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[Infectious burdens of reproduction of female dogs].

The results of gynecological investigations in 142 bitches were evaluated and the complexity of infectious affections is discussed. High proportions of infectious cases were found in cases of limitation of fertility (67.5%), in vaginal discharge in the estrus (60.8%), in cases of mastitis/pseudopregnancy (61.5%) and in mortality of newborn puppies. St. aureus and E. coli were often isolated. There is a high etiological correlation in epidemiology of diseases of the reproductive tract in the single bitch and especially in kennel bitches.

Animals↗

The ecological distribution of reproductive mode in oribatid mites, as related to biological complexity.

The high incidence of asexuality in oribatid mites presents an unusual opportunity for examining hypotheses for the maintenance of sex. There is a presumed range in age of asexual species: many oribatid species are phylogenetically clustered, occurring in speciose early-derivative families or genera without sexual species, while others are phylogenetically isolated from other asexual species, occurring in later derivative taxa with sexual congeners. We examined the distribution of oribatid mite reproductive mode in soil of corn fields, grassy and shrub fields, and forests in central New York State (three replicate plots of each type, with 25 samples per plot), to test three ecological predictions from current theory. (1) If overall biotic uncertainty, as generated by competitors and predators, mediates the ecological distribution of oribatid mites, then the proportion of asexual oribatid mites should be negatively correlated with biological diversity; we examine this prediction using literature data as well. (2) If Muller's Ratchet (the stochastic loss of best genotypes, which is independent of environment), mediates the success of asexuality, then no ecological pattern should exist. (3) If general purpose genotypes are characteristic of asexual oribatid mites, their habitat distribution should be broader than that of sexual species. For each plot the level of asexuality was compared to indices of overall biotic diversity, as calculated from the pooled oribatid mite (competitors) and mesostigmatid mite (predator) communities. We found no negative correlation in this relationship in our own data or in the literature analysis of 290 faunal surveys from 50 literature sources, so we reject biotic uncertainty as an important determinant of reproductive mode distribution. When only data on phylogenetically clustered asexuals are considered, there is instead a positive correlation between asexuality and diversity that is not explained. Because of the latter pattern we tentatively reject Muller's Ratchet as the primary factor maintaining reproductive mode in these mites, but cannot reject it for isolated asexual species. Niche breadth in sexual and asexual oribatid mites provides no support for widespread general purpose genotypes but broad patterns in the literature suggest that the idea needs further investigation. Possible complicating or unknown factors that are discussed include historical disturbance in the study area, the relationship between parasitism and general biotic diversity, and the level and source of genetic diversity in asexual oribatid mites.

Animals↗

Putative rat sperm lipid-binding protein: isolation and partial characterization.

Previous work has identified a prominent 22-24-kD protein that is present in rat male reproductive tissues, including epididymis and testis (Brooks, 1985; Jones and Brown, 1987; Moore et al., 1987). Using a monoclonal antibody (designated mAb-B109) against this 24-kD antigen (referred to as B109), we have isolated the protein using a combination of chromatofocusing and electroelution from SDS-PAGE gels, and reverse phase HPLC. B109 (pI = 4.8) is amino-terminal blocked. To obtain internal amino acid sequences, the isolated protein was cleaved either with cyanogen bromide in 70% formic acid or with TLCK-treated chymotrypsin. With cyanogen bromide treatment, two peptides, 17.8 kD and 11.9 kD, were isolated and partial amino acid sequences obtained. Chymotryptic peptides were isolated by reverse-phase HPLC and two were chosen for sequence analysis. A computer search for sequence homology through the protein identification resource (PIR) matched B109 to a basic 21-kD cytosolic protein (pI = 7.4) found in bovine brain (> 80% homology). When peptide sequence differences obtained in the present study were substituted into the 21-kD cytosolic protein sequence obtained from the PIR using Intelligenetics software, the calculated pI dropped from 7.4 to 5.8, suggesting that pI differences between the bovine and rat molecules are the result of amino acid substitutions in the testis protein and not tissue-specific posttranslational processing. It has been postulated that the 21-kD bovine brain protein is associated with phospholipid transport, although the function of B109 is unknown.

