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Activin, a member of the transforming growth factor-beta superfamily, is an important modulator of follicle-stimulating hormone synthesis and secretion in the pituitary and plays autocrine/paracrine roles in the regulation of ovarian follicle development. From a microarray study on mouse ovarian granulosa cells, we discovered that the estrogen receptor beta (ERbeta) is inducible by activin. We previously demonstrated that estrogen suppresses activin gene expression, suggesting a feedback relationship between these two follicle-regulating hormones. The purpose of this study was to investigate fully activin A regulation of ER expression. Real time reverse transcription-PCR assays on cultured granulosa cells showed that both ERalpha and ERbeta mRNAs were induced by activin A at 4, 12, and 24 h in a dose-responsive manner. Western blots confirmed an increase in their protein levels. Consistent with increased ERalpha and ERbeta expression, activin A stimulated estradiol-induced estrogen response element promoter activity. Activin A stimulation of ER expression was a direct effect at the level of gene transcription, as it was not abolished by cycloheximide but was abolished by actinomycin D, and in transfected granulosa cells activin A stimulated ERalpha promoter activity. To investigate the effect of activin in vivo and, thus, its biological significance, we examined ER expression in inhibin transgenic mice that have decreased activin expression and discovered that these mice had decreased ERalpha and ERbeta expression in the ovary. We also found that ER mRNA levels were decreased in Müllerian inhibiting substance promoter (MIS)-Smad2 dominant negative mice that have impaired activin signaling through Smad2, and small interfering RNAs targeting Smad2 or Smad3 suppressed ERalpha promoter activation, suggesting that Smad2 and Smad3 are involved in regulating ER levels. Therefore, this study reveals an important role for activin in inducing the expression of ERs in the mouse ovary and suggests important interplay between activin and estrogen signaling.

The mechanisms and physiology of reproductive function have fascinated scientists throughout time. Recent cellular and molecular level structural studies have provided unprecedented insights into reproductive systems and signaling networks. This ‘cutting edge’ editorial provides a recent example in each of these areas, namely, the anatomical integrity of the follicle, the molecular structure of activin with its binding partners and the molecular regulation of inhibin. These three examples of structure informing function help explain reproductive health and may provide solutions to reproductive disease.

Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (kA) was modeled for the two groups of animals, and consistent with the empirical data, the kA for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4–6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.

The MtT/S somatotroph cell line should be a growth hormone-releasing hormone (GHRH)-responsive model system for the study of physiological control of growth hormone (GH) transcription because GH secretion from these cells is stimulated by GHRH. To examine the GH transcriptional activity of these cells, endogenous GH mRNA levels were measured using a ribonuclease protection assay following treatment under a variety of hormonal conditions. While omission of serum led to reduction of GH mRNA to 22% of control levels by 2 days and to 8% by 5 days (P0.05 for both), GH mRNA levels were maintained at control values in serum-free medium containing 5 nM dexamethasone and 30 pM triiodothyronine (TDM). However, the addition of 10 nM GHRH under any treatment condition did not significantly alter GH mRNA levels. Characterization of the MtT/S cells showed that GHRH-receptor (GHRH-R) mRNA was detectable by reverse transcription-polymerase chain reaction (RT-PCR) amplification. Measurement of extracellular cAMP showed that the MtT/S cells have basal levels ofor =20 nmol/10(6) cells per h in both serum-containing and serum-free media, and that GHRH had no effect on cAMP levels, suggesting constitutive activation. To rule out the possibility of autocrine stimulation by GHRH produced endogenously, GHRH mRNA was not detectable in MtT/S cells using RT-PCR amplification. The stimulatory G-protein alpha subunit, mutations of which are known to activate adenylate cyclase constitutively in acromegaly, was sequenced but found not to differ from normal pituitary in the regions most commonly mutated. Finally, treatment with 10 microM forskolin, to directly activate adenylate cyclase, increased GH mRNA to 140% of controls in TDM, and to 163% in serum-free medium after 2 days, and to 166% in TDM-treated cells and 174% in serum-free culture after 5 days (all P0.05). Taken together, these data indicate that although MtT/S cells express the GHRH-R, GHRH cannot stimulate adenylate cyclase to increase GH transcription due to constitutive elevation of cAMP levels, by a means that may be similar to that in cases of acromegaly not caused by oncogenic gsp mutations.

Aging is associated with a variety of alterations in circadian rhythms, including changes in the response to environmental stimuli. The underlying causes for these age-related changes in the circadian system remain unknown. Recent studies have demonstrated that light induces the expression of Fos and phosphorylation of the cyclic-AMP response element-binding protein in the rodent suprachiasmatic nuclei, the location of a master circadian pacemaker in mammals, suggesting that these transcription factors may mediate the effects of light on the circadian clock. The purpose of this study was to determine the effects of aging upon light-induced phase-shifting of circadian locomotor activity rhythms, Fos protein expression and cyclic-AMP response element-binding protein phosphorylation in the suprachiasmatic nuclei. Young (three to four months) and old (18-22 months) male golden hamsters free-running in constant darkness were exposed to 5-min monochromatic light pulses of different irradiance levels, at circadian time 19, after which either steady-state phase shifts of locomotor activity rhythms were measured, or else immunocytochemistry for Fos or for phospho-cyclic-AMP response element-binding protein was performed. Old hamsters were approximately 20 times less sensitive to the phase-shifting effects of light on the activity rhythm, and the photic irradiance threshold for Fos-like immunoreactivity induction in the suprachiasmatic nuclei was elevated when compared to young animals. Aging was also associated with a deficit in cyclic-AMP response element-binding protein phosphorylation by light. These data indicate that there are dramatic changes in light-activated molecular responses in the suprachiasmatic nuclei of old hamsters, and suggest that these molecular changes may underlie age-related changes in the effects of light on the circadian clock system.

Inhibins and activins exhibit a broad spectrum of biologic activities, many of which affect the reproductive axis. Within the ovary and testis, the synthesis of the inhibin and activin alpha and beta subunits is regulated by circulating hormones, such as the pituitary gonadotropins, and by diverse paracrine factors. Considerable progress has been made in establishing patterns of inhibin and activin subunit gene expression in the gonads and in cultured gonadal cells. Analysis of the inhibin and activin subunit genes is now providing insight into the signaling pathways and molecular mechanisms by which inhibin and activin subunit gene expression is modulated in the ovary and testis. Genetic manipulation of the inhibin and activin subunit genes promises additional revelations on the biologic functions of these intriguing hormones.

The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.

Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter.