Abstract: Transcription factors belonging to the Per/Arnt/Sim (PAS) domain family are highly conserved and many are involved in circadian rhythm regulation. One member of this family, aryl hydrocarbon receptor (AhR), is an orphan receptor whose physiological role is unknown. Recent findings have led to the hypothesis that AhR has a role in circadian rhythm, which is the focus of the present investigation. First, time-of-day-dependent mRNA expression of AhR and its signaling target, cytochrome p4501A1 (Cyp1a1), was determined in C57BL/6J mice by quantitative RT-PCR. Circadian expression of AhR and Cyp1a1 was observed both in the suprachiasmatic nucleus (SCN) and liver. Next, the circadian phenotype of mice lacking AhR (AhRKO) was investigated using behavioral monitoring. Intact AhRKO mice had robust circadian rhythmicity with a similar tau under constant conditions compared to wild-type mice, but a significant difference in tau was observed between genotypes in ovariectomized female mice. Time to reentrainment following 6-h advances or delays of the light/dark cycle was not significantly different between genotypes. However, mice exposed to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 microg/kg of body weight) displayed decreased phase shifts in response to light and had altered expression of Per1 and Bmal1. These results suggest that chronic activation of AhR may affect the ability of the circadian timekeeping system to adjust to alterations in environmental lighting by affecting canonical clock genes. Further studies are necessary to decipher the mechanism of how AhR agonists could disrupt light-induced phase shifts. If AhR does have a role in circadian rhythm, it may share redundant roles with other PAS domain proteins and/or the role of AhR may not be exhibited in the behavioral activity rhythm, but could be important elsewhere in the peripheral circadian system.

Abstract: Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play an important role in reproductive function. These cells originate in the nasal compartment and migrate into the basal forebrain in association with olfactory/vomeronasal nerves in embryonic life in rodents. Here, we studied the role of neuropilins and their ligands, semaphorins, in the development of the olfactory-GnRH system. We focused on Neuropilin-2 knock-out (Npn-2(-/-)) mice, because they are known to display defasciculation of olfactory nerves and reduced fertility. We found a significant decrease in the number of GnRH neurons in the hypothalamus and a marked reduction in their gonadal size. We then observed an abnormal increase of GnRH neurons in the noses of Npn-2(-/-) mice, indicating that these cells failed to migrate into the forebrain. However, because neuropilins and semaphorins are involved in events of neuronal migration in the brain, we asked whether the observed reduction in GnRH neurons was directly attributable to the action of these molecules. Using fluorescence-activated cell sorting and reverse transcription-PCR on mRNA derived from embryonic green fluorescent protein (GFP)-GnRH transgenic mice, we found expression of class 3 semaphorins and their receptors (neuropilin-1/2 and plexin-A1) in GnRH neurons. Furthermore, double-immunofluorescence experiments showed that migrating GnRH neurons, as well as associated olfactory fibers, express Npn-2 in the nasal region. We then used a line of immortalized GnRH neurons (GN11 cells) that display the same expression patterns for semaphorins and their receptors as GFP-GnRH cells and found that class 3 semaphorins and vascular endothelial growth factors modulate their migratory activity. These studies provide support for the direct involvement of neuropilins and their ligands in the establishment of the GnRH neuroendocrine system.