Amino Acid Sequence↗

Completion of the full-length genome sequence of Menangle virus: characterisation of the polymerase gene and genomic 5' trailer region.

Menangle virus (MenV), isolated in 1997 from stillborn piglets during an outbreak of reproductive disease at a large commercial piggery, is the only new paramyxovirus to be identified in Australia since Hendra virus in 1994. Following partial characterisation of the MenV genome, we previously showed that MenV is a novel member of the genus Rubulavirus. Here we report the characterisation of the large (L) polymerase gene and the adjacent 5' trailer region of MenV, which completes the full-length genome sequence of this novel paramyxovirus (15,516 nucleotides), and thereby confirm its taxonomic position within the family Paramyxoviridae.

Amino Acid Sequence↗

Non-concerted evolution of the RET76 satellite DNA family in Reticulitermes taxa (Insecta, Isoptera).

The evolutionary dynamics of satellite DNA is most often studied in canonical mating systems, where bisexuality and panmixis are the rule. In eusocial termites, the limited number of reproducers starting a new colony and the maintenance of the colony through few neotenics act as bottle-necks both in space and time. No data on repetitive DNA are available for Isoptera and for their peculiar reproductive strategy. Here we present the first satellite DNA family isolated in European Reticulitermes. RET76 is a G+C rich satellite embodying two sub-families with a 76 bp monomer. RET76 sequences are highly variable (sequence homology is lower than 80% within sub-families and lower than 68% in the entire family) and this variability is equally distributed among the eight analysed taxa, thus depicting a pattern of non-concerted evolution. The absence of variant fixation--together with the strict monomer length conservation--may be explained at the molecular level as due to functional constraints acting on these sequences, and/or at the organismic level by considering the involvement of eusociality in preventing or greatly reducing variant fixation, somehow mimicking an unisexual strategy.

Animals↗

Melamine resins and their application in electron microscopy.

Melamine resins are derived from the heterocyclic compound triaminotriazine, C3H6N6. Similar to proteins in structure and reactivity, water-soluble melamine resins can be used as water-embedding media for electron microscopy (Bachhuber and Frösch, 1983). The idea behind this approach was to study some of the artefacts of traditional embedding techniques and to work out conditions to eliminate as far as possible denaturing of proteins and extraction of lipids. Sectioned cells and tissues processed in the melamine resin Nanoplast show remarkable preservation of ultrastructure. Because they can be sectioned extremely thinly, melamine resins are particularly suitable for dark-field and electron spectroscopic imaging of unstained molecular suspensions providing in this way an unusually clear reproduction of ultrastructural detail such as the helical structure of isolated unstained double-strand DNA molecules (Frösch et al., 1987b). In 1988, the melamine resin Nanostrat was introduced as an EM-compatible prolific substrate foil for cell culture (Westphal et al., 1988). Cells or bacteria cultivated on this material can be processed for various kinds of follow-up techniques like TEM, SEM, vertical sectioning and immunocytochemistry.

Animals↗

Expression profile of the tumorigenic factors associated with tumor size and sex steroid hormone status in uterine leiomyomata.

