Unifying Concepts Learned from Methyl Farnesoate for Invertebrate Reproduction and Post-Embryonic Development

Since the discovery that methyl farnesoate (MF), the unepoxidatedform of the insect juvenile hormone (JHIII), is produced bymandibular organs of numerous crustaceans, extensive evidencehas accumulated that this compound appears to perform similarfunctions in the Crustacea as JH performs in insects. A majorfunction of MF appears to be in enhancing reproductive maturation.This was first shown by indirect experimentation with eyestalkablation, which augmented MF production. Subsequently, directtreatments of several species of crustacea with MF showed thatreproductive maturation was enhanced. A second function of MF, similar to that of the JH of insects,is in the maintenance of juvenile morphology. This is especiallytrue in the late larval transformations into juveniles, whereMF plays an inhibitory role, as well as during the transformationof juveniles into adults. These results were inferred from eyestalkremoval experiments. In the case of the larval-juvenile transition,inhibitory results were also obtained with MF by direct hormonetreatments. However, the transition from very early larval stages,such as one nauplius stage proceeding to the next, which inmany cases also involves morphogenetic changes, may be occurringin the presence of MF. Indeed, MF appears to be stimulatoryto early postembryonic larval stages of Crustacea. Again, thisfunction of MF in Crustacea appears to be similar to functionsof JH in early postembryonic insects. However, it should bepointed out that there are many more "early" stages in Crustaceathan there are in insects, and very few of these cases havebeen investigated. When considering the animal kingdom and larval metamorphosis,the question may be raised whether there are other members ofthe JH family regulating metamorphosis and reproduction. Oneplausible example appears to be among certain annelids. Thetrochophores of Capitella respond to various juvenoids, butare most responsive, within one hour, to MF and eicosatrienoicacid. This latter compound is present also in adult annelids,where it has been named "Sperm Maturation Factor," since itseems to function in the maturation of sperm in Arenicola. Therefore,eicosanoids perform in annelids two functions performed in insectsby JHs. In conclusion, it seems that there are morphogenesis promotingresponses to JHs in early larval development in crustaceans,annelids, and possibly other forms, which differ from thoseMF effects in later larvae of Crustacea where MF retards morphogenesis.Such early responses as noted here have recently also been describedfor insects. Furthermore, it is clear that the polyunsaturated8,11,14-eicosatrienoic and aracidonic acids seem to be juvenoids,and appear to function as such in annelids, and may also befunctionally active in insects and crustaceans. It seems reasonableto conclude therefore that new and novel juvenoids exist, whileothers still await discovery.     

Albert Léon Charles Calmette (1863-1933) and the antituberculous BCG vaccination

Albert Léon Charles Calmette was the first person to develop an anti-venom serum. His work revolutionized the treatment of snakebite in men and domestic animals. He is also well known for his development of the Bacillus Calmette-Guérin (BCG) vaccine for the prevention of tuberculosis. This article reviews the life experiences of this pioneer bioscientist, the current status of BCG in preventing tuberculosis, and the potential of BCG in immunotherapy for cancer.     

Preservation of mouse sperm by convective drying and storing in 3-O-methyl-D-glucose

With the fast advancement in the genetics and bio-medical fields, the vast number of valuable transgenic and rare genetic mouse models need to be preserved. Preservation of mouse sperm by convective drying and subsequent storing at above freezing temperatures could dramatically reduce the cost and facilitate shipping. Mouse sperm were convectively dried under nitrogen gas in the Na-EGTA solution containing 100 mmol/L 3-O-methyl-D-glucose and stored in LiCl sorption jars (Relative Humidity, RH, 12%) at 4°C and 22°C for up to one year. The functionality of these sperm samples after storage was tested by intracytoplasmic injection into mouse oocytes. The percentages of blastocysts produced from sperm stored at 4°C for 1, 2, 3, 6, and 12 months were 62.6%, 53.4%, 39.6%, 33.3%, and 30.4%, respectively, while those stored at 22°C for 1, 2, and 3 months were 28.8%, 26.6%, and 12.2%, respectively. Transfer of 38 two- to four-cell embryos from sperm stored at 4°C for 1 year produced two live pups while 59 two- to four-cell embryos from sperm stored at 22°C for 3 months also produced two live pups. Although all the pups looked healthy at 3 weeks of age, normality of offspring produced using convectively dried sperm needs further investigation. The percentages of blastocyst from sperm stored in the higher relative humidity conditions of NaBr and MgCl(2) jars and driest condition of P(2)O(5) jars at 4°C and 22°C were all lower. A simple method of mouse sperm preservation is demonstrated. Three-O-methyl-D-glucose, a metabolically inactive derivative of glucose, offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of cell membrane.     