Abstract: The aryl hydrocarbon receptor (AhR) mediates adverse effects of dioxins, but its physiological role remains ambiguous. The similarity between AhR and canonical circadian clock genes suggests potential involvement of AhR in regulation of circadian timing. Photoproducts of tryptophan (TRP), including 6-formylindolo[3,2-b]carbazole (FICZ), have high affinity for AhR and are postulated as endogenous ligands. Although TRP photoproducts activate AhR signaling in vitro, their effects in vivo have not been investigated in mammals. Because TRP photoproducts may act as transducers of light, we examined their effects on the circadian clock. Intraperitoneal injection of TRP photoproducts or FICZ to C57BL/6J mice dose dependently induced AhR downstream targets, cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 mRNA expression, in liver. c-fos mRNA, a commonly used marker for light responses, was also induced with FICZ, and all responses were AhR dependent. A rat-immortalized suprachiasmatic nucleus (SCN) cell line, SCN 2.2, was used to examine the direct effect of TRP photoproducts on the molecular clock. Both TRP photoproducts and FICZ-increased CYP1A1 expression and prolonged FICZ incubation altered the circadian expression of clock genes (Per1, Cry1, and Cry2) in SCN 2.2 cells. Furthermore, FICZ inhibited glutamate-induced phase shifting of the mouse SCN electrical activity rhythm. Circadian light entrainment is critical for adjustment of the endogenous rhythm to environmental light cycle. Our results reveal a potential for TRP photoproducts to modulate light-dependent regulation of circadian rhythm through triggering of AhR signaling. This may lead to further understanding of toxicity of dioxins and the role of AhR in circadian rhythmicity.

Abstract: Time-of-day-dependent variation in neuronal ischemia is well documented. Whether this results from changes in time-of-day variation in susceptibility or from other causative factors remains unclear. We hypothesize that hippocampal cells exhibit variation in activation of cell death predictive markers in response to ischemia induced at different times-of-day. Changes in hippocampal circadian clock gene rhythmicity may also be associated with ischemia. Transient global ischemia was induced in rats at three times of day and animals were sacrificed 24 h later. Hippocampal caspase-3, -8 and -9 transcripts and active proteins and calbindin protein were measured in the CA1 region of the hippocampus. In a second study, 24-h rhythms of circadian regulatory transcripts were determined in hippocampus after global ischemia. Caspase-3, -8 and -9 transcripts and active protein levels were increased substantially when ischemia occurred in early night (ZT14); smaller changes were observed in late night (ZT20, or day ZT6). Calbindin levels decreased following ischemia, especially at ZT14. Ischemia shifted the rhythm of the Per1 transcript; peak expression occurred 6 h earlier following ischemia. Rhythms of Cry1 and Bmal1 were not altered. Greater induction of caspases and decline of calbindin when ischemia was performed at ZT14 than at ZT20 or ZT6 support the concept of increased hippocampal susceptibility to ischemia at ZT14. Alteration of the Per1 transcript suggests a potential role for the circadian clock in this process. Notably, ZT14 represents the beginning of the rats' nocturnal period of activity, corresponding to the time when humans experience the greatest neuronal ischemic damage from stroke.

Abstract: A molecular device that measures time on a daily, or circadian, scale is a nearly ubiquitous feature of eukaryotic organisms. A core group of clock genes, whose coordinated function is required for this timekeeping, is expressed both in the central clock and within numerous peripheral organs. We examined expression of clock genes in the rat ovary. Transcripts for core oscillator elements (Arntl, Clock, Per1, Per2, and Cry1) were present in the ovary as indicated by quantitative real-time RT-PCR. Rhythmic expression patterns of Arntl and Per2 transcripts and protein products were out of phase with respect to the central oscillator and in complete antiphase to each other. Expression of Arntl was significantly elevated after the LH surge on the day of proestrus. Finally, hCG treatment induced cyclic expression of both Arntl and Per2 gene products in hypophysectomized, immature rats primed with eCG. Collectively, these data suggest that the core underpinnings of the transcriptional/translational feedback loop that drives circadian rhythmicity is present in the rat ovary. Furthermore, the study identifies LH as a potential regulator of circadian clock gene rhythms in the ovary.

Abstract: The recent identification of specific genes responsible for the generation of endogenous circadian rhythmicity in the suprachiasmatic nucleus presents a new level of investigation into endogenous rhythmicity and mechanisms of synchronization of this circadian clock with the environmental light?dark cycle. This article describes techniques that employ antisense and decoy oligodeoxynucleotides (ODN) to determine the roles of specific molecular substrates both in endogenous rhythmicity and in regulating the effects of light on the mammalian circadian clock. Application of antisense ODN technology has revealed a role for timeless (Tim) in the core clock mechanism and established that induction of period1 (Per1) is required for light responsiveness. Likewise, a decoy ODN designed to sequester activated CREB protein definitively demonstrated a requirement for CRE-mediated transcription in light signaling. Experiments designed with these molecular tools offer new insights on the interaction of cellular processes and signaling with the molecular clockworks.