OBJECTIVE: To use tissue microarray in combination with dendrogram cluster analysis to characterize some potential tumorigenic factors in association with tumor size and sex steroid hormone status in uterine leiomyomata. DESIGN: Expression analysis of 21 selected potential tumorigenic factors in 60 patients with uterine leiomyomata. SETTING: University clinical research laboratory. PATIENT(S): Hysterectomy specimens from 60 patients with uterine leiomyomata of different ages and tumor sizes. INTERVENTION(S): Tissue cores from normal myometrium and leiomyomata were examined by immunohistochemistry. MAIN OUTCOME MEASURE(S): Semiquantitative immunointensity was scored and analyzed by net gain and loss between normal myometrium and leiomyomata and integrated into dendrogram cluster tree view. RESULT(S): We found that upregulation of estrogen and progesterone receptors was reverse associated with tumor size. Upregulation of some factors (HMGA2, sex steroid receptor cofactors, proteins in insulin pathway, and CD24) were identified in a group of patients in their later forties, were associated with large fibroids, and were weakly affected by the SSH status (illustrated by endometrial phases and menopause). Downregulation of tuberin and glucocorticoid receptor was mostly isolated in a second group of women at their late reproductive age. CONCLUSION(S): Analyses of the sex steroid hormone receptors and the nonsex steroid hormone factors in leiomyomata and the matched myometrium showed different expression patterns in different tumor sizes and patients' ages. A group of patients in their late forties with the larger leiomyomata contributes largely by upregulation of nonsex steroid hormone factors. Adenomyosis is a protective factor preventing large leiomyomata.

Aged↗

Ecological characterization of Steinernema anatoliense (Rhabditida: Steinernematidae).

Our study describes the basic ecological characteristics of the entomopathogenic nematode Steinernema anatoliense including its response to temperature, moisture, and host range. The effect of temperature and soil moisture on the infection of Galleria mellonella larvae by S. anatoliense was determined. The temperature range for infectivity was greater than that for development. The optimal temperature for infection and development was 25 degrees C. Although S. anatoliense infected the hosts at 10 degrees C, no reproduction occurred at this temperature. This nematode species that was isolated from a cold region of Turkey exhibited warm-adapted temperature characteristics. Optimum water content of the soil for S. anatoliense to infect the host was 10%.

Animals↗

Trichinella nativa in a black bear from Plymouth, New Hampshire.

A suspected case of trichinellosis was identified in a single patient by the New Hampshire Public Health Laboratories in Concord, NH. The patient was thought to have become infected by consumption of muscle larvae (ML) in undercooked meat from a black bear killed in Plymouth, NH in October 2003 and stored frozen at -20 degrees C fro 4 months. In January 2004, a 600 g sample of the meat was thawed at 4 degrees C, digested in hydrochloric acid and pepsin, and larvae were collected by sedimentation. Intact, coiled, and motile ML were recovered (366 larvae per gram (l pg) of tissue), which were passed into mice and pigs. Multiplex PCR revealed a single 127 bp amplicon, indicative of Trichinella nativa. The Reproductive Capacity Index (RCI) for the T. nativa-Plymouth isolate in mice was 24.3. Worm burdens in the diaphragms of two 3-month-old pigs given 2,500 ML were 0.05 and 0.2l pg by 35 days post-inoculation, while 2.2 and 0.75 l pg were recovered from two 3-month-old pigs given 10,000 ML; no larvae were recovered from four 1-year-old pigs given 2,500 ML (n=2) or 10,000 ML (n=2). Viable larvae were also recovered from frozen black bear meat harvested at two additional locations, one in southern Ontario, Canada, and one in upstate New York, USA. Multiplex PCR using genomic DNA from these parasite samples demonstrated that both isolates were T. nativa. This is the first report of the freeze-resistant species, T. nativa, within the continental United States.

Animals↗

The apoptotic profile of human cumulus cells changes with patient age and after exposure to sperm but not in relation to oocyte maturity.

OBJECTIVE: To determine the expression of apoptosis-associated molecules on cumulus cells removed from individual oocytes of different maturity, inseminated oocytes and to investigate the possibility of an age-dependent expression. DESIGN: Analysis of apoptosis in cumulus cells isolated from oocytes of different stages of maturity. SETTING: Assisted reproductive technology program of the Birmingham Women's Hospital, Birmingham, UK. PATIENT(S): Patients undergoing intracytoplasmic sperm injection or IVF cycles. MAIN OUTCOME MEASURE(S): Percentage of positive cumulus cells when assessed for nuclear DNA damage using the terminal deoxyuridine nucleotide end-labeling assay or stained with antibodies [Fas, Fas ligand, the antiapoptotic protein Bcl-xl, and the RNA-binding protein (TIAR)]. RESULT(S): Cumulus cells collected from mature oocytes showed no significant difference in the percentage of apoptotic markers compared to those recovered from immature oocytes, whereas those from patients >/=38 years differed significantly. When cumulus cells were exposed to sperm the levels of apoptotic markers altered significantly from those not exposed to sperm. CONCLUSION(S): The results show that the cumulus cells of human oocytes are equipped with a mechanism to undergo apoptosis and that patient age and the exposure of cumulus cells to sperm can alter their profiles of apoptotic markers.

