Ultrasound-guided transfundal uterine sperm recovery from Macaca fascicularis

Previous studies from this center have indicated that the cynomolgus macaque (Macaca fascicularis) may serve as a model for human sperm interaction with the cervix and uterus. In some macaque species, transcervical aspiration of the uterine contents carries a significant risk of disturbing the cervical milieu due to the serpentine nature of the cervix. The only alternatives have been surgical procedures such as laparotomy or laparoscopy. In this paper, we report our experience with a new technique for ultrasound-guided sampling of spermatozoa in the macaque uterus. Twenty adult female cynomolgus macaques were monitored for menses (first day of menses = day l), and one mating per cycle was allowed on day 10, 11, or 12. In one group of ten animals, cervical mucus was sampled at 3 or 18 hr postcoitus (pc) and ultrasound-guided uterine aspiration was performed at 24 hours pc. In a second group of ten monkeys, uterine aspiration was at six hr pc and sperm numbers and motility were counted in the uterine fluid. Uterine fluid was obtained from fourteen of twenty monkeys. Pregnancy occurred in ten of the twenty experimental cycles. Ultrasound-guided uterine aspiration appears to be a reliable method for the evaluation of sperm transport in female macaques. The correlations between uterine sperm recovery and cervical mucus sperm populations arc discussed. The high conception rate in treatment cycles indicates that this procedure can be performed without apparent risk to pregnancy.     

Gletscherwinde und Ihr Einfluß auf die Pflanzenwelt

Ohne Zusammenfassung     

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.     

The emergence of ecological engineering as a discipline

Pioneering efforts in ecological engineering (a precedent setting engineering and applied science discipline in which the self-engineering capabilities of ecosystems are managed for the benefit of the environment and humankind) research and practice have proven to be tremendous strides toward establishing a new engineering discipline with a science base in ecology. Case studies, demonstrations and applications pertaining to restoration, rehabilitation, conservation, sustainability, reconstruction, remediation and reclamation of ecosystems using ecological engineering techniques are numerous. This has brought the field to the current level where many scientists and engineers adequately support the concept of, and need for, ecological engineering, and generally agree that ecological engineering has been sufficiently defined. There is also general agreement that full emergence as an engineering discipline remains a difficult task. Certain general characteristics of existing engineering disciplines can guide the emergence of ecological engineering and thus are a vital context covered in this paper. From the context of engineering practice, three concepts are evident including: (1) establishment of formal foundations for ecological engineering research and development; (2) development of core ecological engineering sciences and curricula; and (3) certification in ecological design. These elements are important components of a formal approach to develop ecological engineering as a principled, quantitative, recognized, practical, novel, and formal engineering discipline that coalesces past and future research and practice into cohesive underpinnings.     

Separate effects of caffeine and dbcAMP on macaque sperm motility and interaction with the zona pellucida

Capacitation of macaque sperm with caffeine and dbcAMP is required for fertilization in vitro. This study determined the separate effects of caffeine and dbcAMP on sperm-zona pellucida binding and the acrosome reaction of zona bound sperm. Semen from 6 cynomolgus macaques was washed through 60% Percoll, resuspended, and washed with BWW media and incubated for 2.5 hr. Caffeine, dbcAMP (2 mM each), or both (1 mM each) were added to aliquots of the sperm suspensions. Immature macaque oocytes were placed into drops of sperm suspensions, coincubated with sperm for 30 sec, and either fixed immediately or removed to sperm-free media and incubated 1 hr before fixation. There were no significant diffences between groups in the percentage of live, acrosome-reacted sperm in suspension. Treatment with caffeine and dbcAMP or with caffeine alone, significantly increased the number of sperm bound to each zona pellucida (96 ± 16 and 81 ± 17, respectively) compared to control and dbcAMP treatment (15 ± 4 and 28 ± 13). However, treatment with dbcAMP, alone and with caffeine, resulted in a higher percentage of acrosome-reacted sperm on the zona (15.2 ± 2.1 and 9.0 ± 0.6) than control or caffeine treatment (3.0 ± 1.4 and 2.4 ± 0.5). Effects on sperm motility consistent with hyperactivation were detected only when both caffeine and dbcAMP were present. Although both caffeine and dbcAMP are presumed to increase or to produce the same effects as increased intracellular cAMP levels, these compounds have different effects on the ability of sperm to bind to the zona and to undergo the acrosome reaction. © 1994 Wiley-Liss, Inc.     