Abstract: Circadian clocks comprise a cyclic series of dynamic cellular states, characterized by the changing availability of substrates that alter clock time when activated. To determine whether circadian clocks, like the cell cycle, exhibit regulation by key phosphorylation events, we examined endogenous kinase regulation of timekeeping in the mammalian suprachiasmatic nucleus (SCN). Short-term inhibition of PKG-II but not PKG-Ibeta using antisense oligodeoxynucleotides delayed rhythms of electrical activity and Bmal1 mRNA. Phase resetting was rapid and dynamic; inhibition of PKG-II forced repetition of the last 3.5 hr of the cycle. Chronic inhibition of PKG-II disrupted electrical activity rhythms and tonically increased Bmal1 mRNA. PKG-II-like immunoreactivity was detected after coimmunoprecipitation with CLOCK, and CLOCK was phosphorylated in the presence of active PKG-II. PKG-II activation may define a critical control point for temporal progression into the daytime domain by acting on the positive arm of the transcriptional/translational feedback loop.

Abstract: The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, we examined clock-controlled protein kinase G (PKG) regulation in the mammalian SCN. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occurred at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro was delayed by approximately 3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (alphaODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKG-necessary window in both rat and mouse depended on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms showed the same temporal sensitivities and qualitative responses, we predict that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state.

Abstract: Light is a prominent stimulus that synchronizes endogenous circadian rhythmicity to environmental light/dark cycles. Nocturnal light elevates mRNA of the Period1 (Per1) gene and induces long term state changes, expressed as phase shifts of circadian rhythms. The cellular mechanism for Per1 elevation and light-induced phase advance in the suprachiasmatic nucleus (SCN), a process initiated primarily by glutamatergic neurotransmission from the retinohypothalamic tract, was examined. Glutamate (GLU)-induced phase advances in the rat SCN were blocked by antisense oligodeoxynucleotide (ODN) against Per1 and Ca(2+)/cAMP response element (CRE)-decoy ODN. CRE-decoy ODN also blocked light-induced phase advances in vivo. Furthermore, the CRE-decoy blocked GLU-induced accumulation of Per1 mRNA. Thus, Ca(2+)/cAMP response element-binding protein (CREB) and Per1 are integral components of the pathway transducing light-stimulated GLU neurotransmission into phase advance of the circadian clock.

Abstract: Despite a central circadian role in Drosophila for the transcriptional regulator Timeless (dTim), the relevance of mammalian Timeless (mTim) remains equivocal. Conditional knockdown of mTim protein expression in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms, and altered levels of known core clock elements. Full-length mTim protein (mTIM-fl) exhibited a 24-hour oscillation, where as a truncated isoform (mTIM-s) was constitutively expressed. mTIM-fl associated with the mammalian clock Period proteins (mPERs) in oscillating SCN cells. These data suggest that mTim is required for rhythmicity and is a functional homolog of dTim on the negative-feedback arm of the mammalian molecular clockwork.

Abstract: We investigated a role for cAMP/protein kinase A (PKA) in light/glutamate (GLU)-stimulated state changes of the mammalian circadian clock in the suprachiasmatic nucleus (SCN). Nocturnal GLU treatment elevated [cAMP]; however, agonists of cAMP/PKA did not mimic the effects of light/GLU. Coincident activation of cAMP/PKA enhanced GLU-stimulated state changes in early night but blocked light/GLU-induced state changes in the late night, whereas inhibition of cAMP/PKA reversed these effects. These responses are distinct from those mediated by mitogen-activated protein kinase (MAPK). MAPK inhibitors attenuated both GLU-induced state changes. Although GLU induced mPer1 mRNA in both early and late night, inhibition of PKA blocked this event only in early night, suggesting that cellular mechanisms regulating mPer1 are gated by the suprachiasmatic circadian clock. These data support a diametric gating role for cAMP/PKA in light/GLU-induced SCN state changes: cAMP/PKA promotes the effects of light/GLU in early night, but opposes them in late night.

