Identification and characterization of a novel postlarval hemolymph protein from Manduca sexta

The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [³⁵S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of M_r ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ～8.6). Electrophoresis under denaturing conditions reveals a subunit M_r ≈ 50,000 while the native protein has an apparent M_r ～ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.     

Regulation of follicular development by diethylstilboestrol in ovaries of immature rats

Immature female rats received either one injection of 2 mg diethylstilboestrol (DES)/rat subcutaneously and were killed 12 h later or received two injections of DES at 0 and 24 h and were killed at 24, 36 and 48 h after the initial injection. The ovarian follicles were released by enzymic digestion with collagenase and separated into those of small, medium and large diameter (less than 200 microns, 200-400 microns and greater than 400 microns) by filtration through graded Teflon sieves and granulosa cells were extracted from these follicles. The ovaries of immature rats treated with pregnant mares' serum gonadotrophin (PMSG) were used for comparative purposes. Incorporation of [3H]thymidine into granulosa cell DNA was augmented by DES and by PMSG. Small follicles were more strongly stimulated by DES at 12 h than those of other sizes, but rates increased significantly in medium and large follicles at 48 h. Aromatase activity in the DES-treated group was low at all times and in all follicles. Rates of oestrogen and progesterone production in response to 36 h of exposure to follicle-stimulating hormone (FSH) in vitro were significantly lower than in the PMSG-treated group. FSH-stimulated steroid production in the DES group at 36-48 h was lower, particularly in the medium follicles. A significant rise in serum FSH, luteinizing hormone (LH) and progesterone concentrations was noted only at 36 h after DES treatment, while serum and follicular fluid oestrogen values remained unchanged. When these changes were compared with those in PMSG-treated rats, there were obvious differences. The pattern of thymidine incorporation and aromatase activity differed with time and follicle size. Serum FSH and LH values were not affected by PMSG treatment, but serum and follicular fluid oestradiol values increased with time. The PMSG-treated animals ovulated in response to human chorionic gonadotrophin, but the DES-treated rats did not ovulate in spite of the presence of some large antral follicles in the ovaries. These findings show that initial exposure of follicles to high concentrations of oestrogen results in follicles which fail to respond to subsequent gonadotrophin surges and are thereby restricted in their ability to differentiate fully.     

Inhibition by diethylstilboestrol of proliferative potential of follicles of different sizes in immature rat ovaries

Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter less than 200 microns (small), 200-400 microns (medium) and greater than 400 microns (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells. Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentrations are highest.     

Comets and the formation of biochemical compounds on the primitive Earth – A review

Thirty years ago it was suggested that comets impacting on the primitive Earth may have represented a significant source of terrestrial volatiles, including some important precursors for prebiotic synthesis (Oró, 1961,Nature 190: 389). This possibility is strongly supported not only by models of the collisional history of the early Earth, but also by astronomical evidence that suggests that frequent collisions of comet-like bodies from the circumstellar disk around the star Pictoris are taking place. Although a significant fraction of the complex organic compounds that appear to be present in cometary nuclei were probably destroyed during impact, it is argued that cometary collisions with the primitive Earth represented an important source of both free-energy and volatiles, and may have created transient, gaseous environments in which prebiotic synthesis may have taken place.     

Expectations of assisted conception for infertility.

OBJECTIVE--To provide reliable prognostic information for couples seeking assisted conception. DESIGN--Analysis of four years'' practice (1988-91). SETTING--Private university service linked with NHS reproductive medicine services. PATIENTS--804 couples with various causes of subfertility, median duration five years, median age of women 34 years. INTERVENTIONS--1280 completed cycles: 950 in vitro fertilisation, 144 gamete intrafallopian transfer, and 186 intrauterine insemination and superovulation. MAIN OUTCOME MEASURES--Pregnancy and birth rates per cycle and cumulative pregnancy and take home baby rates per couple. RESULTS--In women under 40 years and men with normal sperm, whatever the cause of infertility, results with in vitro fertilisation improved steadily reaching a pregnancy rate per cycle of 30% (95% confidence interval 26% to 35%) during 1990-1 and birth rate per cycle of 29% (23% to 35%) in 1990. Pregnancy and birth rates for gamete intrafallopian transfer were 36% (28% to 44%) and 26% (17% to 37%) and for intrauterine insemination 18% (12% to 24%) and 16% (10% to 22%). After six cycles cumulative probability of pregnancy was 82% and cumulative take home baby rate 70%. Considering only in vitro fertilisation and gamete intrafallopian transfer after four cycles the pregnancy rate was 78% (66% to 91%). CONCLUSIONS--Conception is less likely in women over 40 and men with sperm dysfunction. For other couples the prognosis for a live birth is at least as good as for fertile couples if they persist with treatment.     

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.     

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.     

Molecular Genetic Characterization of a Locus That Contains Duplicate Adh Genes in DROSOPHILA MOJAVENSIS and Related Species

Drosophila mojavensis and other species of the mulleri subgroup contain a duplicate gene encoding the enzyme alcohol dehydrogenase (ADH). Studies on the genetic relationship of the two genes using electrophoretic variants show them to be closely linked. We have cloned a 13.5-kb fragment of D. mojavensis DNA into the lambda vector, Charon 30. This fragment contains both Adh genes separated by approximately 2 kb of DNA. The clone hybridized to a single position on chromosome 3 in D. mojavensis following in situ hybridization. It is likely that the genes are tandemly arranged in the genome. One of the two genes shows a complexity in its structure that suggests the close linkage of a pseudogene or part of a gene. The structure of the Adh locus in five species of the mulleri subgroup have been compared by constructing restriction maps of genomic DNA. Two of these species D. arizonensis and D. mojavensis express Adh-1 in the ovaries; the others do not. In comparing these species it is evident that there has been one or two insertions into the region between the Adh genes. It is possible that one of these structural changes is related to the change in Adh tissue-specific expression that has occurred during the evolution of these species.