, and the reuniens and paraventricular nuclei of the thalamus, each independently becomes refractory to melatonin. Small implants of Mel that were left in place for 40 wk and that act locally on these brain nuclei, induced testicular regression within 6 wk in male Siberian hamsters; 12 wk later Mel implants no longer suppressed reproduction and gonadal recrudescence ensued. Hamsters that were then given a systemic Mel infusion s.c. immediately initiated a second gonadal regression, implying that neurons at each site become refractory to Mel without compromising responsiveness of other Mel target tissues. Refractoriness occurs locally and independently at each neural target tissue, rather than in a separate "refractoriness" substrate. Restricted, target-specific actions of Mel are consistent with the independent regulation by day length of the several behavioral and physiological traits that vary seasonally in mammals.

Colestipol-induced hepatotoxicity.

A 65-year-old man with type IIa dyslipidemia who received flavored colestipol granules 2 scoops/day for 3 months developed asymptomatic hepatotoxicity. Several of his liver enzymes were elevated 10 times the upper limit of normal. One week after discontinuing colestipol, serum transaminases fell dramatically, with some returning to normal limits. Four weeks after colestipol was discontinued, all liver function tests were normal. Rechallenge was not attempted. Other potential causes of hepatocellular injury were evaluated. Bile acid-binding resins commonly are administered to treat type IIa dyslipidemia. Despite extensive use of the resins, significant elevations of transaminase levels are rare. Because the exact mechanism of bile acid resin-induced hepatotoxicity is unknown, high-risk patients may require liver function test monitoring and education on hepatotoxic side effects.

Low ambient temperature accelerates short-day responses in Siberian hamsters by altering responsiveness to melatonin.

Exposure to low ambient temperatures (Ta) accelerates appearance of the winter phenotype in Siberian hamsters transferred from long to short day lengths. Because melatonin transduces the effects of day length on the neuroendocrine axis, the authors assessed whether low Ta promotes the transition to winterlike traits by accelerating the onset of increased nocturnal melatonin secretion or by enhancing responsiveness to melatonin in short day lengths. Male hamsters were transferred from 16L (16 h light/day) to 8L (8 h light/day) photoperiods and held at 5 degrees C or 22 degrees C. Locomotor activity was recorded continuously, and body mass, testis size, and pelage color were determined biweekly for 8 weeks. The duration of nocturnal locomotion (alpha), a reliable indicator of the duration of nocturnal melatonin secretion, lengthened significantly earlier in hamsters exposed to a Ta of 5 degrees C than 22 degrees C. Cold exposure increased the proportion of hamsters that were photoresponsive: gonadal regression in short days increased from 44% at 22 degrees C to 81% at 5 degrees C (p < 0.05); low Ta did not, however, accelerate testicular regression in animals that were photoresponsive. Nonphotoresponsive animals at 5 degrees C temporarily had longer alphas during the first 4 weeks in short days and significant decreases in body mass and testicular size that were reversed during the ensuing weeks when alpha decreased. In a 2nd experiment, pinealectomized male hamsters infused for 10 h/day with melatonin for 2 weeks had significantly lower body and testes masses when maintained at 5 degrees C but not 22 degrees C. Low-ambient temperature appears to accelerate the appearance of the winter phenotype primarily by increasing target tissue responsiveness to melatonin and to a lesser extent by augmenting the rate at which the duration of nocturnal melatonin secretion increases in short day lengths.

Testicular development in Siberian hamsters depends on frequency and pattern of melatonin signals.

