Neuroendocrine regulation of reproduction in Atlantic cod (Gadus morhua): Evidence of Eya3 as an integrator of photoperiodic cues and nutritional regulation to initiate sexual maturation.

Evidence from mammals and aves alludes to a possibly conserved seasonal photoperiod induced neuroendocrine cascade which stimulates subsequent sexual maturation however our understanding of this mechanism in teleosts is lacking. Unlike all teleosts studied to date, the Atlantic cod (Gadus morhua) is a short day breeder with the reduction in day-length from the summer solstice stimulating gametogenesis. Cod specific orthologues of eya3, tshß and dio2 were identified and their expression was monitored in the brain and pituitary of cod held under either stimulated or inhibited photoperiod conditions. While no differential expression was apparent in brain dio2 & tshß and pituitary tshß, there was significant temporal variation in expression of pituitary eya3 under the SNP treatment, with expression level elevating in association with active gametogenesis. Under the LL treatment, sexual maturation was inhibited and there was a corresponding suppression of eya3 expression. In a second study the impact of size/energetic status on the initiation of sexual maturation was investigated. In the feed restricted population maturation was significantly suppressed (5% sexually mature) compared to the ab libitum fed stock (95% sexually mature) with there being a concomitant significant suppression in pituitary eya3 expression. Overall, these results suggest that pituitary eya3 has the potential to act as an integrator of both environmental and energetic regulation of sexual maturation of cod. Being the first account of eya3 induction in a short day breeding teleost, the conserved association with stimulation of reproduction and not seasonal state indicates that the upstream drivers which initiate the pathway differ among vertebrates according to their breeding strategies, but the pathway itself and its role in the reproductive cascade appears to be conserved across the vertebrate clade.

Effect of size on spawning time in the lesser sandeel Ammodytes marinus.

Ovarian development was examined in relation to size and temperature in late pre-spawning Ammodytes marinus over 5 years. Oocyte diameter was positively related to length indicating that larger females spawned earlier. Age and temperature, whilst accounting for the effect of length, were not found to affect oocyte development, although the thermal range examined was only 1·3° C.

Analysing migrations of Atlantic cod Gadus morhua in the north-east Atlantic Ocean: then, now and the future.

The application of data storage tags bears the potential for a quantum leap in the research on fish migrations, because not only first-capture and recapture positions are known, but at least theoretically, the migration path during the period at large can be reconstructed. Position, however, cannot be measured directly but has to be estimated using the available data on light, temperature, pressure and salinity. The reconstructed locations based on advanced estimation techniques have been termed geolocations. Examples are discussed which illustrate the applicability of geolocations in individual path descriptions, separation of reproductively isolated populations, timing and areas of spawning, tidal transport and use of protected areas. The examples are based on archival tag data from the North Sea, the Baltic Sea, the Barents Sea and Faroese and Icelandic Waters. Besides presenting the state-of-the-art geolocations for cod Gadus morhua in the north-east Atlantic Ocean, the major aim of this review is to raise awareness of gaps in knowledge and to identify ideas for new research.

Effect of simulated stages of the canine oestrous cycle on Escherichia coli binding to canine endometrium.

Pyometra, a prevalent infectious uterine disease that affects intact middle-aged bitches, is typically associated with Escherichia coli. Our hypotheses were (i) that bacterial adhesion to canine endometrium differs between different stages of the oestrous cycle and (ii) that the adhesin FimH facilitates this adhesion. Twelve post-pubertal, ovariectomized greyhound bitches were treated with exogenous hormones to simulate different stages of the oestrous cycle. Tissue samples from each uterus were incubated with a pathogenic E. coli strain carrying the fimH gene, but no other adhesin genes (P4-wt)--or an E. coli strain in which fimH was insertionally inactivated (P4-∆fimH::kan)--or with phosphate-buffered saline as a negative control. After washing, tissue samples were homogenized for quantification of adherent bacteria. The differences in binding to canine endometrium at different stages of the oestrous cycle were not significant. However, the mean difference in binding of the P4-wt and the P4-∆fimH::kan across all stages of the simulated oestrous cycle was significant (p < 0.001 by paired t-test on geometric means). Individual differences in numbers of P4-wt bacteria bound between dogs might suggest genetic variations or epigenetic differences in FimH receptor expression by the endometrium, unrelated to the stage of the oestrous cycle.