Are blastocyst prostaglandins produced endogenously?

This study was undertaken to determine whether preimplantation mouse and rabbit blastocysts possess cyclooxygenase activity and therefore are able to metabolize arachidonic acid (AA) to prostaglandins (PGs) and thromboxane B2. Single rabbit blastocysts or groups of 100 mouse blastocysts were incubated for 4 h in the presence of 5 muCi/ml [3H] AA (sp. ac. 61 Ci/mmol) or for 24 h and 18 h with 0.2 and 0.1 muCi/ml [14C] AA (sp. ac. 56.5 Ci/mmol), respectively. Incubated blastocysts were subsequently exposed for 10 min to 24 h to the Ca2+ specific ionophore, X-537A (10 microM), to stimulate release of radiolabeled AA from the phospholipid pool. After incubation, incorporation of AA into the phospholipid and neutral lipid pools, and metabolism of the fatty acid to PGs and thromboxane B2, were determined using thin-layer chromatographic (TLC) and liquid scintillation spectroscopic techniques. In addition, spent incubation media were analyzed for radiolabeled PG content. In blastocysts of both species, incorporation of AA into phospholipids was greater than that into neutral lipids (mouse: 1.0 vs. 0.7 pmol/blastocyst; rabbit: 159.7 vs. 56.9 pmol/blastocyst). The ionophore stimulated the release of AA from the phospholipid, and to a lesser extent, from the neutral lipid pool, of both blastocysts. No newly synthesized PGs were detected in either mouse blastocysts or their spent incubation media after stimulation with X-537A. Radiolabeled PGs (PGE2, PGF2 alpha, PGD2, PGA2) and thromboxane B2 were present in the media of rabbit blastocyst incubations, however, but were undetectable in the tissue extracts. Increases in metabolism of AA into each compound were observed with an increase in time of exposure to ionophore, and meclofenamic acid (2 microM) partially inhibited the synthesis of all compounds during a 24-h incubation. The results are discussed with regard to the role of blastocyst PGs in the events of implantation.     

Hematologic and enzymatic analysis of Ctenopharyngodon idella x Hypophthalmichthys nobilis F1 hybrids

1. Erythrocyte counts, hemoglobin concentrations and hematocrit values were determined for diploid and triploid Ctenopharyngodon idella X Hypophthalmichthys nobilis hybrids and the parental species. 2. Comparisons of diploid and triploid hybrids with the parental species revealed low erythrocyte counts for triploids, high mean corpuscular hemoglobin values for triploids, elevated hematocrits for diploids and triploids and similar hemoglobin concentrations for all fish. 3. Alkaline phosphatase, aldolase, and lactate dehydrogenase specific activities were determined spectrophotometrically. Levels of specific activity of these enzymes in the hybrids were consistently elevated above that of the parental species. These higher levels of enzyme activities in hybrids were probably the result of a breakdown in gene regulation.     

Noninvasive measurement of glucose uptake by two populations of murine embryos

Glucose uptake was measured by a noninvasive fluorescence technique on a total of 165 morula- and blastocyst-stage murine embryos in two different culture media. Eighty-four embryos were tested in M2 medium, and the remaining 81 embryos were tested in M16. Embryos assayed in M2 took up significantly less glucose over the 4-h assay period than did embryos assayed in M16. The lower uptake of glucose by embryos in M2 corresponded with a decrease in the quality of embryos cultured overnight in M2 as judged by morphological criteria. Embryos that were judged to be degenerate or had gross abnormalities took up significantly less glucose than did normal embryos. Glucose uptake in both populations of embryos covered a wide range of values and was normally distributed. A significant effect between mothers was noted in glucose uptake for embryos assayed in both M16 and M2 media. The possible uses of noninvasive measures of glucose uptake as a test of embryo viability or for optimizing culture conditions are discussed.