We investigated the impact of frequency and pattern of melatonin signals on reproductive development in Siberian hamsters. Juvenile males gestated in short day lengths and housed in constant illumination to suppress melatonin secretion were infused with melatonin for 5 h either once or twice per day for 20 days. Melatonin infusions at either frequency produced equivalent increases in testes and body weights that exceeded those of animals infused with saline but were indistinguishable from those of hamsters transferred to long day lengths. The reproductive system appears to be maximally stimulated by a single short melatonin signal each day. Other animals kept from birth in a short photoperiod were treated 6 h after onset of darkness with the beta-adrenergic receptor antagonist DL-propranolol to shorten melatonin secretion on the night of injection but not on subsequent nights. This permitted interpolation of short nightly melatonin signals of 4-5 h duration against a background of long melatonin signals of 10-12 h duration on other nights. Treatment regimes that maintained a 1:1 ratio of short to long melatonin signals for 8 wk stimulated reproductive development; a 1:2 signal ratio, in each of three different patterns, was uniformly ineffective. The number of successive short melatonin signals had little influence on the interval across which successive melatonin signals were summated to influence photoperiodic traits. The neuroendocrine axis appears more responsive to short melatonin signal frequency than pattern for development of the summer phenotype.

Cytotoxic effects of cigarette smoke alkaloids inhibit the progesterone production and cell growth of cultured MA-10 Leydig tumor cells.

Inhibition of corpus luteum progesterone synthesis by cigarette smoke alkaloids might, in part, explain the generally poorer outcome of pregnancy in smoking women. The present experiments evaluate the effects of cigarette smoke alkaloids on progesterone biosynthesis and cell growth. Studies were based using the MA-10 Leydig tumor cell line. The steroid pathway in MA-10 cells has only two specific enzymatic steps. The cholesterol passes to the cholesterol side-chain cleavage enzyme and then metabolizes the resulting pregnenolone to progesterone and partly to 20alpha-dihydroprogesterone. Incubation of MA-10 cells with nicotine, cotinine, anabasine, all of these alkaloids, or an aqueous extract of cigarette smoke resulted in dose-dependent inhibition of progesterone and 20alpha-dihydroprogesterone synthesis. The number of cells in the treated dishes seemed less than the control. This latter finding prompted experiments evaluating the short-term effects of the alkaloids on cell growth. Growth of MA-10 cells influenced with alkaloids or smoke extract was also inhibited. All of the inhibitory effects of nicotine, cotinine, anabasine and cigarette smoke extract on MA-10 cells were explained by cytotoxicity. The cytotoxic effect could reduce the fertilization, implantation, and early human development. This mechanism entails the consequence of impaired placental growth, disorder in the placental vascular architecture and placental circulation, and small-for-date babies.

Temperature-independence of circannual variations in circadian rhythms of golden-mantled ground squirrels.

In golden-mantled ground squirrels, phase angles of entrainment of circadian locomotor activity to a fixed light-dark cycle differ markedly between subjective summer and winter. A change in ambient temperature affects entrainment only during subjective winter when it also produces pronounced effects on body temperature (Tb). It was previously proposed that variations in Tb are causally related to the circannual rhythm in circadian entrainment. To test this hypothesis, wheel-running activity and Tb were monitored for 12 to 14 months in castrated male ground squirrels housed in a 14:10 LD photocycle at 21 degrees C. Animals were treated with testosterone implants that eliminated hibernation and prevented the marked winter decline in Tb; these squirrels manifested circannual changes in circadian entrainment indistinguishable from those of untreated animals. Both groups exhibited pronounced changes in phase angle and alpha of circadian wheel-running and Tb rhythms. Seasonal variation in Tb is not necessary for circannual changes in circadian organization of golden-mantled ground squirrels.

Barbiturates inhibit progesterone synthesis in cultured Leydig tumor cells and human granulosa cells.

Screening drugs used in obstetrical practice for effects on steroid hormone synthesis revealed that phenobarbital inhibited progesterone synthesis in MA-10 Leydig tumor cells. The inhibition was apparently a drug class effect since it could be reproduced by other barbiturates. Barbiturate blockade was reversible and could be bypassed in the MA-10 cells by using 22-hydroxycholesterol. Human granulosa cell progesterone synthesis was also inhibited in a dose dependent fashion by phenobarbital, secobarbital and barbituric acid. Significant inhibition occurred in dose ranges that would be therapeutic for treating epilepsy. From these data we conclude that barbiturates block steroidogenesis by inhibiting cholesterol transport to the cholesterol side chain cleavage enzyme.

