Why so many mammalian spermatozoa--a clue from marsupials?

Mammals generally ejaculate many more spermatozoa than seem to be needed for fertilization. This apparent profligacy has not been explained, but observations made in marsupials may shed light on it. The Virginia opossum, Didelphis virginiana, inseminates only about three million spermatozoa, a very low number. As a corollary, relatively few (ca. 13 X 10(6] are stored in each cauda epididymidis. However, some 5% of the spermatozoa that the opossum ejaculates populate the oviduct about 12 h later when ovulation can be anticipated--a success rate in the female orders of magnitude greater than in eutherian mammals. It is not certain what determines the unusually efficient transport to and the high survival rate of spermatozoa in the oviduct of Didelphis, but two unusual features suggest themselves as possible contributors. Didelphis (and all other American marsupial) spermatozoa undergo a head-to-head pairing in the epididymis by the acrosomal face; this serves to isolate the acrosome of ejaculated spermatozoa from the female milieu until the pairs separate in the oviduct. Secondly, spermatozoa are housed in special crypts in the isthmus of the oviduct. Australian marsupials, which usually lack such features, store spermatozoa in the epididymis in numbers more close to those in comparably sized eutheriam mammals. Exceptions which store very low sperm numbers there can be seen in one Australian Family, the Dasyuridae . The spermatozoa of dasyurids are not paired, but the species examined possess distinctive sperm storage crypts in the oviducal isthmus similar to those in the opossum. The present findings suggest that where mechanisms exist that could protect the acrosome and, or, the whole spermatozoon in the female tract, a much lower level of sperm production can be maintained without compromising fertility. While the number ejaculated typically by any one species is probably determined ultimately by several interacting factors, it therefore seems likely that a most important one in this respect relates to conditions spermatozoa face in the female tract.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

The covalent structure of cartilage collagen. Amino acid sequence of residues 552–661 of bovine α1(II) chains

The covalent structure of the first 111 residues from the N-terminus of peptide α1(II)-CB10 from bovine nasal-cartilage collagen is presented. This region comprises residues 552–661 of the α1(II) chain. The sequence was determined by automated Edman degradation of peptide α1(II)-CB10 and of peptides produced by cleavage with trypsin and hydroxylamine. Comparison of this region of the α1(II) chain with the homologous segment of the α1(I) chain indicated a homology level of 85%, slightly higher than that of 81% reported for the N-terminal region of the α1(II) chain (Butler, Miller & Finch (1976) Biochemistry 15, 3000–3006). The occurrence of two residues of glycosylated hydroxylysine was established at positions 564 and 603, the first present exclusively as galactosylhydroxylysine and the latter as a mixture of galactosylhydroxylysine and glucosylgalactosylhydroxylysine. Also, two residues at positions 648 and 657 were tentatively identified as glycosylated hydroxylysines. The amino acid sequences adjacent to the hydroxylysine residues so far identified in the α1(II) chain were compared with the homologous regions of the α1(I) and α2 chains, but no obvious prerequisite for hydroxylation could be seen. From comparison with the homologous sequence of the α1(I) chain, it appears that the α1(II)-chain sequence presented here contains three more amino acids than that reported for the α1(I) chain. This triplet would be interposed between residues 63 and 64 of the reported sequence of peptide α1(I)-CB7 from calf skin collagen. Data on the purification of the subpeptides and their amino acid compositions have been deposited as Supplementary Publication SUP 50087 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Determination of 2,4-diamino-5-benzylpyrimidines in combination with sulphadiazine in biological fluids using high-performance liquid chromatography

High-performance liquid chromatographic methods for the simultaneous analysis of tetroxoprim (TXP)/sulphadiazine (SDZ) and metioprim (MTP)/SDZ in serum and prostatic secretion (PS) are described. The detection limits in serum and PS were 50 ng TXP per ml and 100 ng SDZ per ml, and 40 ng MTP per ml and 100 ng SDZ per ml, respectively. The intra-assay coefficients of variation were in the range of 2.7–2.19%. Some preliminary data from a pharmacokinetic study in geriatric patients and a distribution study in dogs are presented. These methods enable the investigator to process a large number of TXP/SDZ and MTP/SDZ samples in one working day.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Unexpected oocyte growth after follicular antrum formation in four marsupial species.

During examination of maturing preovulatory marsupial oocytes we noted that oocyte diameters were invariably about 50% greater than the figures reported in earlier histological studies. As all previous investigations were limited to small follicles (at most 25% the size of the ovulating follicle), the present study was initiated to examine oocyte growth during the whole period of follicular development. Oocyte and follicle diameters were measured for three Australian (Trichosurus vulpecula, Macropus eugenii and Bettongia penicillata--fresh nonfixed material) and one American marsupial species (Monodelphis domestica--histological sections) in which multiple follicle development had been induced by exogenous gonadotrophin treatment. In all species oocytes were obtained from follicles ranging from pre-antral to immediately pre-ovulatory (maximum follicle sizes obtained were: T. vulpecula, 4.5 mm; M. eugenii, 4.3 mm; B. penicillata, 2.5 mm; M. domestica, 0.7 mm). In two of the species (T. vulpecula and B. penicillata) ovulated oocytes were also examined. In T. vulpecula and M. eugenii oocytes were found to achieve much greater diameters than previously reported from histological studies of small follicles (< 0.8 mm) and similar patterns of growth were found in the other two species. In the four species oocytes reached diameters about two to three times that found for eutherian mammals. It was concluded that the marsupial oocyte continued to grow after formation of the follicular antrum and that, although the rate of oocyte growth slowed in larger follicles, it continued into the period immediately before ovulation. In B. penicillata the largest oocytes were obtained after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that cortical granule formation is a periovulatory event in marsupials.

Formation of cortical granules was examined in superovulated oocytes from three marsupial species, brushtail possums (Trichosurus vulpecula) tammar wallabies (Macropus eugeniii) and grey short-tailed opossums (Monodelphis domestica) and in oocytes obtained during natural cycles in Macropus eugenii. Superovulation was induced by pregnant mares' serum gonadotrophin/gonadotrophin-releasing hormone (PMSG/GnRH) protocols and natural ovulation by removal of pouch young. Oocytes were collected after ovariectomy or by laparoscopically guided follicle aspiration into Hanks balanced salt solution (HBSS) supplemented with either 2.5% fetal calf serum (FCS) or 2.5% bovine serum albumin (BSA). Ovulated oocytes were collected by removing and flushing the oviducts with HBSS and fixed immediately for electron microscopy. There were no differences in the morphology or timing of formation of cortical granules between superovulated and naturally cycling animals. Cortical granules were absent from germinal vesicle (GV) stage follicular oocytes before the luteinizing hormone (LH) surge in all species. Dark cortical granules, similar in appearance to those seen in the oocytes of eutherian mammals, were found just beneath the plasma membrane (9 per 100 microns of plasma membrane) of preovulatory oocytes at germinal vesicle, metaphase 1 or anaphase 1 stages. In addition, they contained a number of less electron-dense cortical granules (12 per 100 microns plasma membrane). The cortical cytoplasm of preovulatory oocytes was rich in Golgi complexes actively involved in vesicle formation. Large numbers of dark cortical granules (90 per 100 microns plasma membrane) were found only in ovulated oocytes. A small number of cortical granules of lighter electron density were also present in ovulated oocytes. This suggests that the marsupial oocyte is following a very different timetable for cortical granule formation and accumulation from eutherian mammals and that oocytes of marsupials may not achieve cytoplasmic maturity until after ovulation. The significance of these events for fertilization and development remains to be established.     

A comparative study of chiasmata in male and femaleBettongia penicillata (Marsupialia)

The chiasma frequency and distribution does not differ substantially between the sexes in the marsupialBettongia penicillata. This result contrasts with the observations made on other species of marsupials.