SummaryPrevious methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean ± SD) expression of monocyte TF in normal subjects was very low (1.1 ± 0.95%, Mean Fluorescence [Mean FL] 0.20 ± 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 ± 11.2% (Mean FL 0.32 ± 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electroejaculation: 1) a constant-voltage method where stimulus current is a variable and 2) a constant-current method where stimulus current is operator-controlled. The constant-current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The influence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were detrimental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra- and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys. © 1996 Wiley‐Liss, Inc.

Previously we demonstrated high concentrations of desmosterol and docosahexaenoic acid (DHA, 22:6 n-3) in monkey testes and sperm. Desmosterol, a cholesterol precursor, is not present elsewhere in the body. High concentrations of DHA are found elsewhere only in the retina and brain. To examine the distribution of these compounds in the heads and tails of sperm, we separated them and determined their sterol, fatty acid, and phospholipid molecular species composition. Desmosterol predominated in tails (134.4 vs. 1.7 g/10 9 cells in heads). The cholesterol content was also greater in the tails (66.2 vs. 30.3 g/10 9 cells in heads).Sperm tails had more polyunsaturated fatty acids than the heads (34.1 vs. 12.1% of total fatty acids) which resulted mainly from the higher contents of DHA (19.6 vs. 1.1%) and arachidonic acid (20:4 n-6) (6.4 vs. 1.6%) in the tails. These differences in fatty acid composition occur red mainly in phospholipids: phosphatidyl choline and phosphatidyl ethanolamine for n-3 fatty acids and phosphatidylserine and cardiolipin for n-6 fatty acids. Fifteen phospholipid molecular species were identified. Sperm tails had more molecular species containing unsaturated fatty acids than the heads. Our results reveal the large differences in membrane lipid composition between the heads and tails of sperm. Most (99%) of the desmosterol and DHA in sperm is located in the tail. These differences may be responsible for the different functions of these two components of sperm. The large number of double bonds in DHA, six, and in desmosterol, two, may contribute to the membrane fluidity necessary for the motility of the sperm tails.

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