Functions of ectopically transplanted invasive horse trophoblast

Abstract

The invasive and fully antigenic trophoblast of the chorionic girdle portion of the equine fetal membranes has the capacity to survive and differentiate after transplantation to ectopic sites. The objectives of this study were to determine i) the survival time of ectopically transplanted allogeneic trophoblast cells in non-pregnant recipient mares, ii) whether equine chorionic gonadotropin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and iii) whether eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus. Ectopically transplanted chorionic girdle survived for up to 105 days with a mean lifespan of 75 days (95% confidence interval 55-94) and secreted sufficient eCG for the hormone to be measurable in the recipients' circulation. Immunohistochemical labeling of serial biopsies of the transplant sites and measurement of eCG profiles demonstrated that graft survival was similar to the lifespan of equine endometrial cups in normal horse pregnancy. The eCG secreted by the transplanted cells induced corpora lutea formation and sustained systemic progesterone levels in the recipient mares, effects that are also observed during pregnancy. This in turn caused suppression of estrus behavior in the recipients for up to 3 months. Thus, ectopically transplanted equine trophoblast provides an unusual example of sustained viability and function of an immunogenic transplant in a recipient with an intact immune system. This model highlights the importance of innate immunoregulatory capabilities of invasive trophoblast cells and describes a new method to deliver sustained circulating concentrations of eCG in non-pregnant mares.

Fig. 1. Direct detection of transplanted allogeneic trophoblast cells in the non-pregnant mare by biopsy and immunohistochemistry

(A) Chorionic girdle trophoblast cells were transplanted to the vulvar mucosa of non-pregnant nulliparous mares (Group 1 recipients, n=3, Table 1). Labeling was performed on cryostat sections of vulvar biopsies obtained at day 21, 28, 35, 42, 49 and 56 days post-transplantation using a monoclonal antibody against equine trophoblast (mAb 102.1). Images shown are from day 21, 35, 42 and 56 post-transplantation. Black arrows highlight trophoblast cells labeled red. The scale bar represents 100 µm. (B) Negative control section of a day 35 vulvar biopsy of a trophoblast transplant labelled with an isotype control mAb. Black arrow highlights trophoblast cells. The scale bar represents 100 µm. (C) Hematoxylin & Eosin stained formalin fixed section of vulvar biopsy of a trophoblast transplant obtained at day 20 post-transplantation. Large bi-nucleate trophoblast cells are visible in the figure. The scale bar represents 100 µm.

Fig. 2. Indirect detection of transplanted allogeneic trophoblast cells by measurement of secreted eCG

(A) Labeling of section from a day 28 biopsy of trophoblast transplant using anti-equine CG (mAb 67.1). Black arrow highlights eCG secreting trophoblast cells. Scale bar represents 100 µm. (B) Equine CG can be detected in the peripheral blood of biopsied trophoblast transplant recipient mares (Group 1 recipients, n=3, Table 1, ID numbers of mares are indicated).

Fig. 3. The mean lifespan of transplanted allogeneic trophoblast cells in the non-pregnant mare is 75 days

(A) Equine CG can be detected in the peripheral blood of recipient mares that did not have biopsies. Chorionic girdle trophoblast cells were injected into the vulvar mucosa of non-pregnant nulliparous mares (Group 2, Table 1, ID numbers of mares are indicated). (B) Lifespan of ectopically transplanted invasive trophoblast cells. The lifespan of invasive trophoblast cells following transplantation into Group 2 recipient mares was determined indirectly using eCG levels measured in (A) (see Materials and Methods for further details).

Fig. 4. Transplanted chorionic girdle trophoblast cells induce a period of prolonged diestrus in non-pregnant mares

eCG serum levels (left Y axis) and progesterone serum levels (P4) (right Y axis) in Group 2 invasive trophoblast transplant recipients and control recipient mares. Day 0 = day of transplantation for each individual recipient. The accession number of the recipient (from Table 1) is displayed above the graph.

Fig. 5. Transplanted chorionic girdle trophoblast cells modulate the ovarian physiology of non-pregnant mares

(A) The number of luteal structures present on the ovaries of Group 2 chorionic girdle trophoblast recipients (n=4) or control mares (n=4), determined using biweekly transrectal ultrasonography. The data represent the mean ± SEM for each group. Significant p values are shown (* p<0.05 and ** p<0.01) as determined using two-way ANOVA with Bonferroni’s correction. (B) Interovulatory period for chorionic girdle trophoblast recipients (n=4) and control mares (n=4). The number of days between consecutive ovulations was determined for chorionic girdle trophoblast recipients (n=4). The duration between ovulations was averaged over 2–3 cycles for control mares (n=4). Statistical difference in the interovulatory period was determined using Mann-Whitney test.

Fig. 6. Transplanted chorionic girdle trophoblast cells alter the behavior of non-pregnant mares

Transplanted chorionic girdle trophoblast cells modulate the reproductive behavior of non-pregnant mares. Estrous behavior scores in control mares (n=4, solid bars) and chorionic girdle trophoblast recipients (n=4, hatched bars). The data is presented in box and whiskers plots and the error bars represent the 95^th (upper error bars) and 5^th (lower error bars) percentile of the data.