Differentiated structure and function of primary cultures of monkey oviductal epithelium

Summary We have established well-differentiated, polarized cultures of monkey oviductal epithelium. Oviductal epithelial cells were isolated by protease digestion and plated on collagen-coated, porous cell culture inserts. About 5 d after plating, cells developed detectable transepithelial electrical resistance of up to 2000 Ω.cm² (an index of tight junction formation) and transepithelial voltages of up to 20 mV (an index of vectorial transepithelial ion transport). Measurements of short-circuit current in Ussing chambers indicated that active secretion of Cl was the major transepithelial active ion transport process, and that this was stimulated by elevation of either cAMP or Ca. Furthermore, estimates of the volume of mucosal liquid were consistent with Cl secretion mediating fluid secretion. Various microscopical methods showed that the cultures were densely ciliated and contained mature secretory cells. Transport across the oviductal epithelium determines the composition of the oviductal fluid, and the study of the relevant transport processes will be greatly enhanced by well-differentiated cultures of oviductal epithelium of the kind established here.     

Beta-defensin 126 on the cell surface protects sperm from immunorecognition and binding of anti-sperm antibodies

Beta-defensin 126 (DEFB126), formerly known as epididymal secretory protein 13.2 (ESP13.2), coats the entire primate sperm surface until completion of capacitation, and it is a candidate for providing immune protection in the female reproductive tract. To further examine the potential role of DEFB126 as a means of protection from immune recognition, cynomolgus macaque sperm were exposed to a number of treatments that are known to alter sperm surface coats, including capacitation. We used a novel in vivo assay to determine immune recognition: aldehyde-fixed whole sperm injections into rabbits. Following booster injections, immunoblot analyses of whole sperm prepared in various manners was conducted. On Days 60 and 80 post-initial immunization, the antisera showed a remarkably strong reaction to a single 34-36 kDa protein, which was shown to be DEFB126. Sera from rabbits that were immunized with sperm washed more rigorously using Percoll gradients showed an increase in the number and intensity of proteins recognized on whole sperm Western blots, although DEFB126 was still the major immune response. When capacitated sperm, from which most DEFB126 had been released, were used as the immunogen, there was a dramatic increase in the immune recognition to a variety of protein bands. Sperm treated with neuraminidase to remove sialic acid on DEFB126 before fixation were shown to still possess DEFB126, but lacked the sialic acid component of the glycoprotein. These sperm were as immunogenic as capacitated sperm even though the desialylated DEFB126 still covered the entire cell surface. These sperm lost their highly negative charge (the isoelectric point of DEFB126 shifted from pI 3.0 to pI 6.4). Experiments using different sperm plasma membrane protein-specific Igs showed that recognition did not occur when DEFB126 was present, but following capacitation these Igs readily recognized the exposed sperm membrane. Our data suggest that DEFB126 protects the entire primate sperm surface from immune recognition and that the sialic acid moieties are responsible for the cloaking characteristic of this unique glycoprotein.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Temporal pattern in swimming activity of two fish species (Danio rerio and Leucaspius delineatus) under chemical stress conditions

Circadian periodicity of swimming activity was investigated in two fish species, the zebrafish (Danio rerio) and the sunbleak (Leucaspius delineatus) under sublethal long-term exposure to the cyanobacteria toxin microcystin-LR (nominal concentrations of 0.5 μg l^- 1, 5 μg l^- 1, 15 μg l^- 1, 50 μg l^- 1) in 15-litre tanks. Swimming activity of fish was monitored continuously by using an automated video-monitoring and object-tracing system over a period of 17 days. Influenced by long-term exposure to microcystin-LR, Leucaspius delineatus reversed their significant diurnal swimming activity and the fish became statistically significant nocturnal. Danio rerio remained diurnal active, but a significant phase shift was registered. In both Danio rerio and Leucaspius delineatus analysis of time series by cosinor regression revealed microcystin-LR induced dose-dependent alterations of the mean of oscillation, amplitude, acrophase and period length in a different extent. For Danio rerio the periodogram analysis revealed a significant circadian component of swimming activity for control as well as exposure groups, whereby the spectral amplitude clearly decreased at microcystin-LR concentrations of 15 and 50 μg l^- 1. For Leucaspius delineatus the amplitude of circadian rhythm was decreased at all exposure concentrations of MC-LR. Furthermore the dominance of circadian rhythm was clearly reduced, whereas the rate of ultradian rhythms increased at elevated MC-LR concentrations of 5 μg l^- 1, 15 μg l^- 1 and 50 μg l^- 1. The studied temporal aspects of behaviour clearly indicated stress symptoms in both fish species, therefore it proved to be a relevant method to characterise the impact of toxic substances in the environment and for biomonitoring.