The biology of the stoat (Mustela erminea) in the National Parks of New Zealand IV. Reproduction

Abstract

The gonads of 528 female and 821 male stoats were examined. The weights of ovaries and testes in adults peaked simultaneously in October, the season of births and of post-partum oestrus. Of 73 females, 78% had even numbers of nipples, mostly 8 or 10. The mean number of embryos in 13 pregnancies was 8.8 (6–13), and embryo weights ranged from 0.005 g to 2.9 g. Of 11 pregnant females, 8 contained fewer embryos than corpora lutea, and there was evidence of transuterine migration of blastocysts in 6. All but 2 of 451 females caught in December–July inclusive carried corpora lutea of delay. Few adult and no young females were found in oestrus in September–October, though adult males were fertile from August to February (no first-year males were fertile). There was some evidence that the breeding season started later at more southerly latitudes in both males and females. The mean number of corpora lutea per female was 9.7 (n = 439), and there was a significant inverse correlation between counts for the 2 ovaries of one individual. There was generally no significant variation in fecundity of females with age, body weight, or year. Of 11 females which were considered to have lost their litters, 10 were collected in beech (Nothofagus) forests. Four females and 9 males had abnormal gonads; the most severe abnormality was an ovarian teratoma of unknown pathology.     

Selective oxygenation of the endocannabinoid 2-arachidonylglycerol by leukocyte-type 12-lipoxygenase

The endogenous cannabinoid system appears to serve vascular, neurological, immunological, and reproductive functions. The identification of 2-arachidonylglycerol (2-AG) as an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors has prompted interest in enzymes capable of modifying or inactivating this endocannabinoid. Porcine leukocyte 12-liopoxygenase (12-LOX) oxygenated 2-AG to the 2-glyceryl ester of 12(S)-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE-G). The k(cat)/K(M) for oxygenation of 2-AG was 40% of the value for arachidonic acid. In contrast to the results with leukocyte 12-LOX, 2-AG oxygenation was not detected with platelet-type 12-LOX. Among a series of structurally related arachidonyl esters, 2-AG served as the preferential substrate for leukocyte 12-LOX. 12(S)-Hydroxyeicosa-5,8,10,14-tetraenoic acid glyceryl ester (12-HETE-G) was produced following addition of 2-AG to COS-7 cells transiently transfected with leukocyte 12-LOX. These results demonstrate that leukocyte-type 12-LOX efficiently oxidizes 2-AG in vitro and in intact cells, suggesting a role for this oxygenase in the endogenous cannabinoid system.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Gas chromatography/mass spectrometry assays for the determination of debrisoquine and sparteine metabolites in microsomal fractions of rat liver.

Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 micrograms of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.     

Effects of rainfall on the use of foliar analysis for diagnosing boron toxicity in field-grown wheat

The effect of rain on foliage elemental composition, especially B, was assessed using samples of wheat collected at three harvests from a field trial conducted in soil containing excessive levels of B. Moderate rainfall substantially decreased both the B concentration and content of whole shoots and young leaves. The change in B concentration due to rain suggests that foliar analysis is unreliable for diagnosing B toxicity. For the other elements examined (Ca, Cu, Mg, Mn, Mg, P, S, Zn), rainfall had little effect.     

Production of monoclonal antibodies against the B700 murine melanoma antigen and their antimetastatic properties.

Two unique murine melanoma antigens, termed B700 and B50, have been identified and isolated from several different murine melanoma cell lines. Both antigens can be detected on the cell surface, are actively shed in culture, and are often found in close association intracellularly. In previous studies, the antigen B700, which is related to serum albumin by biochemical and immunological criteria, was shown to function as a melanoma-specific tumor rejection antigen. We have also shown that animals sensitized to irradiated JB/RH melanoma cells produce antibodies which recognize B700 and/or B50, with B700 evoking the stronger humoral response. Animals testing positive by ELISA for antibody production to B700 or B50 were used for preparation of hybridomas and four different murine monoclonal antibodies have been produced whose specificities should facilitate epitope mapping. Clones have been used to generate ascites fluid in nude mice; the antibodies specifically recognize B700 and intact murine melanoma cells, but not B50. Two of these monoclonal antibodies have been administered systemically to C57Bl/6 mice bearing 5 day pulmonary metastases of the JB/MS melanoma, and significant inhibition of metastatic growth was observed for both antibodies.