Adult↗

Morphology and function of rooster efferent ductule epithelial cells in culture.

Regulation of the excurrent ducts of the testis is not well understood, particularly in avian species. To investigate the role of steroid hormones in the male reproductive tract, we developed a primary cell culture of epithelia isolated from rooster ductuli efferentes (efferent ductules). Efferent ductules of the avian testis comprise 77% of the epididymal region and form a mass of tubules containing a heavily folded epithelium enmeshed in connective tissue. The epididymal region was separated by microdissection and small epithelial plaques isolated by serial digestion with collagenase, elastase and repeated pipetting. Isolated cell plaques were cultured in a bicameral chamber on Millicell-CM inserts coated with two layers of basement membrane matrix, consisting primarily of laminin and Types I and IV collagen. Active ciliary beat was observed before plating and this activity was maintained for 14 days in culture. Cell plaques attached within 24 h and outgrowths formed a confluent monolayer by 5-6 days. The epithelial nature of cultured cells was demonstrated by immunocytochemical staining for cytokeratin. Light and electron microscopy confirmed that morphology and polarity of the original epithelial cells were maintained in culture. Cultured efferent ductal epithelium was cuboidal in shape and maintained many of the cytoplasmic organelles typical of these cells in vivo. The uptake of cationic ferritin indicated the endocytotic activity of these cultured cells was maintained. Estrogen receptor mRNA expression was maintained in cultured cells. These data demonstrate avian efferent ductal epithelium can be isolated and grown in defined culture medium for the purpose of determining the role of hormones and other factors in regulating the function of the epididymal region in the bird.

Animals↗

Evaluation of toxicity of pesticides and their biodegradation products using human cells.

Juvenoids are biologically active compounds, of relatively low toxicity to humans, that efficiently inhibit the fertility of insects. However, little attention has been paid to the stability and toxicity of products that may be generated by their biodegradation in the ecosystem. This study describes a simple comparison of the toxicity of the active compound and its degradation products generated by aerobic soil microbial isolates. Surprisingly we have found that toxicity of a biologically active carbamate juvenoid N-[2-[4-(2,2-ethylenedioxy-1-cyclohexylmethyl)-phenoxylethyl]carbamate (W328) was comparable with that of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The toxic effect was evaluated using the determination of the ATP/ADP content and viability of HeLa S3 cells exposed to various concentrations of the chemicals tested for various durations. DDT was used as a reference compound. Its toxicity was compared with two juvenile hormone analogs. The original compound, W328, was found to be the most toxic. The major product (W329) generated both by yeast isolates and the mixture of moulds lost its activity on reproduction of the tested insect. Its toxicity towards human cells was also decreased. Another two W328 degradation HPLC fractions exhibited significantly reduced toxicity compared to W328.

Adenosine Diphosphate↗

Effects of the presence of rams during pregnancy on lambing performance in ewes.

