Pulsatile gonadotropin-releasing hormone stimulation of gonadotropin subunit transcription in rat pituitaries: evidence for the involvement of Jun N-terminal kinase but not p38.

We investigated whether Jun N-terminal kinase (JNK) and p38 mediate gonadotropin subunit transcriptional responses to pulsatile GnRH in normal rat pituitaries. A single pulse of GnRH or vehicle was given to female rats in vivo, pituitaries collected, and phosphorylated JNK and p38 measured. GnRH stimulated an increase in JNK phosphorylation within 5 min, which peaked 15 min after GnRH (3-fold). GnRH also increased p38 phosphorylation 2.3-fold 15 min after stimulus. Rat pituitary cells were given 60-min pulses of GnRH or media plus the JNK inhibitor SP600125 (SP, 20 microM), p38 inhibitor SB203580 (20 microM), or vehicle. In vehicle-treated groups, GnRH pulses increased LHbeta and FSHbeta primary transcript (PT) levels 3-fold. SP suppressed both basal and GnRH-induced increases in FSHbeta PT by half, but the magnitude of responses to GnRH was unchanged. In contrast, SP had no effect on basal LHbeta PT but suppressed the stimulatory response to GnRH. SB203580 had no effect on the actions of GnRH on either LH or FSHbeta PTs. Lbeta-T2 cells were transfected with dominant/negative expression vectors for MAPK kinase (MKK)-4 and/or MKK-7 plus a rat LHbeta promoter-luciferase construct. GnRH stimulated a 50-fold increase in LHbeta promoter activity, and the combination of MKK-4 and -7 dominant/negatives suppressed the response by 80%. Thus, JNK (but not p38) regulates both LHbeta and FSHbeta transcription in a differential manner. For LHbeta, JNK is essential in mediating responses to pulsatile GnRH. JNK also regulates FSHbeta transcription (i.e. maintaining basal expression) but does not play a role in responses to GnRH.

A test of founder effect speciation using multiple loci in the auklets (Aethia spp.).

Whether speciation results more frequently from the genetic consequences of founder events or from gradual genetic divergence of large populations is a matter of debate. In this study, multiple analyses were applied to data from three loci (cytochrome b, alpha-enolase intron VIII, and MHC class II B) to test for founder effects associated with speciation in Aethia (Aves: Alcidae), a genus of seabirds thought to have undergone a rapid founder-induced radiation. Effective population sizes (N(e)) were derived from estimators of based on allelic diversity and the coalescent and from data on trans-species polymorphism. Results indicated that N(e) has been on the order of 10(5)-10(6) individuals throughout the evolutionary histories of least and crested auklets (A. pusilla and A. cristatella, respectively) and that N(e) of the ancestral species was at least 16,000 individuals. Computer simulations of MHC evolution indicated that a single-generation bottleneck at speciation could not have involved <85 individuals for each species. More moderate simulation scenarios indicated that population size could not have dropped below 2000 individuals at the time of species founding. Demographic history appears to have been stable for the auklets throughout the past several million years, and a founder effect associated with their speciation is unlikely.

Testosterone stimulates follicle-stimulating hormone beta transcription via activation of extracellular signal-regulated kinase: evidence in rat pituitary cells.

This study investigated whether estradiol (E2) or testosterone (T) activate extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent kinase II (Ca/CaMK II), as indicated by enzyme phosphorylation in rat pituitaries. In vivo studies used adult female rats given E2, T, or empty silastic capsules (vehicle controls). Twenty-four hours later, the rats were given a single pulse of GnRH (300 ng) or BSA-saline (to controls) and killed 5 min later. GnRH stimulated a two- to three-fold rise in activated Ca/CaMK II, and E2 and T had no effect on Ca/CaMK II activation. In contrast, both GnRH and T stimulated threefold increases in ERK activity, with additive effects seen following the combination of GnRH+T. E2 had no effect on ERK activity. In alpha T3 clonal gonadotrope cells, dihydrotestosterone did not activate ERK alone but enhanced and prolonged the ERK responses to GnRH, demonstrating direct effects on the gonadotrope. Thus, the ERK response to GnRH plus androgen was enhanced in both rat pituitary and alpha T3 cells. In vitro studies with cultured rat pituitary cells examined the effect of GnRH+/-T in the presence of the mitogen-activated protein (MAP) kinase kinase inhibitor, PD-098059 (PD). Results showed that PD suppressed ERK activational and FSH beta transcriptional responses to T. These findings suggest that one site of T regulation of FSH beta transcription is through the selective stimulation of the ERK pathway.

POLYTOMIES AND THE POWER OF PHYLOGENETIC INFERENCE.

Although phylogenetic hypotheses can provide insights into mechanisms of evolution, their utility is limited by our inability to differentiate simultaneous speciation events (hard polytomies) from rapid cladogenesis (soft polytomies). In the present paper, we tested the potential for statistical power analysis to differentiate between hard and soft polytomies in molecular phytogenies. Classical power analysis typically is used a priori to determine the sample size required to detect a particular effect size at a particular level of significance (a) with a certain power (1 - ß). A posteriori, power analysis is used to infer whether failure to reject a null hypothesis results from lack of an effect or from insufficient data (i.e., low power). We adapted this approach to molecular data to infer whether polytomies result from simultaneous branching events or from insufficient sequence information. We then used this approach to determine the amount of sequence data (sample size) required to detect a positive branch length (effect size). A worked example is provided based on the auklets (Charadriiformes: Alcidae), a group of seabirds among which relationships are represented by a polytomy, despite analyses of over 3000 bp of sequence data. We demonstrate the calculation of effect sizes and sample sizes from sequence data using a normal curve test for difference of a proportion from an expected value and a t-test for a difference of a mean from an expected value. Power analyses indicated that the data for the auklets should be sufficient to differentiate speciation events that occurred at least 100,000 yr apart (the duration of the shortest glacial and interglacial events of the Pleistocene), 2.6 million years ago.

Intron variation in marbled murrelets detected using analyses of single-stranded conformational polymorphisms.

Combination of the targeted amplification of nuclear introns and the analysis of single-stranded conformational polymorphisms has the potential to provide an inexpensive, rapid, versatile and sensitive genetic assay for evolutionary studies and conservation. We are developing primers and protocols to analyse nuclear introns in vertebrates, and are testing them in a population genetic study of marbled murrelets Brachyramphus marmoratus. Here we present protocols and results for introns for aldolase B, alpha-enolase, glyceraldehyde-3-phosphate dehydrogenase and lamin A. Results suggest that this approach presents a potentially powerful method for detecting genetic variation within and among local populations and species of animals: (i) a variety of genes can be surveyed, including genes of special interest such as those involved in disease resistance; (ii) assays are rapid and relatively inexpensive; (iii) large numbers of genes can be assayed, enabling accurate estimation of variation in the total genome; (iv) almost any mutation can be detected in the genes amplified; (v) the exact nature of variation can be investigated by sequence analysis if desired; (vi) statistical methods previously developed for proteins and/or sequence data can be used; (vii) protocols can be easily transferred to other species and other laboratories; and (viii) assays can be performed on old or degraded samples, blood or museum skins, so that animals need not be killed. Results of analyses for murrelets support earlier evidence that North American and Asiatic subspecies represent reproductively isolated species, and that genetic differences exist among murrelets from different sites within North America.