Abstract: Circadian rhythms in Drosophila melanogaster depend on a molecular feedback loop generated by oscillating products of the period (per) and timeless (tim) genes. In mammals, three per homologs are cyclically expressed in the suprachiasmatic nucleus (SCN), site of the circadian clock, and two of these, mPer1 and mPer2, are induced in response to light. Although this light response distinguishes the mammalian clock from its Drosophila counterpart, overall regulation, including homologous transcriptional activators, appears to be similar. Thus, the basic mechanisms used to generate circadian timing have been conserved. However, contrary to expectations, the recently isolated mammalian tim homolog was reported not to cycle. In this study, we examined mRNA levels of the same tim homolog using a different probe. We observed a significant (approximately threefold) diurnal variation in mTim expression within mouse SCN using two independent methods. Peak levels were evident at the day-to-night transition in light-entrained animals, and the oscillation persisted on the second day in constant conditions. Furthermore, light pulses known to induce phase delays caused significant elevation in mTim mRNA. In contrast, phase-advancing light pulses did not affect mTim levels. The mTim expression profile and the response to nocturnal light are similar to mPer2 and are delayed compared with mPer1. We conclude that temporal ordering of mTim and mPer2 parallels that of their fly homologs. We predict that mTIM may be the preferred functional partner for mPER2 and that expression of mTim and mPer2 may, in fact, be driven by mPER1.

Abstract: The tiny suprachiasmatic nucleus (SCN) of the hypothalamus plays a central role in the daily programming of organismic functions by regulating day-to-day oscillations of the internal milieu and synchronizing them to the changing cycles of day and night and of body state. This biological clock drives the daily expression of vital homeostatic functions as diverse as feeding, drinking, body temperature, and neurohormone secretion. It adaptively organizes these body functions into near-24-hour oscillations termed circadian rhythms. The SCN imposes temporal order 1) through generating output signals that relay time-of-day information, and 2) through gating its own sensitivity to incoming signals that adjust clock timing. Each of these properties, derived from the timebase of the SCN's endogenous near-24-hour pacemaker, persists when the SCN is maintained in a hypothalamic brain slice in vitro. Single-unit recording experiments demonstrate a spontaneous peak in the electrical activity of the ensemble of SCN neurons near midday. By utilizing this time of peak as a "pulse" of the clock, we have characterized a series of time domains, or windows of sensitivity, in which the SCN restricts its own sensitivity to stimuli that are capable of adjusting clock phase. Pituitary adenylyl cyclase-activating peptide (PACAP) and cAMP comprise agents that reset clock phase during the day time domain; both PACAP and membrane-permeable cAMP analogs cause phase advances only when applied during the day. In direct contrast to PACAP and cAMP, acetylcholine and cGMP analogs phase advance the clock only when applied during the night. Sensitivity to light and glutamate arises concomitant with sensitivity to acetylcholine and cGMP. Light and glutamate cause phase delays in the early night, by acting through elevation of intracellular Ca2+, mediated by activation of a neuronal ryanodine receptor. In late night, light and glutamate utilize a cGMP-mediated mechanism to induce phase advances. Finally, crepuscular domains, or dusk and dawn, are characterized by sensitivity to phase resetting by the pineal hormone, melatonin, acting through protein kinase C. Our findings indicate that the gates to both daytime and nighttime phase resetting lie beyond the level of membrane receptors; they point to critical gating within the cell, downstream from second messengers. The changing patterns of sensitivities in vitro demonstrate that the circadian clock controls multiple molecular gates at the intracellular level, to assure that they are selectively opened in a permissive fashion only at specific points in the circadian cycle. Discerning the molecular mechanisms that generate these changes is fundamental to understanding the integrative and regulatory role of the SCN in hypothalamic control of organismic rhythms.

