Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development.

The ability of an oocyte to support early embryonic development requires both nuclear and cytoplasmic maturation. We have investigated the effects of brain-derived neurotrophic factor (BDNF) on maturation of the bovine oocyte and embryo development after parthenogenetic activation. By RT-PCR and immunohistochemistry, cumulus and oocytes were shown to express mRNA and protein for BDNF and the p75 common neurotrophin receptor. However, mRNA for the BDNF-specific full length and truncated isoforms of the TrkB receptor are only detected in cumulus, suggesting that oocytes and cumulus differ in their capacity to respond to neurotrophin signalling. In in vitro maturation experiments, the proportion of cumulus oocyte complexes maturing to metaphase II was not altered by BDNF in groups lacking fetal calf serum (FCS), but was significantly lower than the positive control containing 10% FCS (P < 0.01). However, after maturation, the proportion of parthenogenetically activated oocytes forming blastocysts was highest for 10 ng/ml BDNF (24%, n = 95) followed by 100 ng/ml BDNF (18%, n = 91) and 10% FCS (15%, n = 103), which in turn were greater than no serum (10%, n = 83; P < 0.01). Maturation in the presence of a BDNF blocking antibody resulted in a blastocyst yield that was comparable to the absence of serum, and lower than in the presence of BDNF (P < 0.01). Similar effects on progression to metaphase II and blastocyst formation were observed using oocytes matured without cumulus. Together, these results provide the first evidence for a role for neurotrophins in promoting oocyte cytoplasmic competence to support embryonic development, despite being insufficient in the absence of serum to enhance nuclear maturation.

Steroidogenesis in bovine granulosa cells: the effect of short-term changes in dietary intake.

The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-I) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford x Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period (17 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-I or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.

Decreased muscle cell proliferation in chicks with a deletion in the GH receptor gene.

The increase in muscle weight in neonatal animals is a consequence of increased protein accretion and DNA content. GH increases protein accretion but direct effects of GH on myogenic cell proliferation have not been demonstrated. Sex-linked dwarfism in the chick is caused by mutation or deletion in the GH receptor gene and has provided a useful model to study the physiological consequences of GH insensitivity. This study determined the consequences of GH receptor gene mutation on muscle cell proliferation in vivo. Northern and Southern blotting and PCR analysis revealed restriction fragment length polymorphism patterns and a 1.7 kb deletion of the intracellular domain of the GH receptor gene in commercial dwarf broiler chicks, similar to the Connecticut strain in which there is a dysfunctional GH receptor. Cell proliferation was measured in muscle sections from normal and dwarf chicks after incorporation of 5-bromo-2'-deoxyuridine (BrdU; 25 mg/kg) in vivo at 2, 5 and 13 days of age. Incorporation of BrdU into nuclei was measured in frozen sections, counterstained with propidium iodide to estimate the total number of nuclei by quantitative image analysis, and the labelling index was calculated. Paraffin-embedded sections of breast muscle were stained using an anti-human IGF-I polyclonal antibody. Expression of IGF-I mRNA in muscle from each genotype at 5 days of age was measured by RNAse protection assay. The labelling index was similar in 2-day-old chicks from both genotypes (normal, 20.14 +/- 2.39%; dwarf, 19.79 +/- 5.83%). By day 5 the labelling index had decreased but was significantly higher (P < 0.02) in normal (12.53 +/- 3.36%) compared with the dwarf (6.25 +/- 1.39%). By 13 days of age, there was a further decrease in labelling index but no difference between the groups (normal, 4.92 +/- 1.28%; dwarf, 4.96 +/- 1.51%). IGF-I mRNA was expressed and IGF-I peptide was identified in muscle sections but there was no difference between genotypes. The results show that cell division in breast muscle in vivo is high in neonatal chicks but it declines with increasing age. The absence of a functional GH receptor in the dwarf is associated with a greater decline in DNA synthesis and suggests that GH may directly affect a proportion of cells, since there was no difference in IGF-I mRNA or peptide.

Antibodies to normal and Alzheimer human brain structures from non-immunised mice of various ages.

Supernatants from mouse spleen hybridoma lines established without previous immunisation were screened immunohistochemically against cryostat sections of human temporal cortex and found to stain a variety of brain structures, including Alzheimer plaques and tangles. The age of the mice had no effect on antibody production.

Effects of alpha- and beta-tigogenin cellobiosides on cholesterol absorption.

We have synthesized alpha- and beta-anomers of tigogenin cellobioside and have determined their effects on intestinal absorption of [1,2-3H]cholesterol in rats. We demonstrated that the loss of tritium label likely to occur in the conversion of cholesterol to coprostanone was minimal. Dose response studies showed that both anomers depressed intestinal absorption of cholesterol but the depression was greater with the beta-anomer.

Synthesis and antimicrobial activity of 3H-1,2,4-thiadiazolo fused heterocycles.

Various fused 3H-1,2,4-thiadiazoles were prepared. Significant in vitro Gram-positive antibacterial and antifungal activities were observed fro certain members of the series.

Agents for alkylating steroid hormone receptors. 3. Omega-substituted esters of 17-alpha-hydroxyprogesterone.

PIP: The synthesis and Clauberg activity of the 8-carbomethoxyoctanoate (IIe), 9-carbomethoxynonanoate (IIf), 9-carboethoxynonanoate (IIg), 9-carbo-tert-butoxynonanoate (IIh), 8-carboxyoctanoate (IIId), 9-oxo-10-diazodecanoate (IVd), 5-oxo-6-hydroxyhexanoate (Va), and 6-oxo-7-hydroxyheptanoate (Vb) esters of 17-alpha-hydroxyprogesterone are reported in this article. Esters IIa-IIe and IIe-IIh were tested by the McPhail modification of the Clauberg assay. The progestational activity increased with chain length for the glutarate ester IIa through the pimelate ester IIe. It then decreased slightly for the azelate ester IIe and decreased further for the methyl and tert-butyl sebacates IIf and IIh.^ieng

Agents for alkylating steroid hormone receptors. 2. 16-alpha substituted progesterone derivatives.

PIP: A series of diazo ketones derived from esters of 17-alpha-hydroxyprogesterone were studied in order to find an agent capable of selectively alkylating the Clauberg receptor. In continuation of the search, derivatives of 16-alpha-prophyl-, 16-alpha-butyl, and 16-alpha-pentylprogesterone were synthesized and assayed for Clauberg activity. The 3-carbon side chain was derivatized as omega alcohol, mesylate, tosylate, bromoacetate, and para-fluorosulfonylbenzoate; the 4-carbon side chain as omega alcohol, mesylate, and bromoacetate; and the n-pentyl chain as omega alchol, mesylate, tosylate, bromoacetate, para-fluorosulfonylbenzoate, toluate, and bromide.^ieng