Ultrasonographic assessment of the endometrium in rhesus monkeys during the normal menstrual cycle

This study was undertaken to determine whether cyclical changes in the endometrium of the rhesus monkey could be observed by using ultrasound. Three indices of endometrial size were examined: the antero-posterior (or ventro-dorsal), longitudinal, and transverse diameters. Changes in the ultrasonic reflectivity of the endometrium were also assessed. We have attempted to correlate these endometrial parameters with the hormonal status of the animal. Ultrasonography was performed for an average of 12 consecutive days during 19 menstrual cycles. All ultrasonic recordings were normalized to the day of the estradiol (E2) peak (Day 0). We found that the reflectivity of the endometrium was dependent on the stage of the cycle: during the follicular phase, the endometrium appeared less echogenic (darker) compared to the myometrium; in the luteal phase, the endometrium was more echogenic (lighter). During the follicular phase (Days -9 to 0), there was a linear increase in the antero-posterior (p less than 0.001), longitudinal (p less than 0.05), and transverse (p less than 0.001) diameters. In the luteal phase (Days 1-15), no significant changes were observed in these diameters. An estimated endometrial volume (EEV) was obtained by the product of the antero-posterior, longitudinal, and transverse diameters. Each animal observed during the follicular phase (n = 14) exhibited a peak in the EEV, which correlated with the day of the E2 peak (p less than 0.01). From this study, we conclude that the sonographic appearance of the endometrium of the rhesus monkey reflects the cyclical changes that occur during the menstrual cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of blastocysts derived from in vitro-fertilized rhesus monkey ova

Six in vitro-fertilized ova were cultured for 10 to 13 days in vitro. All six had formed blastocysts with cavities, five had hatched from the zona pellucida, and one had attached to the substrate. After fixation and preparation for examination by light and electron microscopy, it was determined that all but the youngest blastocysts had developed substantial amounts of syncytial trophoblast, which morphologically resembled the syncytial trophoblast present in the first 2 days of implantation in vivo. One of the smaller blastocysts had developed syncytial trophoblast but had not hatched from the zona. All of the blastocysts showed indications of loss or inadequate development of the inner cell mass constituents, indicating that the culture conditions were suboptimal for these constituents. Apparent C-type virus particles were abundant, budding from the basal surface of the syncytial trophoblast. Because the type of trophoblast formed was that normally associated with epithelial invasion and formation of the trophoblast plate, it is suggested that such blastocysts would be useful for in vitro implantation studies as well as studies of formation of syncytial trophoblast. However, other methods should be developed for study of postimplantation embryo development. It is also noted that the inadequate differentiation of the epiblast and endoderm would not have been apparent without microscopic examination.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

A cell surface block to polyspermy occurs in golden hamster eggs

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique (R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148-154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the "unfusing" of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.     

Mitochondria in stem cells

The current status of knowledge about mitochondrial properties in mouse, monkey and human embryonic, adult and precursor stem cells is discussed. Topics include mitochondrial localization patterns, oxygen consumption and ATP content in cells as they relate to the maintenance of stem cell properties and subsequent differentiation of stem cells into specific cell types. The significance of the perinuclear arrangement of mitochondria, which may be a characteristic feature of stem cells, as well as the expression of mitochondrial DNA regulatory proteins and mutations in the mitochondrial stem cell genome is also discussed.     

Interactions between embryos and the culture milieu

Although in vitro production of embryos up to the blastocyst stage is now possible in numerous species, the quality and quantity of embryos are still not satisfactory. Clearly, culture conditions do not yet replace all of the benefits of development within the female reproductive tract. Analysis of the interactions between embryos and the components of culture media provides insights into regulatory mechanisms and how they are perturbed in vitro, and also offers some clues about the nature of the support provided to early embryos by the female tract. Further elucidation of these events and their underlying regulation will be helpful for improving culture media formulations to support normal embryo development in vitro.