Ewes of the Préalpes-du-Sud breed (n = 112) were mated with fertile rams and were used to investigate the effect of the presence of vasectomized rams during pregnancy on reproductive outcomes. Ewes in the control group (n = 56) were isolated from rams during the whole period of pregnancy, whereas those in the experimental group (n = 56) were kept with vasectomized rams from day 10 post-mating until lambing. At day 10 post-mating, a series of blood samples was collected every 15 min for 8 h from five control ewes and five experimental ewes to determine their patterns of LH secretion. The introduction of the ram was associated with a rapid increase of pulsatile LH release. The lag between the introduction of the ram and the onset of the first episodic LH release was less than 15 min. The mean(+/- sem) number of LH pulses/4 h after the introduction of the ram (2.8 +/- 0.4) was significantly higher (P < 0.01) than that observed/4 h before the introduction of the ram (1.4 +/- 0.2). Although more ewes were pregnant in the control group (87.5%) than in the ram-exposed group (82.1%), the difference was not significant. The presence of rams did not affect gestation length (145.8 days), overall lamb mortality (3.5%) or birth weights of single (3.96 kg), twin (3.24 kg) or triplet (2.59 kg) lambs. The proportion of ewes with multiple births in the control group (69.4%) was significantly greater (P < 0.05) than that in the ram-exposed ewes (47.8%). The ewes in the control group had significantly more (P < 0.01) twin lambs born alive (72.3%) than the ewes in the ram-exposed group (50.0%). In conclusion, the presence of vasectomized rams during early pregnancy affected the incidence of multiple births but did not affect pregnancy rate or gestation length. The altered fertility of ewes exposed to vasectomized rams may reflect changes in embryonic loss during early pregnancy.

Animals↗

Effects of bull exposure on postpartum ovarian activity of dairy cows.

The effects of bull exposure on postpartum reproduction have been previously studied in beef, but not in dairy cattle. The objective of this experiment was to investigate effects of bull exposure on postpartum reproductive characteristics during early lactation in high-producing Holstein dairy cattle. Forty-five multiparous dairy cows (9764 +/- 140 kg milk/lactation) were randomly grouped at the time of calving. Treatment 1 (NBE) was never exposed to the bull, treatment 2 (2 x BE) experienced fenceline contact with a mature Holstein bull twice daily during routine detection of oestrus, and treatment 3 (CBE) was in continuous proximity to the bull. All cows were observed for oestrous behaviour twice daily for 1/2 h following milking. Blood samples were collected every other day for progesterone (P4) assay. Postpartum ovarian reactivation, indicated by the occurrence of the first sustained rise in P4, was later (P = 0.02) during the postpartum period in both bull-exposed (2 x BE, CBE) treatments compared to the NBE treatment. Time postpartum until completion of the first ovarian cycle tended (P = 0.08) to be shorter in NBE cows compared to bull-exposed cows. Peak P4 concentrations were higher (P = 0.05) in NBE cows compared to bull-exposed cows prior to the second postpartum ovulation. Results indicate an extended period to ovarian reactivation in dairy cattle exposed to a bull, but bull exposure had no effect on long-term reproductive performance of exposed cows.

Animals↗

Comparison of antibody responses and virus shedding following administration of trivalent oral poliomyelitis vaccines prepared either in monkey or human diploid cell substrates.

Nineteen (22.9%) of 83 sera collected before vaccination from adult volunteers aged 21-64 years were without neutralizing antibody to poliomyelitis at levels of 0.15 i.u./ml for types I and II and 0.1 i.u./ml for type III. Some correlations were found between the history of previous vaccination and the presence of antibody but these were not well defined. Vaccination with a single dose of trivalent oral polio vaccine elicited fourfold or greater antibody responses to one or more poliomyelitis types in 53 (63.9%) volunteers, the percentage antibody resposnes being inversely related to the titre of antibody present before vaccination. Types I, II or III poliomyelitis virus were recovered from 76.8% of faecal samples collected 1 week after vaccination. The percentage recovery progressively declined thereafter until virus was recovered from 10.5% of samples collected 6 weeks after vaccination. Type for type, the titres and percentages of antibody responses and virus shedding in faeces were similar following trivalent oral poliomyelitis vaccines whether prepared in monkey or human diploid cell substrates. Some change in reproductive capacity temperature (r.c.t./40) marker was found in faecal isolates from volunteers vaccinated with monkey kidney and human diploid grown vaccines but no change in 'd' marker was found.

Adult↗

Production of meiosis-activating sterols from metabolically engineered yeast.

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.

Cholestadienols↗

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