Timing of the maturation transition in haddock Melanogrammus aeglefinus.

The timing of maturation in haddock Melanogrammus aeglefinus was examined using changes in gonad development, follicle stimulating hormone ß (FSH-ß) transcript expression profile, growth and condition of 1 year old females held under a common environment between the summer and winter solstices. The circumnuclear ring, cortical alveolus and vitellogenic oocyte stages were first observed in August, October and November, respectively. FSH-ß transcript levels did not change significantly until September but increased markedly thereafter in maturing fish. A combined analysis of the mean oocyte diameter of the leading cohort, histological staging and FSH-ß transcript profile provided evidence of a commitment to maturation by October or November. Contrary to that previously proposed for gadoid species, histological analysis of field-caught immature M. aeglefinus during the spawning season indicated that cortical alveolar, rather than circumnuclear ring, stage oocytes provided definitive evidence of maturation. A decrease in relative liver size following the summer solstice suggested a possible link between energy status and maturation.

Electronic tags reveal behaviour of captured and discarded fish.

Internally implanted data storage tags (DST) recording hydrostatic pressure (depth) and temperature in Atlantic cod Gadus morhua were used to track the behaviour of nine individuals during fishing operations. Gadus morhua showed identifiable and characteristic behavioural responses to different capture processes. In a unique observation, one fish survived being discarded and upon return to the seabed exhibited a cessation of activity comparable to observations on stressed fish under laboratory conditions.

Understanding temperature effects on recruitment in the context of trophic mismatch.

Understanding how temperature affects the relative phenology of predators and prey is necessary to predict climate change impacts and recruitment variation. This study examines the role of temperature in the phenology of a key forage fish, the lesser sandeel (Ammodytes marinus, Raitt) and its copepod prey. Using time-series of temperature, fish larval and copepod abundance from a Scottish coastal monitoring site, the study quantifies how thermal relationships affect the match between hatching in sandeel and egg production of its copepod prey. While sandeel hatch time was found to be related to the rate of seasonal temperature decline during the autumn and winter through effects on gonad and egg development, variation in copepod timing mostly responded to February temperature. These two temperature relationships defined the degree of trophic mismatch which in turn explained variation in local sandeel recruitment. Projected warming scenarios indicated an increasing probability of phenological decoupling and concomitant decline in sandeel recruitment. This study sheds light on the mechanisms by which future warming could increase the trophic mismatch between predator and prey, and demonstrates the need to identify the temperature-sensitive stages in predator-prey phenology for predicting future responses to climate change.

Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA.

The simian virus 40 A protein (T antigen) recognized and bound to the consensus sequence 5'-GAGGC-3' in DNA from many sources. Sequence-specific binding to single pentanucleotides in randomly chosen DNA predominated over binding to nonspecific sequences. The asymmetric orientation of protein bound to nonorigin recognition sequences also resembled that of protein bound to the origin region of simian virus 40 DNA. Sequence variations in the DNA adjacent to single pentanucleotides influenced binding affinities even though methylation interference and protection studies did not reveal specific interactions outside of pentanucleotides. Thus, potential locations of A protein bound to any DNA can be predicted although the determinants of binding affinity are not yet understood. Sequence-specific binding of A protein to cellular DNA would provide a mechanism for specific alterations of host gene expression that facilitate viral function.

The influence of exogenous progestin on the occurrence of proestrous or estrous signs, plasma concentrations of luteinizing hormone and estradiol in deslorelin (GnRH agonist) treated anestrous bitches.