Abstract: Circadian clocks are complex biochemical systems that cycle with a period of approximately 24 hours. They integrate temporal information regarding phasing of the solar cycle, and adjust their phase so as to synchronize an organism's internal state to the local environmental day and night. Nocturnal light is the dominant regulator of this entrainment. In mammals, information about nocturnal light is transmitted by glutamate released from retinal projections to the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Clock resetting requires the activation of ionotropic glutamate receptors, which mediate Ca2+ influx. The response induced by such activation depends on the clock's temporal state: during early night it delays the clock phase, whereas in late night the clock phase is advanced. To investigate this differential response, we sought signalling elements that contribute solely to phase delay. We analysed intracellular calcium-channel ryanodine receptors, which mediate coupled Ca2+ signalling. Depletion of intracellular Ca2+ stores during early night blocked the effects of glutamate. Activators of ryanodine receptors induced phase resetting only in early night; inhibitors selectively blocked delays induced by light and glutamate. These findings implicate the release of intracellular Ca2+ through ryanodine receptors in the light-induced phase delay of the circadian clock restricted to the early night.

Abstract: Anatomical studies have suggested that the germinal disc (GD) region (GDR; GD plus overlying granulosa cells) is the growth center of the avian preovulatory follicle. The objective of this study was to characterize the physiology of the GDR by comparing the functions of two morphologically distinct populations of granulosa cells. The three markers of the physiology of individual granulosa cells examined were 1) proliferation, 2) production of plasminogen activator (PA), and 3) production of progesterone. The effect of LH on each of these functions was also evaluated. Sections 8 mm in diameter were obtained from granulosa cells associated with the GD (GD granulosa cells) and from granulosa cells on the layer distal to the GD (nonGD granulosa cells) from the five largest preovulatory follicles (F5-F1, F1 designated the largest) 12-14 h (before the LH surge) or 2 h (after the LH surge) before ovulation. Proliferation was measured using [3H]thymidine incorporation. PA activity was measured using the chromogenic substrate S-2251. Progesterone was measured by RIA. Incorporation of [3H]thymidine was very high in GD and nonGD granulosa cells from F5 and F4 follicles and decreased dramatically as the follicle progressed through the hierarchy, but remained significantly higher in GD granulosa cells compared to nonGD granulosa cells at all stages of development examined (F5-F1). Exposure of follicles to LH in vivo inhibited [3H]thymidine incorporation by GD granulosa cells in all follicles except the F5. In contrast, in vivo exposure to LH had no effect on [3H]thymidine incorporation by nonGD granulosa cells. PA production by GD granulosa cells was high throughout the stages of maturation studied (F5-F1), whereas PA production by nonGD granulosa cells decreased as follicles matured from F5 to F1. Interestingly, LH stimulated PA production by F5 GD granulosa cells, had no effect on PA production by F3 GD granulosa cells, and inhibited PA production by F1 GD granulosa cells. In contrast, LH inhibited PA production by nonGD granulosa cells in F3 and F1 follicles. Progesterone production by GD granulosa cells was low in F3 and F1 follicles. Progesterone production by nonGD granulosa cells increased as the follicle matured from the F3 to F1 stage and was stimulated significantly by LH. These data indicate that physiological differences in granulosa cell function are dependent upon the location of granulosa cells relative to the GD. GD granulosa cells are less mature, proliferate more rapidly, and produce more PA than nonGD granulosa cells, which produce more progesterone and less PA. Differences in granulosa cell function may be due to the influence of the GD, providing physiological evidence that the GDR may be the growth center of the follicle.

Abstract: Plasminogen activator (PA) is hypothesized to be important in the remodeling of the extracellular matrix during follicular growth. The granulosa layer produces high amounts of PA in response to a stimulatory factor, produced by the theca layer, that is inhibited by LH. To determine the site and mechanism by which LH inhibits PA production, we asked 1) whether LH acts on the granulosa layer and/or the theca layer to inhibit PA production by the largest preovulatory follicle (F1), and 2) whether LH affects PA production by acting at the mRNA or protein level. Sections (10 mm in diameter) of granulosa layers obtained from the F1 follicle before (14 h before ovulation) or after (2 h before ovulation) the LH surge were incubated (24 h at 37 degrees C) in theca-conditioned medium; this medium had been prepared by incubation of 10-mm-diameter sections of theca layers, obtained before (14 h before ovulation) or after (2 h before ovulation) the LH surge, in Dulbecco's Modified Eagle's Medium for 24 h at 37 degrees C. PA production in culture medium was measured with use of the chromogenic substrate S-2251. PA production was high when granulosa layers obtained before the LH surge were incubated in theca-conditioned medium obtained before the LH surge; it was also high when granulosa layers obtained before the LH surge were incubated in theca-conditioned medium obtained after the LH surge. PA production was low when granulosa layers obtained after the LH surge were incubated in theca-conditioned medium obtained before the LH surge, and was also low when granulosa layers obtained after the LH surge were incubated in theca-conditioned medium obtained after the LH surge. Northern and Western blots and activity assays performed on granulosa layer homogenates indicated that PA mRNA, protein, and activity were high before the LH surge and low after the LH surge. Production of the stimulatory factor by the theca layer is apparently unaffected by LH. After exposure to LH, the granulosa layer is no longer capable of producing PA, even in the presence of the theca-derived stimulatory factor. We conclude that the granulosa layer is the site of mRNA and/or protein regulation of PA production by LH.

Abstract: Microscopic analysis of ovarian follicles in the domestic hen has revealed differences in the cellular structure of granulosa cells that are dependent upon the location of granulosa cells relative to the germinal disc, which contains the female gamete. These differences appear as a morphological gradient, which implies variations in granulosa cell function. This observation prompted us to hypothesize that the germinal disc region (GDR) of the avian preovulatory follicle participates in the process of follicular growth by producing factors that act in a paracrine manner to stimulate proliferation of and inhibit steroidogenesis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we asked two questions: 1) Are physiological gradients of proliferation and steroidogenesis present within the granulosa layer of a preovulatory follicle? 2) Does the GDR secrete factors that affect granulosa cell proliferation and/or steroidogenesis? Incorporation of 3H-thymidine was used as a measure of proliferation, and production of progesterone was used as a measure of steroidogenesis. In the first experiment, 8-mm-diameter sections were obtained from three morphologically distinct regions of the granulosa monolayer: 1) the GDR, 2) granulosa cells distal to the GDR (distal granulosa) and 3) granulosa cells midway between the GDR and distal granulosa cells (proximal granulosa cells). The GDR incorporated the most 3H-thymidine and produced the least progesterone. Distal granulosa cells incorporated the least 3H-thymidine and produced the most progesterone. Proximal granulosa cells incorporated an intermediate amount of 3H-thymidine and produced an intermediate amount of progesterone. To answer the second question, conditioned medium was prepared from GDRs and distal granulosa cells (control) obtained from the F1 (largest preovulatory follicle) and F3 (the third-largest preovulatory follicle) follicles. Sections (8-mm in diameter) of the distal granulosa layer (F3 for 3H-thymidine incorporation, F1 for progesterone production) were incubated in GDR-conditioned medium or granulosa cell-conditioned medium to determine whether factors secreted into the medium by the GDR and distal granulosa cells affect granulosa cell proliferation and/or steroidogenesis. Certain samples of GDR-conditioned medium and granulosa cell-conditioned medium were boiled, protease-treated or charcoal-stripped. F3 and F1 GDRs produced heat- and protease-sensitive factors that promoted proliferation and inhibited progesterone production by granulosa cells. These data indicate that diametrically opposed gradients of proliferation and steroidogenesis are present within the granulosa layer of an individual preovulatory follicle. Furthermore, the GDR produces proliferation-stimulating and steroidogenesis-inhibiting factors that may act in an autocrine or paracrine manner to influence proliferation and steroidogenesis in granulosa cells.

