Binding of LH and FSH to porcine granulosa cells during follicular maturation.

The uptake of 125I-labelled LH by equal numbers of granulosa cells from small, medium or large follicles was greater by cells from large follicles. In contrast, granulosa cells obtained from small follicles bound much more 125I-labelled FSH per cell than did cells obtained from medium and large follicles. Competition studies with unlabelled hormones indicated that porcine granulosa cells have specific receptors for LH and FSH. The addition of diethylstilboestrol enhanced the binding of 125I-labelled LH and inhibited the binding of 125I-labelled FSH to granulosa cells harvested from small and medium-sized follicles, but had no effect on those from large follicles.     

Riding tandem: circadian clocks and the cell cycle

The circadian clock, which governs metabolic and physiological rhythms in diverse organisms, shares common features with the cell cycle. Yet, these two oscillatory systems seem to be fully independent of each other. Recent studies now reveal that some essential regulatory elements are common to both the cell cycle and circadian clock.     

Period responses to Zeitgeber signals stabilize circadian clocks during entrainment

Circadian clocks with characteristic period (τ) can be entrained to light/dark (LD) cycles by means of (i) phase shifts which are due to D/L “dawn” and/or L/D “dusk” transitions, (ii) period changes associated with long-term light exposure, or (iii) by combinations of the above possibilities. Based on stability analysis of a model circadian clock it was predicted that nocturnal burrowing mammals would benefit less from period responses than their diurnal counterparts. The model further predicted that maximal stability of circadian clock is reached when the clock slightly changes both its phase and period in response to light stimuli. Analyses of empirical phase response curve (PRC) and period response curve (τRC) of some diurnal and nocturnal mammals revealed that PRCs of both diurnal and nocturnal mammals have similar waveform while τRCs of nocturnal mammals are of smaller amplitude than those of diurnal mammals. The shape of the τRC also changes with age and with increasing strength of light stimuli. During erratic fluctuations in light intensity under different weather conditions, the stability of phase of entrainment of circadian clocks appears to be achieved by an interplay between phase and period responses and the strength of light stimuli.     

Cellular clocks: coupled circadian and cell division cycles

Gating of cell division by the circadian clock is well known, yet its mechanism is little understood. Genetically tractable model systems have led to new hypotheses and questions concerning the coupling of these two cellular cycles.     

Local regulation of granulosa cell maturation

Fluid from small antral follicles inhibits several functions of porcine granulosa cells from 3-10-mm follicles in vitro, whereas fluid from large follicles stimulates cells from small follicles. Local factors may be needed in vivo to enable granulosa cells to fully respond to gonadotrophins. Only those follicles containing local stimulators may develop while those containing inhibitors may become arrested in development or become atretic. We have compared the actions of GnRH analogs and chondroitin sulfate (CS) on porcine granulosa cell steroidogenesis with actions of follicular fluids. GnRH agonist mimicked follicular fluid inhibition of progesterone secretion but GnRH antagonist did not antagonize follicular fluid's inhibitory actions. GnRH antagonist mimicked follicular fluid enhancement of basal and LH-stimulated progesterone secretion, but did not mimic follicular fluid enhancement of FSH action or stimulation of estrogen secretion. GnRH agonist blocked the enhancement of LH-stimulated progesterone secretion by both GnRH antagonist and stimulatory follicular fluid. CS inhibited basal and LH-stimulated progesterone secretion but did not inhibit pregnenolone utilization, aromatase activity or estrogen secretion. GnRH-like molecules and CS may be partially responsible for follicular fluid actions on granulosa cells. The actions of other molecules are needed to explain the total effects of follicular fluids on granulosa cells.     

Effect of adiponectin on bovine granulosa cell steroidogenesis,oocyte maturation and embryo development

Background  

Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.     

Microfilaments and FSH stimulation of rat granulosa cell steroidogenesis in vitro

The secretion of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5?IU/animal; T3 group-100?μg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.     

The circadian clocks of plants and cyanobacteria

Classical research on the circadian rhythms of plants helped to demonstrate that all living organisms utilize circadian clocks to adapt their day–night cycles and that the clock is the basis for photoperiodic time measurements. Molecular models for the circadian oscillator have now been elucidated in Drosophila, Neurospora, mice and cyanobacteria. All share a similar feedback structure, but key proteins in each of the oscillators are different. A plant clock model has yet to be proposed, but clock mutants of Arabidopsis are expected to reveal key proteins in the mechanism. Here we discuss how a self-sustained oscillation is established in eukaryotic and prokaryotic models, and the polyphyletic evolution of these clock systems.     

Role of ovarian theca and granulosa cell interaction in hormone productionand cell growth during the bovine follicular maturation process

We have investigated the possible role of theca and granulosa cell interaction in the control of the hormone-producing activity and growth of granulosa and theca cells during bovine ovarian follicular development, using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. When follicular cells were isolated from small follicles (3-5 mm), theca cells reduced estradiol, progesterone, and inhibin production by granulosa cells to 14 +/- 5%, 64 +/- 6%, and 27 +/- 4%, respectively, of the production by granulosa cells cultured alone. On the other hand, when the cells were isolated from large follicles (15-18 mm), theca cells increased these levels to 253 +/- 34%, 156 +/- 24%, and 287 +/- 45%, respectively. Theca cells did not affect the growth of granulosa cells. Androstenedione production by theca cells was augmented by granulosa cells to 861 +/- 190% (in small follicles) and 1298 +/- 414% (in large follicles), respectively. The growth of theca cells was also augmented by granulosa cells (small follicle, 210 +/- 43%, and large follicle, 194 +/- 24%, respectively). These results indicate that theca cells secrete factor(s) inhibiting the differentiation of immature while promoting that of matured granulosa cells; they also suggest that granulosa cells secrete factor(s) promoting both the differentiation and growth of theca cells throughout the follicular maturation process.     

