Ultrastructural studies of glycan changes in the apical surface of the uterine epithelium during pre-ovulatory and pre-implantation stages in the marmoset monkey

Abstract

It has been postulated that carbohydrates are involved in a variety of cell-cell interactions including blastocyst implantation. In primates, there are only limited investigations on the ultrastructural localisation of the cyclic changes in uterine epithelial surface carbohydrates. Our aim was to investigate such changes during the pre-ovulatory and pre-implantation stages of the reproductive cycle in the marmoset monkey.After fixation of endometrial tissues, avidin-ferritin lectin cytochemistry was employed for apical surface glycan detection at the ultrastructural level. Five lectins were used including Canavalia ensiformis (Con A), Lotus tetragonolobus (LTA), Glycine max (SBA), Phytolacca americana (PWM) and Triticum vulgaris (WGA). Morphometry was used to quantitate changes in the intensity of lectin staining by determining the total number of ferritin particles per unit length of membrane. Surface and intra-cytoplasmic vesicles, stained by the lectins, were also examined.Quantitative ferritin assessment showed that 1 day before presumed implantation (days 11 to 12 after ovulation in the marmoset monkey) there was a significant increase in Con A, LTA and SBA staining on the apical uterine epithelial plasma membrane compared to the pre-ovulatory phase and earlier stages of pregnancy (days 4-8 after ovulation). A significant increase in PWM was also detected from early pregnancy to preimplantation stages. All lectins except WGA produced reproducible staining within reproductive cycle groups. The greatest variation and intensity of epithelial surface staining was observed with WGA and the weakest with LTA. The patchy staining with LTA compared with thick coverage by WGA indicated the complexity of the carbohydrate arrangement in the glycocalyx of the uterine surface plasma membrane. Reduction of WGA reactivity after neuraminidase treatment suggested that the lectin binding might be related to the presence of heavily sialylated apical uterine membrane glycoconjugates.This is the first high-resolution study in primates to report quantitative cyclic changes in fucosyl, galactosyl, glucosyl, and mannosyl sugar residues of the apical uterine epithelial glycocalyx. The findings support the concept Abbreviations: DC, NЈN diacetyl chitobiose; Fuc, fucose; Glc, glucose; Man, mannose; GalNAc, N-acetyl galactosamine; GlcNAc, N-acetyl glucosamine; NeuNAc, neuraminic acid (sialyl residues); NЈdase, neuraminidase; sWGA, succinyl wheatgerm; WGA, native wheatgerm; nil, not applied; 0 to 7, lectin staining on a scale from 0 (negative) to 7 (heavy).

Characteristics of the uterine luminal surface epithelium at preovulatory and preimplantation stages in the marmoset monkey

“…As previously described (Niklaus et al, 1999), luteolysis and the initiation of a new reproductive cycle (day 1 of proliferative phase) was induced by an intramuscular injection of 0.5 g Estrumate (I.C.I., PitmanMoore, Melbourne, Australia). Ovulation (approximately 9 -10 days after luteolysis) was determined by a decrease in plasma progesterone levels to less than 10 ng/ml (Hodges et al, 1987).…”

“…Embryos were recovered in approximately 70% of cases at day 11 of pregnancy. At appropriate stages of the reproductive cycle, animals were anaesthetized by administering 0.8 ml of Saffan (Intervet, Melbourne, Australia) intramuscularly and uterine tissue was collected by hysterectomy or curettage as described previously (Niklaus et al, 1999). Three stages of the marmoset reproductive cycle were examined; early-late proliferation (preovulatory), days 4 -8 of pregnancy, and day 11 of pregnancy.…”

“…Hysterectomy specimens were obtained from five animals each at the respective stages: preovulatory (n ϭ 5), days 4 -8 of pregnancy (n ϭ 5), and day 11 of pregnancy (n ϭ 5). Most of these tissues had been previously used for lectin histochemistry studies of the apical luminal epithelium (Niklaus et al, 1999).…”

“…Random photos of surface epithelium were taken at a magnification of 28,000ϫ by using the microscope camera frame as a guide as described previously (Niklaus et al, 1999). A total of 30 electron photomicrographs from at least six blocks of tissue, enlarged to 90,000ϫ, were used per animal for MV analysis.…”

“…Chavez (1990) also mentioned that the uterine luminal epithelial surface of mice stained uniformly with CF and certain lectins but that individual cells showed heterogeneity. It is also interesting that the uterine luminal surface of the marmoset, reacted variably from cell to cell to a range of lectins, despite overall homogeneity across a large area of sampled epithelium (Niklaus et al, 1999). Other than protocol variation or species differences, this variability may be explained by the variation in the content and acidity of the uterine glycocalyx.…”