Abstract: The stages of follicular maturation of a preovulatory follicle in the hen can be divided into an extended proliferative phase (prior to LH surge) and a brief ovulatory phase (after LH surge). Previous studies suggest involvement of plasminogen activator (PA) in both the proliferative and ovulatory phases. The goals of the present study were 1) to determine whether PA production by granulosa and theca is dependent upon interaction of the two cell layers; 2) to investigate whether the structural difference of the stigma (site of follicular rupture) and nonstigma regions of the theca layer affect PA production; 3) to determine whether there is a change in the ability of the granulosa layer and stigma or nonstigma regions of the theca layer to produce PA as the follicle makes the transition from the proliferative to the ovulatory phase; and 4) to characterize the type(s) of PA produced by the hen follicle. Equal proportions of the granulosa layer (10-mm diameter) and stigma or nonstigma regions of the theca layer (10 mg) obtained from the F1 preovulatory follicle 8 h before ovulation (before LH surge) or 2 h before ovulation (after LH surge) were incubated alone or in combination for 24 h. PA was measured in tissue homogenates and medium by use of the chromogenic substrate S-2251. The granulosa layer or stigma or nonstigma regions of the theca layer incubated alone and obtained either 8 h or 2 h before ovulation had very low amounts of PA activity in the medium and tissue homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Previous work has suggested that the germinal disc and the overlying layer of granulosa cells located near the germinal disc, collectively referred to as the germinal disc region (GDR), is a primary "growth center," regulating granulosa cell proliferation within follicles in the rapid growth phase. This study was designed to determine whether the presence of a viable GDR is required for the completion of follicular maturation and ovulation of the avian follicle. Twelve or 24 h before the expected time of ovulation, the GDR of the largest preovulatory follicle (F1) was destroyed by applying solid CO2 (2 x 2 mm) for 20 sec. A region of the follicle wall equal in size to but opposite the GDR was destroyed in control birds. Blood samples were taken 24 h and 6 h before and at the time of ovulation. The effects of GDR destruction on plasma progesterone (P4), LH, and ovulation were determined. Destruction of the F1 GDR 24 h before ovulation resulted in an absence of the preovulatory rise in plasma P4, attenuation of the LH surge, blocked ovulation, and atresia of the F1 follicle. Controls displayed typical preovulatory profiles of plasma P4 and LH and ovulated at the expected time. In contrast to the data collected after destruction of the GDR 24 h before ovulation, destruction of the GDR 12 h before ovulation did not disrupt ovulation. Furthermore, destruction of the GDR 24 h before ovulation had no effect on basal or LH-stimulated P4 production by granulosa cell cultures prepared 12 h after destruction of the GDR.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Progesterone conjugated to bovine serum albumin (BSA) was used as a probe to study sex differences and the effects of hormonal status on binding of progesterone to crude synaptosomal membrane preparations (P2) derived from the mediobasal hypothalamic-anterior hypothalamic-preoptic area or the corpus striatum. Binding of 125I-labeled BSA linked to progesterone at the 11 position of the steroid (P-11-BSA) was decreased by competition with unlabeled P-11-BSA or P-3-BSA (in which progesterone is bound to BSA at the 3 position). P-3-BSA displayed higher affinity than P-11-BSA. Hypothalamic and striatal preparations from adult females show high specific binding (60-80%) to the progesterone-BSA conjugate. Specific binding was reduced more than 80% 14 days after ovariectomy. Estrogen treatment (10 micrograms per rat for 4 days) of 14-day ovariectomized rats restored specific binding to levels equivalent to intact females. In contrast, adult males displayed drastically reduced or no specific binding in either tissue. No specific binding was detected after orchidectomy. Estrogen treatment of orchidectomized animals induced specific binding sites similar to those in intact females. Additionally, an affinity probe was developed by linking primary amines on the P-3-BSA conjugate to agarose activated aldehydes in an AminoLink column. A digitoxin-solubilized fraction from female rat P2 cerebellum preparations yielded a single major band after affinity purification with an estimated molecular mass of 40-50 kDa in an SDS/PAGE system after silver stain. These results show a reversible sex difference in the specific binding of progesterone to synaptosomal membrane sites in the central nervous system of male and female rats which is dependent on estrogen.