Timing to perfection: the biology of central and peripheral circadian clocks

The mammalian circadian system, which is comprised of multiple cellular clocks located in the organs and tissues, orchestrates their regulation in a hierarchical manner throughout the 24?hr of the day. At the top of the hierarchy are the suprachiasmatic nuclei, which synchronize subordinate organ and tissue clocks using electrical, endocrine, and metabolic signaling pathways that impact the molecular mechanisms of cellular clocks. The interplay between the central neural and peripheral tissue clocks is not fully understood and remains a major challenge in determining how neurological and metabolic homeostasis is achieved across the sleep-wake cycle. Disturbances in the communication between the plethora of body clocks can desynchronize the circadian system, which is believed to contribute to the development of diseases such as obesity and neuropsychiatric disorders. This review will highlight the relationship between clocks and metabolism, and describe how cues such as light, food, and reward mediate entrainment of the circadian system.     

Regional pattern of cell maturation and progesterone biosynthesis in the avian granulosa cell layer

The objective of the present study was to compare the structural and functional features of cells derived from histologically different regions of the granulosa cell layer of hen preovulatory follicles. Granulosa cells were isolated from a 0.8-1.5-cm diameter region of the granulosa layer overlying the germinal disc (GD) or from the remainder of the granulosa layer peripheral to the disc region (GP). In the first study, the isolated cells were prepared from each region of the five largest preovulatory follicles; fixed; stained with fluorescent dyes for DNA, total protein, and RNA; and analyzed by use of multiparameter flow cytometry. A greater percentage of cells from the GD region than from the GP region were in proliferative (S and G2/M) stages of the cell cycle in the four largest follicles. In addition, GD cells had lower relative protein content than GP cells in the two largest follicles. In the second study, progesterone biosynthesis in response to treatment with luteinizing hormone (LH) or forskolin was examined in granulosa cells from the GD and the GP regions of the largest preovulatory follicles. GP cells had greater responsiveness to the treatments than GD cells. In addition, conversion of 25-hydroxy-cholesterol to progesterone was greater in GP cells than in GD cells. There were no differences in cyclic adenosine 3',5'-monophosphate (cAMP) production by GD and GP cells in response to LH or forskolin or in the ability of cells from each region to convert pregnenolone substrate to progesterone via 3 beta-hydroxysteroid dehydrogenase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone-induced desensitization of cultured rat granulosa cells to FSH

Monolayers of granulosa cells (GC) derived from immature hypophysectomized diethylstilbestrol-treated rats became refractory in terms of FSH-stimulable cyclic AMP production following exposure to the homologous hormone. In the presence of ovine FSH (5 μg/ml), maximal refractoriness was attained after 4 h of incubation. Upon removal of the FSH from the medium, the cells regained their full responsiveness within 24 h. The extent of desensitization was dependent upon the dose of FSH, and could not be overcome by increasing the dose of the hormone during the challenge period. Exposure of GC monolayers for 2–4 h to the protein synthesis inhibitors actinomycin D (8 μg/ml) and cycloheximide (5 μg/ml) on their own enhanced FSH-stimulable cyclic AMP production. When added together with FSH, the inhibitors did not prevent the process of desensitization to the hormone. The results suggest that the initial phases of FSH-induced desensitization do not require de novo protein synthesis.     

Retinal circadian clocks and control of retinal physiology

Retinas of all classes of vertebrates contain endogenous circadian clocks that control many aspects of retinal physiology, including retinal sensitivity to light, neurohormone synthesis, and cellular events such as rod disk shedding, intracellular signaling pathways, and gene expression. The vertebrate retina is an example of a "peripheral" oscillator that is particularly amenable to study because this tissue is well characterized, the relationships between the various cell types are extensively studied, and many local clock-controlled rhythms are known. Although the existence of a photoreceptor clock is well established in several species, emerging data are consistent with multiple or dual oscillators within the retina that interact to control local physiology. A prominent example is the antiphasic regulation of melaton in and dopamine in photoreceptors and inner retina, respectively. This review focuses on the similarities and differences in the molecular mechanisms of the retinal versus the SCN oscillators, as well as on the expression of core components of the circadian clockwork in retina. Finally, the interactions between the retinal clock(s) and the master clock in the SCN are examined.     

Environmental stimulus perception and control of circadian clocks

Circadian rhythms are regulated by clocks located in specific structures of the central nervous system, such as the suprachiasmatic nucleus (SCN) in mammals, and by peripheral oscillators present in various other tissues. Recent discoveries have elucidated the control of central and peripheral clocks by environmental signals. The major synchroniser in animals is light. In mammals, a subset of retinal ganglion cells receive light signals that are transmitted to the SCN via the retinohypothalamic tract. Photoreception is probably elicited by a novel opsin, melanopsin, although cryptochromes may also play a role. These signals feed directly to the SCN master clock, which then provides timing cues to peripheral clocks. In contrast to mammals, peripheral tissues in the fly and in the fish are directly photoreceptive. However, alternative routes exist. Some peripheral clocks in mammals can be specifically entrained in an SCN-independent manner by restricting food during the light period.     

A blend of two circadian clocks, seasoned to perfection

The daily activity of the fruit fly Drosophila is controlled by both a "morning" and an "evening" circadian clock. In this issue Stoleru et al. (2007) demonstrate that day length determines which clock dominates the neural circuitry governing circadian behavior. Thus, these findings suggest a mechanism by which the system for circadian timing adapts to changes in the seasons to impose appropriate rhythms of daily activity.     

Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.