Behavior of stabled horses provided continuous or intermittent access to drinking water.

OBJECTIVE: To compare quantitative measures and clinical assessments of behavior as an indication of psychologic well-being of stabled horses provided drinking water continuously or via 1 of 3 intermittent delivery systems. ANIMALS: 22 Quarter Horse (QH) or QH-crossbred mares and 17 Belgian or Belgian-crossbred mares (study 1) and 24 QH or QH-crossbred mares and 18 Belgian or Belgian-crossbred mares (study 2). PROCEDURE: Stabled horses were provided water continuously or via 1 of 3 intermittent water delivery systems in 2 study periods during a 2-year period. Continuous 24-hour videotaped samples were used to compare quantitative measures and clinical assessments of behavior among groups provided water by the various water delivery systems. RESULTS: All horses had clinically normal behavior. Significant differences in well being were not detected among groups provided water by the various delivery systems. CONCLUSIONS AND CLINICAL RELEVANCE: Various continuous and intermittent water delivery systems can provide adequately for the psychologic well-being of stabled horses.

Clinical, biochemical, and hygiene assessment of stabled horses provided continuous or intermittent access to drinking water.

OBJECTIVE: To compare health, hydration status, and management of stabled pregnant mares provided drinking water continuously or via 1 of 3 intermittent delivery systems. ANIMALS: 22 Quarter Horse (QH) or QH-crossbred mares and 18 Belgian or Belgian-crossbred mares (study 1); 24 QH or QH-crossbred mares and 18 Belgian or Belgian-crossbred mares (study 2). PROCEDURE: Stabled horses were provided water continuously or via 1 of 3 intermittent water delivery systems in 2 study periods during a 2-year period. Body temperature, attitude, appetite, water intake, and urine output were recorded daily. Hygiene of each horse and the stable were assessed weekly. Clinical and biochemical measures of hydration were determined 3 times during each study. Clinical measures of hydration included skin turgor, gum moisture, capillary refill time, and fecal consistency. Biochemical measures of hydration included PCV, plasma total protein concentration, serum osmolality, plasma vasopressin concentration, urine specific gravity, and urine osmolality. RESULTS: All horses remained healthy. Stable hygiene was worse when horses had continuous access to water. Clinical and biochemical measures of hydration did not differ among water delivery systems. CONCLUSIONS AND CLINICAL RELEVANCE: Various continuous and intermittent water delivery systems provided adequate amounts of water to stabled horses to maintain health and hydration status. Providing intermittent access to water may be preferable on the basis of stable hygiene.

Influence of nicotine, cotinine, anabasine and cigarette smoke extract on human granulosa cell progesterone and estradiol synthesis.

To reveal the well known effect of smoking on the incidence of early abortion, the possible effects of cigarette alkaloids on progesterone and estradiol synthesis were investigated. A suspected cause for early spontaneous abortion is corpus luteum insufficiency. The present experiments evaluate the effects of cigarette smoke alkaloids on progesterone and estradiol biosynthesis. Human granulosa cells were obtained from patients undergoing in vitro fertilization and embryo transfer treatment because of infertility. Incubation of the granulosa cells with cotinine, anabasine, with the combination of nicotine, cotinine and anabasine, or with an aqueous extract of cigarette smoke resulted in inhibition of progesterone synthesis. The alkaloids and smoke extract decreased the DNA content of the culture dish. These latter findings suggested a cytotoxic effect of the alkaloids. Both cotinine and anabasine slightly stimulated the synthesis of normalized estradiol. However, nicotine, combination of all three alkaloids, and cigarette smoke extract had no significant influence on estradiol production. Taken together, these data would suggest that cigarette alkaloids inhibit cellular progesterone synthesis both by inhibiting progesterone synthesis and by causing less specific toxic effects to the cell. In contrast, cigarette smoke alkaloids slightly stimulated or had no effect on estradiol production. These concomitant actions of cigarette alkaloids partly explain the higher incidence of early abortion in pregnant women who smoke.