The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.

We have cloned and sequenced a cDNA (RPR4) encoding a new member of the secretin/calcitonin/parathyroid hormone (PTH) receptor family. RPR4 was identified by PCR of rat pituitary cDNA, and a full-length clone was isolated from a rat olfactory bulb cDNA library. When RPR4 was functionally expressed in COS 7 cells, cyclic adenosine monophosphate (cAMP) production was stimulated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptides (PACAP-38 and PACAP-27) and helodermin, with equal potency. Peptide histidine isoleucine (PHI) and rat growth hormone releasing hormone (rGHRH) also stimulated cAMP production at lower potency. This suggests that RPR4 encodes a novel VIP receptor which we have designated the VIP 2 receptor. In situ hybridisation showed that mRNA for this receptor was present mainly in the thalamus, hippocampus and in the suprachiasmatic nucleus.Vasoactive intestinal peptide (VIP); G protein-linked receptor; Rat pituitary; Rat olfactory bulb; cDNA cloning

We have shown previously that the reactive oxygen species generated by washed human ejaculates originate from cells which can be isolated in the low density region of discontinuous Percoll gradients. In this study we have used a simplified two-step (40/80%) Percoll gradient to separate human ejaculates (n = 109) into two populations of spermatozoa, exhibiting either a high or a low capacity for reactive oxygen species generation. We have then examined the relationships between this activity and other properties of the isolated fractions, with particular emphasis on the presence of leucocytes, which we have quantified using a monoclonal antibody directed against the common leucocyte antigen. The low-density cells recovered from the 40%/80% interface of the Percoll gradients, differed from the high-density fraction in exhibiting significantly reduced sperm motility, poorer sperm morphology, and a considerably enhanced capacity for reactive oxygen species production (P less than 0.001). In six cases the elevated levels of reactive oxygen species generation were associated with leucocyte concentrations in excess of 1 x 10(6) per 10(7) sperm, suggesting that leucocytes enter the seminal compartment in an activated, oxygen-radical generating, state. However, in the majority of cases exhibiting high levels of reactive oxygen species production, leucocyte numbers were low or absent and the semen profiles were unremarkable, except that seminal sperm concentrations tended to be low. These results suggest that the oxidative stress responsible for defective sperm function involves reactive oxygen species originating from two sources; the sperm and infiltrating leucocytes.

The X-linked gene filamin A (Flna) encodes a widely expressed actin-binding protein that crosslinks actin into orthogonal networks and interacts with a variety of other proteins including membrane proteins, integrins, transmembrane receptor complexes and second messengers, thus forming an important intracellular signalling scaffold. Heterozygous loss of function of human FLNA causes periventricular nodular heterotopia in females and is generally lethal (cause unknown) in hemizygous males. Missense FLNA mutations underlie a spectrum of disorders affecting both sexes that feature skeletal dysplasia accompanied by a variety of other abnormalities. Dilp2 is an X-linked male-lethal mouse mutation that was induced by N-ethyl-N-nitrosourea. We report here that Dilp2 is caused by a T-to-A transversion that converts a tyrosine codon to a stop codon in the Flna gene (Y2388X), leading to absence of the Flna protein and male lethality because of incomplete septation of the outflow tract of the heart, which produces common arterial trunk. A proportion of both male and female mutant mice have other cardiac defects including ventricular septal defect. In addition, mutant males have midline fusion defects manifesting as sternum and palate abnormalities. Carrier females exhibit milder sternum and palate defects and misshapen pupils. These results define crucial roles for Flna in development, demonstrate that X-linked male lethal mutations can be recovered from ENU mutagenesis screens and suggest possible explanations for lethality of human males hemizygous for null alleles of FLNA.