UNLABELLED: The objectives of this study were to confirm: (i) whether progestin treatment suppressed GnRH agonist-induced estrus in anestrous greyhound bitches; and (ii) the site of progestin action (i.e. pituitary, ovary). All bitches received a deslorelin implant on Day 0 and blood samples were taken from -1 h to +6 h. Five bitches were treated with megestrol acetate (2 mg/kg orally once daily) from -7 d to +6 d (Group 1) and 10 bitches were untreated controls (Group 2). Proestrous or estrous signs were observed in 4 of 5 bitches in Group 1, and 4 of 10 bitches in Group 2 (P = 0.28). The plasma LH responses (area under the curve from 0 to 6h after implantation) were higher (P = 0.008) in Group 2 than in Group 1. Plasma LH responses were similar (P = 0.59) in bitches showing signs of proestrus or estrus (responders) and in non-responders. The plasma estradiol responses (calculated as for LH response) were greater in Group 1 than in Group 2 (P = 0.048), and in responders than in non-responders (P = 0.02). IN CONCLUSION: (i) progestin treatment (a) did not suppress the incidence of bitches showing deslorelin-induced proestrus or estrus, and (b) was associated with a reduced pituitary responsiveness and an increased ovarian responsiveness to deslorelin treatment; (ii) the occurrence of proestrous or estrous signs reflected increased ovarian responsiveness to induced gonadotrophin secretion and not increased pituitary responsiveness to deslorelin.

Degeneration and apoptosis of endometrial cells in the bitch.

The relationships between changes in plasma progesterone concentrations, degeneration of the luminal epithelium, the occurrence of apoptosis of endometrial cells and endometrial leucocyte populations in the bitch were determined. Mature bitches (n = 15) were euthanized and necropsied when in diestrus (Days 7-75, n = 12) or in anestrus (Days 10, 32 and 53). Degeneration of the luminal epithelium was observed in bitches in late diestrus (Days 38-75, n = 5) when plasma progesterone concentrations were decreasing and in anestrus (Days 10 and 32, n = 2) when plasma progesterone concentrations were < 0.5 ng/mL. Endometrial leucocyte populations increased after degeneration of the luminal epithelium (around Day 42 of diestrus). Apoptosis was mainly observed in the basal glandular epithelial cells and endothelial cells of blood capillaries in all except anestrous bitches. Very few apoptotic cells were found in the superficial glandular epithelial cells and stromal cells. Higher apoptotic indices were detected in the basal glandular epithelium on Days 12-42 of diestrus than at other stages. Therefore, apoptosis of glandular basal epithelial cells occurred mainly in early diestrus, degeneration of cells of the luminal epithelium occurred from mid-diestrus to early anestrus, and the increase in leucocyte numbers may have been a consequence and not a cause of luminal epithelial degeneration.

The roles of progestagen and uterine irritant in the maintenance of cystic endometrial hyperplasia in the canine uterus.

Cystic endometrial hyperplasia (CEH) was induced in the left uterine horns of 14 mature ovariectomised greyhound bitches with an intra-luminal silk suture (uterine irritant) and treatment with estradiol benzoate and megestrol acetate (to simulate stages of a normal canine estrous cycle). Right uterine horns served as suture-free controls. From Day 30 of simulated diestrus, bitches received treatments of suture removal (n = 4), progestagen withdrawal (n = 5) or both (n = 5). Necropsies were performed 3 or 9 weeks later. At 3 weeks, severe cystic endometrial hyperplasia was present in all (6/6) left horns and in no (0/6) right horns. At 9 weeks, the left horns in 5/6 of bitches subjected to progestagen-withdrawal had recovered (in varying degrees) from cystic endometrial hyperplasia, whereas no recovery was evident in the left horns of bitches (n = 2) that continued to receive progestagen. This study demonstrated that: (i) cystic endometrial hyperplasia was reversible upon withdrawal of progestagen; (ii) progestagen maintained cystic endometrial hyperplasia in the presence or absence of irritant; and (iii) persistent endometrial irritation in the absence of progestagen may not maintain cystic endometrial hyperplasia.

A model for cystic endometrial hyperplasia/pyometra complex in the bitch.

