The mystery of the capsular vesicles: preliminary studies

Abstract

An unexpected experimental finding was the presence of capsular vesicles (CVs) in the uterine flushings of mares 48h after transfer of a punctured Day 8 embryo. These CVs were thought to originate from fragments of capsule, in the same way that trophoblast fragments readily form vesicles in vitro. However, recovery of the capsule with the puncture hole alongside numerous CVs showed this was not the case. A preliminary study was undertaken to see if CVs formed in recipient mares following transfer of trophoblast vesicles (TVs) for 24h and if CV formation was uterine stage specific. Day 11 embryos were recovered from donor mares and the capsule removed before fragmenting the trophoblast. Small fragments of trophoblast were incubated in DMEMF12 (+ 5% FBS and pen/strep) for 48h in a Tri-gas incubator at 38°C. After 48h any degenerate TVs were discarded, and the remainder were washed in H199 (+ 5% FBS + pen/strep). Twenty to 50 TVs were transferred to the uteri of 25 recipient mares divided into 4 groups; G1, Days 3–5 post ovulation (n=6); G2, Days 6–8 (n=7); G3, Days 9–11 (n=6); Days 12–14 (n=6). The recipient mares’ uteri were flushed 24h after TV transfer and the recovered fluid searched. In all groups TVs were recovered with varying degrees of degeneration. Data were analyzed using aOne Way ANOVA on ranks with a post-hoc Dunn's test. The median number of CVs varied between G1–4 (0 vs. 68 vs. 17.5 vs. 6.5, respectively), with G1 versus G2 varying significantly (P<0.05). When adjusted for CVs per transferred TV, median values for G1–4 were (0 vs. 1.6 vs. 0.51 vs. 0.225), with G1 versus G2 remaining significantly different (P<0.05). The highest number of CVs recovered was 148 (2.96 per TV) from a Day 8 recipient, with a trend for a reduction in CV production after Day 8. These results suggest that CVs can form in the uterus following TV transfer providing the uterus is at a stage when an embryo would normally be forming a capsule, since transfers before Day 6 failed to produce CVs. Immunohistochemistry needs to be undertaken to confirm the CVs are identical in composition to in vivo produced capsules. Capsule formation in the mare is believed to involve interaction of the embryo with the endometrium, as in vitro produced embryos fail to produce a proper capsule. In vivo, developmentally regulated changes in composition of the capsule occur as its thickness increases up until at least Day 11. The mechanism of the formation of capsular vesicles remains a mystery as they are free in the uterus and do not appear to form around the trophoblastic vesicles.