The objective of this study was to develop a reliable model for the study of the cystic endometrial hyperplasia and pyometra complex (CEH/P) in the bitch. Greyhound bitches (n = 15) were ovariectomised and allocated into three groups (Group 1, n = 5; Group 2, n = 5; Group 3, n = 10, including 5 used from Group 1). Simulated proestrus, estrus and diestrus were induced by treatment with estradiol benzoate and megestrol acetate. The duration of cervical opening during estrus was determined by the intra-vaginal infusion of radio-opaque medium and subsequent radiography of the uterus (Group 1). One milliliter of a culture of Escherichia coli (with five uro-pathogenic virulence factors as identified by PCR: pap, sfa, hlyA, cnf1 and fim) was inoculated intra-vaginally daily throughout the simulated estrus (Group 2). One milliliter of the culture (n = 6) or sterile Luria-Bertani broth (n = 4) was introduced directly into the uterus on simulated diestrus Days 8 or 12 (Group 3). Necropsies were performed 12 and 7-14 days after the inoculation (Groups 2 and 3). The cervix remained open throughout the duration of simulated estrus (5-6 days) in four out of five bitches, and for a shorter duration (3 days of a 6-day estrus period) in one bitch (Group 1). CEH/P was induced by inoculation of bacteria into the uterus (10/10 bitches) but not into the vagina (0/5 bitches), (P = 0.003). A model for the study of CEH/P has been validated.

Pulsatile administration of gonadotrophin-releasing hormone stimulates, in oestrogen-treated anaesthetized ovariectomized ewes, a surge release of LH qualitatively and quantitatively different from that induced by oestradiol in conscious ovariectomized ewes.

The nature of the gonadotrophin-releasing hormone (GnRH) stimulus of the pituitary necessary for the oestrogen-induced plasma LH surge was studied in ovariectomized ewes. The sheep were treated with oestradiol benzoate (50 micrograms i.m.) at 0 h, and the hypothalamic contribution to the LH surge was blocked by pentobarbitone anaesthesia over the time during which the surge was expected (11-31 h). Pituitary responsiveness to exogenous GnRH (100 ng) administered i.v. in a pulsatile mode (once per hour or once per 20 min) over the period 15-30 h was assessed from plasma concentrations of LH. Neither of the GnRH treatments induced patterns of LH secretion similar to those seen in conscious ovariectomized ewes given oestrogen only. Plasma LH secretion in response to hourly GnRH pulses was less (P less than 0.01) than that associated with oestrogen-induced plasma LH surges in conscious control ewes. With pulses of GnRH administered every 20 min the amount of LH released was greater (P less than 0.05) than that in oestrogen-treated conscious control ewes. In contrast to the single surge induced by oestradiol in conscious ewes, GnRH pulses given every 20 min elicited phasic patterns of LH secretion consisting of two or three distinct surges. The failure of GnRH treatment to elicit an LH surge similar to an oestrogen-induced surge could reflect inappropriate GnRH treatment regimens, and/or inadequate priming of the pituitary with GnRH after induction of anaesthesia but before GnRH treatment.

Changes in the binding characteristics of the mu, delta and kappa subtypes of the opioid receptor in the hypothalamus of the normal cyclic ewe and in the ovariectomised ewe following treatment with ovarian steroids.

The mu, delta and kappa opioid receptor subtypes were measured across the oestrous cycle of the ewe and in ovariectomised (OVX) ewes treated with oestrogen and/or progesterone. We have used a subtype-specific opioid receptor binding assay, in which [3H]diprenorphine non-preferentially labelled each receptor subtype in the presence of blocking concentrations of site-specific opioid analogues. The density and affinity of each receptor subtype was measured in the preoptic area (POA) of the hypothalamus and the mediobasal hypothalamus (MBH). Normally cycling ewes were killed during the luteal phase of the oestrous cycle and at various times after an injection of a synthetic prostaglandin (cloprostenol) to synchronise the onset of the follicular phase. OVX ewes were either untreated as controls (n = 4) or treated with oestrogen (n = 4), progesterone (n = 4) or oestrogen and progesterone combined (n = 4). The total number of opioid receptors did not alter across the oestrous cycle or with steroid hormone treatment. In the POA, the mean (+/- S.E.M.) number of delta receptors was significantly (P < 0.05) greater during the luteal phase than 24 h into the follicular phase (133 +/- 45 vs 35 +/- 8 fmol/mg protein). A significantly (P < 0.05) greater number of delta receptors was also found in the OVX progesterone-treated ewes compared with the control animals (172 +/- 9 vs 39 +/- 4 fmol/mg protein). In the MBH, the number of delta receptors was significantly (P < 0.01) greater in ewes killed 56 h after prostaglandin than luteal-phase ewes (184 +/- 40 vs 51 +/- 7 fmol/mg protein). The number of mu receptors in both the POA and the MBH was also significantly (P < 0.05) higher in the 56-h group than in the 12-h group. A similar trend was also observed in the steroid-treated animals, although differences did not reach statistical significance. The delta:mu ratio in the POA was significantly (P < 0.05) higher in the luteal-phase animals than any of the other groups killed after a cloprostenol injection that causes luteolysis. Similarly the ratio of delta receptor density to mu receptor density was greater (P < 0.05) in the OVX progesterone-treated ewes than in the OVX control ewes. No differences were found in the kappa receptor density across the cycle or with different steroid treatments. These data suggest that the relative proportions of the delta and mu subtypes of the opioid receptor in the hypothalamus change during the oestrous cycle. Regulation appears to be due to the feedback effects of ovarian steroids with progesterone altering the delta:mu ratio. In the MBH, there was a general increase in both delta and mu subtypes during the follicular phase of the oestrous cycle. This may explain, in part, how the responsiveness of the GnRH/LH axis to opioid peptides and antagonists changes across the cycle.

Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen.

The number of pituitary gonadotropin-releasing hormone receptors (GnRH-R) varies across the estrous cycle. We report that there is variable expression of the differently-sized GnRH-R transcripts in cyclic ewes and in an experimental model. During the follicular phase of the cycle, and compared to the luteal phase, there was increased expression of the 1.5, 2.3 and 3.7 kilobase (kb) transcripts with no change in the levels of the 5.6 or the 1.2 kb transcripts. Steady state levels of mRNA for luteinising hormone beta and common alpha subunit were also increased in the follicular phase of the cycle. In hypothalamo-pituitary disconnected ovariectomised ewes given pulsatile GnRH replacement, injection of estrogen increased the 1.5, 2.3 and 3.7 kb, while the levels of the 5.6 and 1.2 kb transcripts were not altered. We conclude that the differential regulation of GnRH-R mRNA occurs through a direct effect of E on the pituitary.

Use of a flavivirus RNA-dependent RNA polymerase assay to investigate the antiviral activity of selected compounds.

We have developed an assay using flavivirus RNA-dependent RNA polymerase to test the inhibitory activity of potential antiviral agents. The effects of antiviral agents on RNA synthesis were examined in this assay using extracts of Vero cells infected with dengue virus type 2 or Kunjin virus. Several different classes of known polymerase inhibitors were tested. The synthesis of double-stranded replicative form RNA was inhibited in a dose-dependent fashion in the presence of the polyoxometalate HPA-23 [(NH4)18(NaW21Sb9O86)17].14 H2O and several structurally related compounds.

Delivery of preventive services to older black patients using neighborhood health centers.

OBJECTIVES: Older black patients are at risk for underutilization of preventive services. Our objectives were to assess the delivery of five preventive services in Title 330-funded health centers in low income neighborhoods in Cleveland, Ohio, and to determine the association of health system factors and health status with the delivery of these services. DESIGN: A cross-sectional study. SETTING: Four neighborhood health centers in low income neighborhoods of Cleveland, Ohio. PARTICIPANTS: A total of 683 black men and women, aged 70 and older, who regarded the health center as their primary source of outpatient care. MEASUREMENTS: Demographic characteristics, independence in basic and instrumental activities of daily living, comorbidity scores, and perceived access were determined by telephone interview. We reviewed charts to determine whether each of five preventive service goals were obtained: influenza vaccination within 1 year; pneumococcal vaccination at any time; mammography within 2 years; Papanicolau screening within 1 year or twice at any time in the past with documentation of normal results; and fecal occult blood testing within 2 years. RESULTS: The defined goals for influenza vaccination, pneumococcal vaccination, mammography, Papanicolau screening, and fecal occult blood testing were achieved for 59%, 64%, 59%, 51%, and 17% of patients, respectively. Influenza and pneumococcal vaccines were obtained more often in persons with greater comorbidity. Mammography and Papanicolau smear were obtained more often in patients without of ADL or IADL impairments. The four clinical sites varied substantially in the delivery of each preventive service. More frequent office visits were associated with greater delivery of all five preventive services. This relationship persisted in multivariable analyses controlling for health status and clinical site. CONCLUSIONS: This study shows that Title 330 federally supported neighborhood health center sites providing primary care to older blacks in Cleveland achieved high rates of performance in four of the five recommended preventive services. In addition, preventive services practices were associated with prognostically relevant health status information. The frequency of office visits was related strongly and consistently to the performance of the various preventive services, indicating that more, not fewer, office visits may be necessary to achieve Healthy People 2000 targets. J Am Geriatr Soc 48:124-130, 2000. Key words: preventive services; blacks; access to care; geriatrics; primary care

Hospital outbreak of Norwalk-like virus.

An outbreak of gastroenteritis caused by Norwalk-like virus occurred in two areas of the hospital: area 1, consisting of three adjacent and interconnected wards, with mostly elderly patients; and area 22, an acute ward in a separate building with elderly patients. In area 1, 40 patients and 20 staff were affected; in area 2, 18 patients and 14 staff were affected. Infection control measures were instituted in consultation with the government health authority. These measures did not appear to affect the course of the outbreak, but may have prevented spreads to the other wards.

Henipaviruses: recent observations on regulation of transcription and the nature of the cell receptor.

Hendra virus (HENV) and Nipah virus (NIPV) are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genetic and biological characteristics that differentiate henipaviruses from other members of the subfamily are summarized. Although they do not display neuraminidase and hemagglutination activities and in that regard resemble viruses in the genus Morbillivirus, several recent observations highlight similarities between henipaviruses and respiroviruses (genus Respirovirus) in structure and replication strategy. First, three-dimensional modeling studies suggest that the external globular head domain of the HENV G protein resembles that of respiroviruses rather than morbilliviruses. Second, the pattern of transcriptional attenuation in HENV-infected cells resembles that observed with Sendai virus, a respirovirus, and differs from that found in cells infected with measles virus, a morbillivirus. Henipaviruses have a broad host range in vitro and in vivo, indicating wide distribution of cellular receptor molecules. The extensive host range has been confirmed in a quantitative in vitro cell-fusion assay using recombinant vaccinia viruses expressing the attachment and fusion proteins of HENV and NIPV. Cell lines of diverse origin and which are permissive in the in vitro cell fusion assay have been identified and the pattern of relative susceptibilities is the same for both HENV and NIPV, implying that both viruses use the same cell receptor. Protease treatment of permissive cells destroys their ability to fuse with cells expressing viral envelope glycoproteins. Virus overlay protein binding assay (VOPBA) and radio-immune precipitation assays confirm that both HENV and NIPV bind to membrane proteins in the 35-50 kD range. Treatment of cell membrane proteins with N-glycosidase eliminates HeV binding activity in VOPBA whereas treatment with neuraminidase has no effect on binding. Thus preliminary evidence suggests that NIPV and HENV bind to the same glycoprotein receptor via a non-sialic acid-dependant mechanism.

Replication of dengue virus type 2 in human monocyte-derived macrophages: comparisons of isolates and recombinant viruses with substitutions at amino acid 390 in the envelope glycoprotein.

The severity of dengue virus infection ranges from mild fever to dengue hemorrhagic fever and shock syndrome. The association of disease severity with virus replication in monocyte-derived macrophages (MDMs) was examined for dengue virus type 2 (DEN-2) isolates from Asia or America. Additionally, we constructed DEN-2 recombinant viruses with substitutions at residue 390 in the envelope glycoprotein (E390) because this residue is linked with the region of virus origin. Comparisons of virus yields of 3 isolates failed to show a correlation with clinical disease. However, the American strain did not replicate as well as the 2 Asian strains. For the recombinant viruses, substitution of Asn (Asian) at E390 with Asp (American) resulted in decreased ability to replicate in MDMs. These results are consistent with the proposal that the lack of association of native American DEN-2 strains with severe disease is linked to reduced ability to replicate in MDMs, and that Asp at E390 may contribute to this reduction.